Abstracts of papers presented

at the 2020 virtual meeting on

GENOME ENGINEERING:
CRISPR FRONTIERS
August 19–August 21, 2020
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 Abstracts of papers presented
at the 2020 virtual meeting on

GENOME ENGINEERING:
CRISPR FRONTIERS
August 19–August 21, 2020

Arranged by

Jennifer Doudna, University of California, Berkeley/HHMI

Maria Jasin, Memorial Sloan Kettering Cancer Center
David Liu, Broad Institute of Harvard and MIT
Jonathan Weissman, Whitehead Institute and MIT/HHMI
Support for this meeting was provided in part by BEX; Cell
Microsystems; IDT; and Millipore Sigma.

Contributions from the following companies provide core support for the
Cold Spring Harbor meetings program.

Corporate Benefactors

Estée Lauder Companies

Regeneron
Thermo Fisher Scientific

Corporate Sponsors

Agilent Technologies
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Genentech, Inc.
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Corporate Partners

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Enzo Life Sciences
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Lundbeck
Novartis Institutes for Biomedical Research
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___________________________________________________________

Cover: This year marks the 100th anniversary of the birth of Rosalind
Franklin, whose work led to the discovery of the structure of DNA. Her
legacy continues to this day, underpinning modern discoveries including the
first 3D structure of a CRISPR-Cas9-guided base editor, ABE8e, (Lapinaite,
Knott et al. Science 2020).

Rosalind Franklin photo by MRC Laboratory of Molecular Biology, CC BY-

SA 4.0.
 GENOME ENGINEERING: CRISPR FRONTIERS
Virtual Meeting
Wednesday, August 19 – Friday, August 21, 2020

Wednesday 10:00 am Rosalind Franklin Tribute

Wednesday 10:15 am 1 CRISPR Biology

Wednesday 2:00 pm 2 Technology I

Thursday 10:00 am 3 COVID

Thursday 2:00 pm 4 CRISPR Structure

Thursday 3:30 pm 5 Technology II

Friday 10:00 am 6 DNA Repair

Friday 2:00 pm 7 Plants

Friday 3:45 pm 8 Therapeutics

Throughout Meeting Virtual Posters

Virtual Bar, Wednesday 5:30 pm

IDT Workshop: Enhancing efficiency and specificity with improved CRISPR

components and a new CRISPR analysis pipeline (p. T-1)
Thursday, 5:30 pm

Cell Microsystems Workshop: Single cell cloning for automated and

efficient CRISPR workflows (p.T-2)
Friday, 5:30 pm

All times shown are US EDT: Time Zone Converter

 

Rosalind Franklin Obituary: BERNAL, J. Dr. Rosalind E. Franklin. Nature 182, 154 (1958).
https://doi.org/10.1038/182154a0. Used with permission.

J.D. Bernal determined the structure of water and pioneered the use of X-ray
crystallography for biological molecules. 
 PROGRAM

WEDNESDAY, August 19—10:00 AM US EDT

Special Tribute
Remembering Rosalind Franklin on her 100th birthday

Fyodor Urnov
Innovative Genomics Institute, Berkeley, California

WEDNESDAY, August 19—10:15 AM US EDT

SESSION 1 CRISPR BIOLOGY

Chairperson: Joshua Modell, Johns Hopkins School of Medicine,

Baltimore, Maryland

CRISPR and coronavirus

Jennifer Doudna.
Presenter affiliation: University of California, Berkeley / HHMI,
Berkeley, California.

Growing diversity of unique CRISPR-Cas-related systems and

Cas1 homologs
Kira S. Makarova, Yuri I. Wolf, Serge A. Shmakov, Yang Liu, Meng Li,
Eugene V. Koonin.
Presenter affiliation: National Institutes of Health, National Center for
Biotechnology Information, Bethesda, Maryland. 1

CRISPR-assisted engineering of bacterial viruses to combat

antibiotic-resistant pathogens
S.M. Nayeemul Bari, Asma Hatoum-Aslan.
Presenter affiliation: The University of Alabama, Tuscaloosa, Alabama. 2

Multiple mechanisms of inhibition of CRISPR-Cas9

Karen Maxwell.
Presenter affiliation: University of Toronto, Toronto, Canada. 3

The ups and downs of CRISPR-Cas systems in Streptococcus

thermophilus
Sylvain Moineau.
Presenter affiliation: Université Laval, Félix d'Hérelle Reference Center
for Bacterial Viruses, Québec City, Canada. 4

v
 Phase-dependent CRISPR evolution
Blake Wiedenheft.
Presenter affiliation: Montana State University, Bozeman, Montana. 5

Killing the messenger The how and why of type III CRISPR
deactivation
Januka S. Athukoralage, Stuart McQuarrie, Sabine Grüschow, Shirley
Graham, Tracey M. Gloster, Malcolm F. White.
Presenter affiliation: Biomedical Sciences Research Complex,
University of St Andrews, St Andrews, United Kingdom. 6

CRISPR vs natural competence Altruistic group defense more

than individual immune system
Robert M. Cooper, Jeff Hasty.
Presenter affiliation: UC San Diego, La Jolla, California. 7

WEDNESDAY, August 19—2:00 PM US EDT

SESSION 2 TECHNOLOGY I

Chairperson: Shengdar Tsai, St. Jude Children's Research Hospital,

Memphis, Tennessee

Genome-wide programmable transcriptional memory by CRISPR-

based epigenome editing
J.K. Nunez, J. Chen, G.C. Pommier, J.Z. Cogan, G.N. Ramadoss, J.M.
Replogle, A.J. Samelson, A.N. Pogson, A. Chung M.D. Leonetti, M.
Kampmann, L.A. Gilbert, J.S. Weissman.
Presenter affiliation: University of California, San Francisco, San
Francisco, California.

Applications of minimal-PAM CRISPR-Cas enzymes

Kathleen A. Christie, Russell T. Walton, Madelynn N. Whittaker,
Benjamin P. Kleinstiver.
Presenter affiliation: Massachusetts General Hospital, Boston,
Massachusetts; Harvard Medical School, Boston, Massachusetts. 8

High-throughput discovery and characterization of human

transcriptional repressor and activator domains
Josh Tycko, Nicole DelRosso, Gaelen Hess, Aradhana, Abhimanyu
Banerjee, Aditya Mukund, Mike Van, Braeden Ego, David Yao, Kaitlyn
Spees, Anshul Kundaje, Michael C. Bassik, Lacramioara Bintu.
Presenter affiliation: Stanford University, Stanford, California. 9

vi
Multiplex enCas12a screens show functional buffering by
paralogs is systematically absent from genome-wide
CRISPR/Cas9 knockout screens
Merve Dede, Megan McLaughlin, Eiru Kim, Traver Hart.
Presenter affiliation: The University of Texas MD Anderson Cancer
Center, Houston, Texas. 10

Functional annotation of gene domains in living cells

Jake Herman, Lucas Carter, Sue Biggins, Patrick Paddison.
Presenter affiliation: Fred Hutchinson Cancer Research Center,
Seattle, Washington. 11

Genetic interaction mapping and exon-resolution functional

genomics with a hybrid Cas9–Cas12a platform
Thomas Gonatopoulos-Pournatzis, Michael Aregger, Kevin R. Brown,
Sha Farhangmehr, Ulrich Braunschweig, Henry N. Ward, Kevin Ha,
Max Billmann, Chad Myers, Ben Blencowe, Jason Moffat.
Presenter affiliation: University of Toronto, Toronto, Canada; National
Cancer Institute (NCI/NIH), Frederick, Maryland. 12

Writing large DNA sequences into the genome with novel

enzymatic machinery
Barrett Steinberg, Yanfang Fu, Donghui Li, Tim Sullivan, Emily Kibbler,
Alexas Dolbee, Sidney Hofmann, Christopher Bartolome, Inna
Shcherbakova, Anne Bothmer, Wes Salomon, Rob Citorik, Stephen
Cleaver, Jacob Rubens, Cecilia Cotta-Ramusino.
Presenter affiliation: Tessera Therapeutics, Cambridge,
Massachusetts. 13

CRISPR RNA-guided integrases for high-efficiency and

multiplexed bacterial genome engineering
Phuc Leo Vo, Carlotta Ronda, Sanne Klompe, Ethan Chen,
Christopher Acree, Harris Wang, Samuel Sternberg.
Presenter affiliation: Columbia University Irving Medical Center, New
York, New York. 14

Improving the chemical synthesis and performance of extended-

length guide RNAs for genome editing and control of gene
expression
Laurakay Bruhn, Bo Curry, Rob Kaiser, Ben Lunstad, Ryan McCaffrey,
Joel Myerson, Subhadeep Roy, Daniel Ryan, Israel Steinfeld, David
Taussig, Suhani Thakker, Doug Dellinger.
Presenter affiliation: Agilent Technologies, Santa Clara, California. 15

vii
 Studying the physiological effects of mutations conferring
insecticide resistance in Drosophila melanogaster and the
application of allelic drive for reverting these mutations in fly
populations
Bhagyashree D. Kaduskar, Raja babu Singh Khushwa, Ankush
Auradkar, Alison Henrique Ferreira Julio, Annabel Guichard, Ethan
Bier.
Presenter affiliation: Tata Institute for Genetics and Society (TIGS-
India), Bangalore, India; Tata Institute for Genetics and Society (TIGS-
UCSD), San Diego, California. 16

CRISPR-Cas from huge phages is a hypercompact genome

editor
Patrick Pausch, Basem Al-Shayeb, Ezra Bisom-Rapp, Connor A.
Tsuchida, Zheng Li, Brady F. Cress, Gavin J. Knott, Steven E.
Jacobsen, Jillian F. Banfield, Jennifer A. Doudna.
Presenter affiliation: University of California, Berkeley, California 17

THURSDAY, August 20—10:00 AM US EDT

SESSION 3 COVID

Chairperson: J. Keith Joung, Massachusetts General Hospital,

Harvard Medical School, Boston, Massachusetts

Harnessing nature’s diversity for gene editing and beyond

Feng Zhang.
Presenter affiliation: Howard Hughes Medical Institute, Chevy Chase,
Maryland; Broad Institute of MIT and Harvard, Cambridge,
Massachusetts; Massachusetts Institute of Technology, Cambridge,
Massachusetts. 18

Direct detection of SARS-CoV-2 with CRISPR Cas13a and a

mobile phone
Melanie Ott.
Presenter affiliation: UCSF, San Francisco, California.

Massively multiplexed nucleic acid detection with CARMEN-

Cas13
Cameron Myhrvold.
Presenter affiliation: Broad Institute of MIT and Harvard, Cambridge,
Massachusetts.

viii
 Detection of SARS-CoV-2 with the CRISPR-based DETECTR
platform
Janice Chen.
Presnter affiliation: Mammoth Biosciences, South San Francisco,
California.

Development of CRISPR as an antiviral strategy to combat SARS-

CoV-2 and RNA viruses
Stanley Qi.
Presenter affiliation: Stanford University, Stanford, California. 19

CRISPR screening for COVID-19

Patrick Hsu.
Presenter affiliation: University of California-Berkeley, Berkeley,
California.

Boostrapping a COVID-19 surveillance program with CRISPR

Max Wilson.
Presenter affiliation: University of California-Santa Barbara, Santa
Barbara, California.

PANEL DISCUSSION

Discussion Leader: Alexis Komor, University of California, San Diego

Panelists

Walter Isaacson, Tulane University

Michael Mina, Harvard University T.H. Chan School of Public Health
Bruce Tromberg, NIH RADx Program, National Institute of Biomedical Imaging
and Bioengineering

CRISPR tools in the time of COVID-19 research Trends from

Addgene
Susanna M. Bachle, Jennifer Tsang, Brook Pyhtila, Joanne Kamens,
Andrew Hempstead.
Presenter affiliation: Addgene, Watertown, Massachusetts. 20

ix
 THURSDAY, August 20—2:00 PM US EDT

SESSION 4 CRISPR STRUCTURE

Chairperson: Audrone Lapinaite, Arizona State University, Tempe

DNA cleavage by class 2 CRISPR-Cas systems

Wei Sun, Zhi Cheng, Xue Huang, Jing Yang, Liang Liu, Jiuyu Wang,
Xueyan Li, Yanli Wang.
Presenter affiliation: Chinese Academy of Sciences, Beijing, China;
University of Chinese Academy of Sciences, Beijing, China. 21

Structure-guided mini and PAM-flex Cas9

Osamu Nureki.
Presenter affiliation: The University of Tokyo, Graduate School of
Science, Tokyo, Japan. 22

Structural insights into the off-target activity of Cas9

Martin Pacesa, Chun-Han Lin, Antoine Cléry, Katja Bargsten, Matthew
J. Irby, Katharina Stengel, Frédéric H.T. Allain, Peter Cameron, Paul
Donohoue, Martin Jinek.
Presenter affiliation: University of Zurich, Zurich, Switzerland. 23

Structures of the Cmr- complex reveal the regulation of the

immunity mechanism of type III-B CRISPR-Cas
Nicholas Sofos, MIngxia Feng, Stefano Stella, Tillmann Pape, Qunxin
She, Guillermo Montoya.
Presenter affiliation: Structural Molecular Biology Group, Novo Nordisk
Foundation Center for Protein Research. University of Copenhagen,
Copenhagen, Denmark. 24

x
 THURSDAY, August 20—3:30 PM US EDT

SESSION 5 TECHNOLOGY II

Chairperson: Alexis Komor, University of California, San Diego

CRISPR-free mitochondrial base editing enabled by an

interbacterial cytidine deaminase toxin
Beverly Y. Mok, Marcos H. de Moraes, Jun Zeng, Dustin E. Bosch,
Anna V. Kotrys, Aditya Raguram, FoSheng Hsu, Matthew C. Radey, S.
Brook Peterson, Vamsi K. Mootha, Joseph D. Mougous, David R. Liu.
Presenter affiliation: Broad Institute of Harvard and MIT / Howard
Hughes Medical Institute, Cambridge, Massachusetts. 25

Cas12a base editors induce efficient and specific editing with low
DNA damage response
Xiao Wang, Chengfeng Ding, Wenxia Yu, Ying Wang, Siting He, Bei
Yang, Xingxu Huang, Zhen Liu, Li Yang, Jia Chen.
Presenter affiliation: ShanghaiTech University, Shanghai, China. 26

Baculovirus vectored highly efficient large cargo DNA docking

and prime editing in genomes
Francesco Aulicino, Martin Pelosse, Christine Toelzer, Julien Capin,
Parisa Meysami, Christiane Berger-Schaffitzel, Mark Dillingham, Imre
Berger.
Presenter affiliation: University of Bristol, Bristol, United Kingdom;
BrisSynBio, Bristol, United Kingdom. 27

PrimeDesign software for rapid and simplified design of prime

editing guide RNAs
Jonathan Y. Hsu, Andrew V. Anzalone, Julian Grünewald, Kin Chung
Lam, Max W. Shen, Karl Petri, David R. Liu, J. Keith Joung, Luca
Pinello.
Presenter affiliation: Massachusetts Institute of Technology,
Cambridge, Massachusetts; Massachusetts General Hospital,
Charlestown, Massachusetts; Harvard Medical School, Boston,
Massachusetts. 28

xi
 Comparative analysis of conventional CRISPR-Cas9 editing and
prime editing of a regulatory element in mice
Pan Gao, Qing Lyu, Amr R. Ghanam, Cicera R. Lazzarotto, Gregory A.
Newby, Wei Zhang, Mihyun Choi, Kevin Holden, John A. Walker,
Anastasia P. Kadina, Rob J. Munroe, Christian M. Abratte, John C.
Schimenti, David R. Liu, Shengdar Q. Tsai, Xiaochun Long, Joseph M.
Miano.
Presenter affiliation: Medical College of Georgia at Augusta University,
Augusta, Georgia. 29

High throughput proteome analysis of gRNA-independent off-

targeting of base editors
Cristina Rocha, Rakesh Chandode, Anna Lina Cavallo, Roberto
Nitsch.
Presenter affiliation: AstraZeneca R&D, Gothenburg, Sweden. 30

Determinants of base editing outcomes from target library

analysis and machine learning
Mandana Arbab, Max W. Shen, Beverly Mok, Chris Wilson, Zaneta
Matuszek, Chris Cassa, David R. Liu.
Presenter affiliation: Broad Institute of MIT and Harvard, Cambridge,
Massachusetts. 31

FRIDAY, August 21—10:00 AM US EDT

SESSION 6 DNA REPAIR

Chairperson: Brittany Adamson, Princeton University, New Jersey

Using CRISPR-based genetic screens to study DNA repair

Michele Olivieri, Alejandro Alvarez-Quilon, Tiffany Cho, Daniel
Durocher.
Presenter affiliation: The Lunenfeld-Tanenbaum Research Institute,
Toronto, Canada. 32

Large-scale phenotypic characterization of genetic variants of the

DNA damage response using CRISPR-dependent base editing
Raquel Cuella-Martin, Samuel B. Hayward, Xiao Fan, Xiao Chen,
Junfei Zhao, Raul Rabadan, Chao Lu, Yufeng Shen, Alberto Ciccia.
Presenter affiliation: Columbia University Irving Medical Center, New
York, New York. 33

xii
Understanding mechanisms of genome editing with high-
resolution functional genomics
Jeffrey A. Hussmann, Purnima Ravisankar, Jun Yan, Albert Xu, Anne
Bothmer, Cecilia Cotta-Ramusino, Jonathan S. Weissman, Britt
Adamson.
Presenter affiliation: UCSF, San Francisco, California. 34

High-throughput approaches to controlling Cas9 and dCas9

outcomes
Lin Lin, Ersin Akinci, Max Shen, Richard Sherwood.
Presenter affiliation: Brigham and Women's Hospital and Harvard
Medical School, Boston, Massachusetts. 35

Mitotic errors and massive chromosome rearrangement are on-

target consequences of genome editing
Mitchell L. Leibowitz, Stamatis Papathanasiou, Phillip A. Doerfler,
Logan J. Blaine, Lili Sun, Yu Yao, Cheng-Zhong Zhang, Mitchell J.
Weiss, David Pellman.
Presenter affiliation: Harvard Medical School, Boston, Massachusetts;
Dana-Farber Cancer Institute, Boston, Massachusetts. 36

Maria Jasin.
Presenter affiliation: Memorial Sloan-Kettering Cancer Center, New
York, New York.

A Rad51-independent pathway promotes single-strand template

repair in gene editing
Danielle N. Gallagher, Nhung Pham, Annie M. Tsai, Abigail N. Janto,
Grzegorz Ira, James E. Haber.
Presenter affiliation: Brandeis University, Waltham, Massachusetts. 37

Comparison of I-SceI and SpCas9 double-strand break end

tethering in vivo
So Jung Lee, Rodney Rothstein.
Presenter affiliation: Columbia University Medical Center, New York,
New York. 38

CRISPR/Cas-mediated chromosome engineering in Arabidopsis

thaliana
Michelle Roenspies, Carla Schmidt, Natalja Beying, Andreas Houben,
Paul Fransz, Holger Puchta.
Presenter affiliation: Karlsruhe Institute of Technology, Karlsruhe,
Germany. 39

xiii
 Precise introduction of small genetic changes without bystander
mutations in vitro and in vivo by scarless gene editing
Brandon W. Simone, Ata Hiro, Han B. Lee, Camden L. Daby, Stephen
C. Ekker, Karl J. Clark.
Presenter affiliation: Mayo Clinic, Rochester, Minnesota. 40

FRIDAY, August 21—2:00 PM US EDT

SESSION 7 PLANTS

Chairperson: Zachary Lippman, Cold Spring Harbor Laboratory,

New York

Targeted insertion of a large DNA fragment using CRISPR-Cas9 in

the model plant rice
Oliver Xiaoou Dong, Shu Yu, Rashmi Jain, Phat Duong, Nan Zhang,
Corinne Butler, Yan Li, Anna Lipzen, Joel Martin, Kerrie Barry, Jeremy
Schmutz, Li Tian, Pamela C. Ronald.
Presenter affiliation: University of California, Davis, Davis, California. 41

Dissecting quantitative trait variation by genome editing

Zach Lippman.
Presenter affiliation: Cold Spring Harbor Laboratory, Cold Spring
Harbor, New York. 42

CRISPR-mediated targeting of epigenetic modifications in plants

Steven E. Jacobsen, Javier Gallego-Bartolome, Ashot Papikian,
Basudev Ghoshal, Jason Gardiner, Wanlu Liu.
Presenter affiliation: HHMI/University of California, Los Angeles, Los
Angeles, California. 43

Overcoming bottlenecks in editing plant genomes

Dan Voytas.
Presenter affiliation: University of Minnesota, St. Paul, Minnesota. 44

Precise gene editing in crops

Yu Mei, John Gierer, Theodore Moll, Gengxiang Jia, Behailu Aklilu,
Presenter affiliation: KWS, St Louis, Missouri. 45

xiv
 FRIDAY, August 21—3:45 PM US EDT

SESSION 8 THERAPEUTICS

Chairperson: Fyodor Urnov, Innovative Genomics Institute, Berkeley,

California

ONE-seq A universal platform for defining off-target profiles of

CRISPR gene editors with unsurpassed sensitivity
Karl Petri, Daniel Y. Kim, Kanae E. Sasaki, Matthew C. Canver, Xiao
Wang, Hyunho Lee, Hina Shah, Joy E. Horng, Kendell Clement,
Sowmya Iyer, Sara P. Garcia, Jimmy A. Guo, Gregory A. Newby, Luca
Pinello, David R. Liu, Martin J. Aryee, Kiran Musunuru, J Keith Joung.
Presenter affiliation: Massachusetts General Hospital, Charlestown,
Massachusetts. 46

Perturbing gene regulatory networks dysregulated in CHD8-

related autism using CRISPR-based transcription factors
Lexi R. Bounds, Charles A. Gersbach.
Presenter affiliation: Duke University, Durham, North Carolina. 47

Modelling mutations and variants that alter gamma globin

expression using genome editing in erythroid cells
Gabriella E. Martyn, Beeke Wienert, Elizabeth S. Stout, Lu Yang,
Manan Shah, Lana C. Ly, Merlin Crossley, Kate G. Quinlan.
Presenter affiliation: UNSW Sydney, Sydney, Australia. 48

Directed evolution of adenine base editors with increased activity

and therapeutic application
Nicole M. Gaudelli, Dieter K. Lam, Holly A. Rees, Noris M. Sola-
Esteves, Luis A. Barrera, David A. Born, Aaron Edwards, Jason M.
Gehrke, Seung-Joo Lee, Alexander J. Liquori, Ryan Murray, Michael
S. Packer, Conrad Rinaldi, Ian M. Slaymaker, Jonathan S. Yen,
Lauren E. Young, Giuseppe Ciaramella.
Presenter affiliation: Beam Therapeutics, Cambridge, Massachusetts. 49

A pooled screen to engineer and optimize Cas RNP variants for

cell-targeted delivery
Akshay Tambe, Rina J. Mepani, Aaron J. Cantor, Sruja S. Iyer,
Angelica F. Castaneda, Hariharan Jayaram.
Presenter affiliation: Spotlight Therapeutics, Hayward, California. 50

xv
Translating adenine base editor technology into a potential
treatment for lung and liver manifestations of alpha-1 antitrypsin
deficiency
Michael S. Packer, Vivek Chowdhary, Genesis Lung, Lo-I Cheng,
Yvonne Aratyn-Schaus, Sarah Smith, Aalok Shah, Delai Chen, Marina
Zieger, Brian Cafferty, Bo Yan, Christian Mueller, Giuseppe
Ciaramella, Francine M. Gregoire.
Presenter affiliation: Beam Therapeutics, Cambridge, Massachusetts. 51

VIRTUAL POSTER SESSION

Assessing the importance of the kinase domain through kinome-

focused CRISPR screening
Ahmad Al Kawam, Alexandra Grassian, Fabien Llambi, Leni Jacob, View
Zhigang Weng, Klaus Hoeflich, Chaoyang Ye. Poster
Presenter affiliation: Blueprint Medicines, Cambridge, Massachusetts. 52

Understanding homology-directed repair in Aedes aegypti

germline engineering
Joshua Ang Xin De, Katherine Nevard, Rebekah Ireland, Lewis View
Shackleford, Estela Gonzalez, Michelle A. Anderson, Luke Alphey. Poster
Presenter affiliation: The Pirbright Institute, Pirbright, United Kingdom. 53

DNA binding by Cas9 interferes with base excision repair

efficiency
Jacob Antony, John Wyrick, John Hinz. View
Presenter affiliation: Washington State University, Pullman, Poster
Washington. 54

Systematic mapping of genetic interactions for metabolic genes

using CRISPR-Cas screens
Michael Aregger, Keith Lawson, Maximilian Billmann, Michael
Costanzo, Thomas Gonatopoulos-Pournatzis, Kevin R. Brown, Anne-
Claude Gingras, Benjamin J. Blencowe, Chad L. Myers, Brenda J.
Andrews, Charles Boone, Jason Moffat. View
Presenter affiliation: University of Toronto, Toronto, Canada; National Poster
Cancer Institute (NCI/NIH), Frederick, Maryland. 55

xvi
Genome-wide identification of quantitative genetic interactions in
human cells using CRISPR/Cas9 screens
Maximilian Billmann, Mahfuzur Rahman, Amy Tong, Katherine Chan,
Michael Costanzo, Henry Ward, Michael Aregger, Matej Usaj,
Catherine Ross, Kevin Brown, Brenda Andrews, Charles Boone,
Jason Moffat, Chad Myers. View
Presenter affiliation: University of Minnesota-Twin Cities, Minneapolis, Poster
Minnesota. 56

Increasing genome editing efficiency with heat treatment in

Arabidopsis thaliana View
Jonas Blomme, Thomas Jacobs. Poster
Presenter affiliation: Ghent University, Ghent, Belgium. 57

Precise genome engineering in Drosophila using prime editing

Justin A. Bosch, Gabriel Birchak, Norbert Perrimon. View
Presenter affiliation: Blavatnick Institute, Harvard Medical School, Poster
Boston, Massachusetts. 58

Reducing Cas9 half-life leads to efficiency enhancement and

reduces mosaicism
Amine Bouchareb, Samy Alghadban, Phalguni Rath, Polinka
Hernandez-Pliego, Daniel Biggs, Chris Preece, Ben Davies. View
Presenter affiliation: Wellcome Trust Centre for Human Genetics, Poster
Oxford, United Kingdom. 59

High-performance CRISPR-Cas12a genome editing for

combinatorial genetic screening
Rodrigo A. Gier, Krista A. Budinich, Niklaus H. Evitt, Zhendong Cao,
Elizabeth S. Freilich, Qingzhou Chen, Jun Qi, Yemin Lan, Rahul M.
Kohli, Junwei Shi. View
Presenter affiliation: Perelman School of Medicine, University of Poster
Pennsylvania, Philadelphia, Pennsylvania. 60

Base-editing mediated targeting of progerin accumulation

Diana Campos-Iglesias, Jose Maria P. Freije, Carlos Lopez-Otin. View
Presenter affiliation: Universidad de Oviedo, Oviedo, Spain; Centro de Poster
Investigación Biomédica en Red de Cáncer, Madrid, Spain. 61

Programmable m6A modification of cellular RNAs with a Cas13-

directed methyltransferase
Christopher Wilson, Peter J. Chen, Zhuang Miao, David R. Liu. View
Presenter affiliation: Harvard University, Cambridge, Massachusetts; Poster
Broad Institute of Harvard and MIT, Cambridge, Massachusetts. 62

xvii
CRISPR-mediated multiplexed live cell imaging of nonrepetitive
genomic loci
Patricia A. Clow, Nathaniel Jillette, Jacqueline J. Zhu, Albert W.
Cheng.
Presenter affiliation: The Jackson Laboratory, Farmington, View
Connecticut; The Jackson Laboratory, Bar Harbor, Maine; University of Poster
Connecticut Health Center, Farmington, Connecticut. 63

JACKIE Enumeration of all single- and multi-copy CRISPR

binding sites in a genome
Jacqueline J. Zhu, Albert W. Cheng.
Presenter affiliation: The Jackson Laboratory, Farmington, View
Connecticut; The Jackson Laboratory, Bar Harbor, Maine; University of Poster
Connecticut Health Center, Farmington, Connecticut. 64

Enhanced precision of base editing with near-PAMless CRISPR-

Cas9 variants
Kathleen A. Christie, Russell T. Walton, Madelynn N. Whittaker,
Benjamin P. Kleinstiver. View
Presenter affiliation: Massachusetts General Hospital, Boston, Poster
Massachusetts; Harvard Medical School, Boston, Massachusetts. 65

A base editing approach to eliminate sickle hemoglobin

S. Haihua Chu, Daisy Lam, Jenny Olins, Alex Liquori, Jeff Marshall,
Jeremy Decker, Tanggis Bohnuud, Luis Barrera, Ian Slaymaker,
Michael Packer, Nicole Gaudelli, Adam Hartigan, Giuseppe View
Ciaramella. Poster
Presenter affiliation: Beam Therapeutics, Cambridge, Massachusetts. 66

C9orf72 frontotemporal dementia (FTD) and amyotrophic lateral

sclerosis (ALS) Using patient cells and CRISPR to reveal
therapeutic approaches
Claire D. Clelland, Sachdev Aradhana, Alisha Birk, Hannah L. Watry,
Luke M. Judge, Bruce R. Conklin. View
Presenter affiliation: Gladstone Institutes, San Francisco, California; Poster
UCSF, San Francisco, California. 67

Standardized quantification of prime editing experiments

Kendell Clement, J. Keith Joung, Luca Pinello. View
Presenter affiliation: Massachusetts General Hospital, Boston, Poster
Massachusetts. 68

xviii
Dominant negative disease, ideal models for allele-specific
therapeutic editing
Bruce R. Conklin, Katie Gjoni, Gokul N. Ramadoss, Juan A. Perez-
Bermejo, Alisha M. Birk, Carissa M. Feliciano, Aradhana R. Sachdev,
Georgia L. Gregory, Hana Y. Ghanim, Hannah L. Watry, Kathleen C.
Keough, Matthew A. Carter, Po-Lin So So, Tessa Dewell, Beeke
Wienert, Zachary S. Nevin, Claire D. Clelland, Luke M. Judge.
Presenter affiliation: Gladstone Institutes, San Francisco, California; View
Innovative Genomics Institute, Berkeley, California; UCSF, San Poster
Francisco, California. 69

The EMBL-EBI Genome Targeting Catalogue

Sybilla Corbett, Thomas Juettemann, Myrto Kostadima, Garth Ilsley,
Fiona Cunningham, Daniel R. Zerbino. View
Presenter affiliation: European Molecular Biology Laboratory, Hinxton, Poster
United Kingdom. 70

Creation of floxed alleles by electroporating two CRISPR RNPs

and two ssODNs
Monica F. Sentmanat, Michael White, Xiaoxia Cui. View
Presenter affiliation: Washington University in St. Louis, St Louis, Poster
Missouri. 71

A homology independent sequence replacement strategy in

human cells using a CRISPR nuclease
Eric W. Danner, Mikhail Lebedin, Kathrin de la Rosa, Ralf Kühn. View
Presenter affiliation: Max Delbrück Center for Molecular Medicine, Poster
Berlin, Germany. 72

CRISPR-Cas9 reprogramming of human stem cells into motor

neurons
Katie Davis-Anderson, Sofiya Micheva-Viteva, Jennifer Harris, Scott
Twary, Rashi Iyer. View
Presenter affiliation: Los Alamos National Laboratory, Los Alamos, Poster
New Mexico. 73

Interrogation of the 6q23 autoimmune disease susceptibility

locus using a mirrored CRISPR chromatin modification screen
James Ding, Kate Duffus, Paul Martin, Gisela Orozco. View
Presenter affiliation: Manchester Academic Health Science Centre, Poster
University of Manchester, Manchester, United Kingdom. 74

xix
Cancer-specific loss of TERT activation sensitizes glioblastoma
to DNA damage
Alexandra Amen, Christof Fellmann, Katarzyna Soczek, Shawn Ren,
Rachel Lew, Gavin Knott, Jesslyn Park, Andrew McKinney, Andrew
Mancini, Jennifer Doudna, Joseph Costello.
Presenter affiliation: University of California, Berkeley, Berkeley, View
California; Gladstone Institutes, San Francisco, California; University Poster
of California, San Francisco, San Francisco, California. 75

RNA editing for repairing point mutations causing Usher

syndrome
Lewis E. Fry, Alun R. Barnard, Michelle E. McClements, Robert E. View
MacLaren. Poster
Presenter affiliation: University of Oxford, Oxford, United Kingdom. 76

Extensive ribose chemical modification of the Cas12a crRNA 5'

handle pseudoknot supports gene editing and modulates cis-
trans activity
Eman A. Ageely, Leonora Abdullahu, Sunit Jana, Ramadevi
Chilamkurthy, Daniel O'Reilly, Philip J. Jensik, Masad J. Damha, Keith View
T. Gagnon. Poster
Presenter affiliation: Southern Illinois University, Carbondale, Illinois. 77

Harnessing the diversity of Cas9 orthologs for genome editing

Giedrius Gasiunas, Joshua K. Young, Darius Kazlauskas, Tomas
Urbaitis, Monika Jasnauskaite, Migle Stitilyte, Sushmitha Paulraj,
Zhenglin Hou, Shane K. Dooley, Clara Alarcon, Doane Chilcoat,
Jennifer L. Curcuru, Megumu Mabuchi, Ryan T. Fuchs, Ezra View
Schildkraut, Brett Robb, Ceslovas Venclovas, Virginijus Siksnys. Poster
Presenter affiliation: CasZyme, Vilnius, Lithuania. 78

A viral guide RNA delivery system for CRISPR-based

transcriptional activation and heritable targeted DNA
demethylation
Basudev Ghoshal, Brandon Vong, Colette L. Picard, Suhua Feng, View
Janet M. Tam, Steven E. Jacobsen. Poster
Presenter affiliation: HHMI/UCLA, Los Angeles, California. 79

Optimized fluorescent CRISPR enzyme fusions allow for

enrichment of edited cells by fluorescence activated cell sorting
Steve E. Glenn, Michael A. Collingwood, Christopher A. Vakulskas, View
Sarah F. Beaudoin, Nicole M. Bode, Mark A. Behlke. Poster
Presenter affiliation: Integrated DNA Technologies, Coralville, Iowa. 80

xx
CRISPOR updates in 2020 More and more genomes, gene model
display and better export options
Maximilian Haeussler, Jean-Paul Concordet. View
Presenter affiliation: University of California at Santa Cruz, Santa Cruz, Poster
California. 81

Using a CRISPR-Cas9-based gene drive to target stress response

genes in Candida albicans View
Viola Halder, Rebecca Shapiro. Poster
Presenter affiliation: University of Guelph, Guelph, Canada. 82

The application of CRISPR/Cas9 for molecular correction therapy

of neuromuscular disorders
Britt Hanson, Sofia Stenler, Anna M. Coenen-Stass, Nina Ahlskog, View
Matthew J. Wood, Thomas C. Roberts. Poster
Presenter affiliation: University of Oxford, Oxford, United Kingdom. 83

Genomic correction of sialic acid storage disease knock-in

human fibroblasts via CRISPR-Cas9 base editing-directed repair
Jerry F. Harb, Elvin P. Morales, Jeffrey Y. Huang, Raymond Y. Wang, View
Shih-Hsin Kan. Poster
Presenter affiliation: CHOC Children's Hospitral, Orange, California. 84

CRISPR-mediated transcriptional activation with synthetic guide

RNAs
Kevin Hemphill, Žaklina Strezoska, Sarah Dickerson Matheny, Elena
Maksimova, Eldon Chou, Maren Mayer Gross, Travis Hardcastle,
Matthew Perkett, Jesse Stombaugh, Emily M. Anderson, Annaleen
Vermeulen, Anja van Brabant Smith. View
Presenter affiliation: Dharmacon, A Horizon Discovery Group Poster
Company, Lafayette, Colorado. 85

Highly efficient “hit-and-run” genome editing with

unconcentrated lentivectors carrying Vpr.Prot.Cas9 protein
produced from RRE-containing transcripts View
Ivana Indikova, Stanislav Indik. Poster
Presenter affiliation: University of Veterinary Medicine, Vienna, Austria. 86

Characterization of synthetic guide RNAs for CRISPR/Cas

genome editing An extensive evaluation of gRNA formats,
purity, and delivery methods View
Ashley Jacobi, Rolf Turk, Nathan Roberts, Garrett Rettig. Poster
Presenter affiliation: Integrated DNA Technologies, Coralville, Iowa. 87

xxi
Enhancement of CRISPR-driven homology directed repair using a
new class of clinical DNA-PK inhibitors
Qingzhou Ji, Jacob T. Lamberth, Fuqiang Chen, Christoph Vahl, View
Thomas Fuchss, Frank T. Zenke, Gregory D. Davis. Poster
Presenter affiliation: MilliporeSigma, St. Louis, Missouri. 88

Massively parallel kinetic profiling of natural and engineered

CRISPR nucleases
Stephen K. Jones Jr, John A. Hawkins, Nicole V. Johnson, Cheulhee
Jung, Kuang Hu, James R. Rybarski, Janice S. Chen, Jennifer A. View
Doudna, William H. Press, Ilya J. Finkelstein. Poster
Presenter affiliation: University of Texas, Austin, Texas. 89

Comparison of Cas9 orthologues for gene suppression activity

Ariel Kantor, Michelle E. McClements, Robert E. MacLaren. View
Presenter affiliation: Nuffield Laboratory of Ophthalmology, University Poster
of Oxford, Oxford, United Kingdom. 90

Genome editing of the Prader-Willi syndrome imprinting center

(PWS-IC) results in loss of transcriptional control for the SNURF-
SNRPN-snoRNA-lncRNA locus in a feline cell culture model
Erik A. Koppes, Marie A. Johnson, Dale W. Lewis, Susanne M. Golin,
Robert D. Nicholls. View
Presenter affiliation: University of Pittsburgh School of Medicine and Poster
UPMC Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania. 91

Controlling endogenous levels of cytidine deaminases by

CRISPRa for eliminating hepatitis B virus
Dmitry Kostyushev, Sergey Brezgin, Anastasiya Kostyusheva,
Ekaterina Bayurova, Ilya Gordeychuk, Lena Soppa, Dieter Glebe,
Vladimir Chulanov. View
Presenter affiliation: National Medical Research Center of Poster
Tuberculosis and Infectious Diseases, Moscow, Russia. 92

Comparing the overall HR/NHEJ ratio of different CRISPR

endonucleases
Sarah L. Krausz, Éva Varga, Hannah N. Stabb, Ervin Welker. View
Presenter affiliation: Research Centre for Natural Sciences, Budapest, Poster
Hungary; Semmelweis University, Budapest, Hungary. 93

xxii
Systematic mapping of genetic interactions in human cancer cells
reveals context dependent cancer signaling pathways
Samson H. Fong, Brent M. Kuenzi, John Lee, Kyle Sanchez, Ana
Bojorquez-Gomez, Kyle Ford, Brenton P. Munson, Katherine Licon,
Jeff Hager, John Paul Shen, Jason F. Kreisberg, Prashant Mali, Trey View
Ideker. Poster
Presenter affiliation: UC San Diego, La Jolla, California. 94

To study the role of DNA topology in regulating LncRNAs

implicated in neural development using CRISPR/Cas9 edited
cortical organoids
Manoj Kumar, Debojyoti Chakraborty.
Presenter affiliation: CSIR-Institute of Genomics and Integrative View
Biology, New Delhi, India; Academy of Scientific and Innovative Poster
Research (AcSIR), Ghaziabad, India. 95

Highly organized, variously patterned accumulation platforms of

transcription activators using Class 1 and Class 2 CRISPR
systems
Atsushi Kunii, Shiho Nakamura, Kazuto Yoshimi, Tomoji Mashimo, View
Takashi Yamamoto, Tetsushi Sakuma. Poster
Presenter affiliation: Hiroshima University, Hiroshima, Japan. 96

Dissecting the role of recurrent hyperploidy in tumorigenesis

Asad A. Lakhani, Vishruth Girish, Christine Scaduto, Jason Sheltzer. View
Presenter affiliation: Cold Spring Harbor Laboratory, Cold Spring Poster
Harbor, New York. 97

CHANGE-seq reveals genetic and epigenetic effects on CRISPR-

Cas9 genome-wide activity
Cicera Lazzarotto, Nikolay Malinin, Yichao Li, Ruochi Zhang, Yang
Yang, GaHyun Lee, Eleanor Cowley, Yanghua He, Kasey Jividen,
Varun Katta, Natalia Kolmakova, Christopher Petersen, Qian Qi,
Samantha Maragh, Giedre Krenciute, Jian Ma, Yong Cheng, Shengdar
Tsai. View
Presenter affiliation: St Jude Children's Research Hospital, Memphis, Poster
Tennessee. 98

xxiii
Investigating proliferation suppressor behavior in whole genome
genetic fitness screens
Walter F. Lenoir, Micaela Morgado, Megan E. McLaughlin, Eiru Kim,
Traver Hart.
Presenter affiliation: The University of Texas MD Anderson Cancer
Center, Houston, Texas; The University of Texas MD Anderson View
Cancer Center UTHealth Graduate School of Biomedical Sciences, Poster
Houston, Texas. 99

CopyCatchers Novel active genetic elements for detecting and

quantifying homolog-directed somatic gene conversion
Zhiqian Li, Valentino M. Gantz, Ethan Bier. View
Presenter affiliation: University of California San Diego, San Diego, Poster
California. 100

A robust method for tagging endogenous genes through

promoter trapping and short homology arms
Xiquan Liang, Narges Anbardar, Sridhar Ranganathan, Mohan c. View
Vemuri, Potter Jason, Jonathan d. Chesnut. Poster
Presenter affiliation: Thermo Fisher Scientific, Carlsbad, California. 101

Enhancing maize grain yield by CRISPR/Cas9 genomic editing of

CLE (CLAVATA3/Embryo-surrounding region) genes for maize
breeding
Lei Liu, Edgar Demesa Arevalo, Richelle Chen, Tara Skopelitis,
Qingyu Wu, David Jackson. View
Presenter affiliation: Cold Spring Harbor Laboratory, Cold Spring Poster
Harbor, New York. 102

Genome editing for FVIII and AAT gene therapy cures

Reka Lorincz, David Curiel. View
Presenter affiliation: Washington University in St Louis, St Louis, Poster
Missouri. 103

Development of an efficient genome editing method in a tilapia

cell line View
Angela L. Man, Tarang K. Mehta, Wilfried Haerty, Federica Di Palma. Poster
Presenter affiliation: The Earlham Institute, Norwich, United Kingdom. 104

Releasing a gRNA from an mRNA transcript for association with

dCas9
Michelle E. McClements, Caroline F. Peddle, Robert E. MacLaren.
Presenter affiliation: University of Oxford, Oxford, United Kingdom; View
Oxford University Hospitals NHS Trust and NIHR Biomedical Research Poster
Centre, Oxford, United Kingdom. 105

xxiv
High-throughput analysis of repaired CRISPR double-stranded
breaks reveals nuclease-specific complexity View
Matthew S. McNeill, Gavin Kurgan, Heng Li, Yu Wang, Mark Behlke. Poster
Presenter affiliation: Integrated DNA Technologies, Coralville, Iowa. 106

in situ generation of CAR T-cells

Samir A. Mendonca, David T. Curiel. View
Presenter affiliation: Washington University in St. Louis School of Poster
Medicine, Saint Louis, Missouri. 107

Assessing efficiency and fidelity of CRISPR based therapies

using molecular combing
Imen Mestiri, Prakhar Bisht, Engin Altunlu, Samira Mzalouat, Aurélien View
Petit, Laurence Maggiorella, Florence Mahé, Aaron Bensimon. Poster
Presenter affiliation: Genomic Vision, Bagneux, France. 108

Efficient production and application of ssDNA for CRISPR/Cas

knockins in human primary T cells
Montse Morell, Tatiana Garachtchenko, Lily Lee, Thomas P. Quinn, View
Michael Haugwtiz, Andrew Farmer. Poster
Presenter affiliation: TakaraBio USA, Mountain View, California. 109

Metal-organic frameworks A novel CRISPR/Cas9 delivery

approach in therapeutic genome editing
Muhammad Naeem, Irshad Ahmad. View
Presenter affiliation: King Fahd University of Petroleum and Minerals Poster
(KFUPM), Dhahran, Saudi Arabia. 110

Extended analysis of amplicon sequencing data with MaChIAto

for prime editing
Kazuki Nakamae, Mitsumasa Takenaga, Shota Nakade, Ichiro
Nazuka, Akinori Awazu, Naoaki Sakamoto, Tetsushi Sakuma, Takashi View
Yamamoto. Poster
Presenter affiliation: Hiroshima University, Hiroshima, Japan. 111

Engineering a miniaturized SaCas9 adenine base editor with

reduced RNA off-targets and increased on-target DNA editing
efficiencies
Minh Thuan Nguyen Tran, Mohd Khairul Nizam Mohd Khalid, Qi Wang,
Jacqueline K. Walker, Predrag Kalajdzic, Grace E. Lidgerwood,
Kimberley Dilworth, Leszek Lisowski, Alice Pebay, Alex W. Hewitt. View
Presenter affiliation: Menzies Institute for Medical Research, The Poster
University of Tasmania, Hobart, Australia. 112

xxv
High-throughput mapping of node of Ranvier and axon initial
segment associated proteins by HiUGE genome editing View
Yuki Ogawa, Matthew Rasband. Poster
Presenter affiliation: Baylor College of Medicine, Houston, Texas. 113

Validation of CRISPR gene knockouts in U2OS cell lines with DIA

MS-based proteomics
Jennifer Oki, Vito Dozio, David Conant, Travis Maures, Michelle Gut, View
Yuehan Feng, Kristina Beeler, Scott Pattison, Nicholas Dupuis. Poster
Presenter affiliation: Synthego Corporation, Menlo Park, California. 114

A gene signal amplifier platform for monitoring the unfolded

protein response
Carlos A. Origel Marmolejo, Bhagyashree Bachhav, Sahiti D.
Patibandla, Alexander L. Yang, Laura Segatori. View
Presenter affiliation: Stanford University School of Medicine, Stanford, Poster
California; Rice University, Houston, Texas. 115

Screening SNP-binding CRISPR/Cas9 gRNAs targeting rhodopsin

as part of an allele-specific strategy to treat autosomal dominant
retinitis pigmentosa View
Caroline F. Peddle, Michelle E. McClements, Robert E. MacLaren. Poster
Presenter affiliation: University of Oxford, Oxford, United Kingdom. 116

Rapid, field-deployable CRISPR diagnostics for any nucleobase

variant in DNA or RNA using a highly specific Cas9 from
Francisellanovicida(FnCas9)
Rhythm Phutela, Mohd Azhar, Dipanjali Sinha, Namrata Sharma,
Manoj Kumar, Debojyoti Chakraborty.
Presenter affiliation: CSIR-Institute of Genomics & Integrative Biology, View
New Delhi, India; Academy of Scientific and Innovative Research, Poster
Ghaziabad, India. 117

CRISPRme Population and patient-specific off-target site

analysis for CRISPR genome editing
Samuele Cancellieri, Elia Dirupo, Anne Shen, Daniel Bauer, Nicola View
Bombieri, Rosalba Giugno, Luca Pinello. Poster
Presenter affiliation: MGH/Harvard/Broad, Boston, Massachusetts. 118

Direct cardiogenic reprogramming of fibroblasts as a promising

therapeutic strategy for injured heart
Volodymyr V. Balatskyi, Anna Myronova, Oksana O. Piven. View
Presenter affiliation: Institute of Molecular Biology and Genetics, Poster
National Academy of Sciences of Ukraine, Kyiv, Ukraine. 119

xxvi
Expanding the scope of plant genome engineering with novel
Cas12a orthologs and highly multiplexable editing systems
Yingxiao Zhang, Qiurong Ren, Xu Tang, Shishi Liu, Aimee A. Malzahn,
Jiaheng Wang, Desuo Yin, Changtian Pan, Yanhao Cheng, Qiudeng View
Que, Yong Zhang, Yiping Qi. Poster
Presenter affiliation: University of Maryland, College Park, Maryland. 120

Large insertions mediated by CRISPR/Cas editing via improved

HDR templates, and comprehensive characterization of editing
events with long read sequencing
Garrett R. Rettig, Mollie Schubert, Gavin Kurgan, Matthew McNeill, View
Jessica Woodley, Mark Behlke. Poster
Presenter affiliation: Integrated DNA Technologies, Coralville, Iowa. 121

Modulating the activity of CRISPR-Cas with chemical

modifications in single-guide RNAs
Daniel Ryan, David Taussig, Suhani Thakker, Israel Steinfeld, Ben
Lunstad, Rob Kaiser, Ryan McCaffrey, Justin Townsend, Patrick View
Chaffey, Bo Curry, Doug Dellinger, Laurakay Bruhn. Poster
Presenter affiliation: Agilent Technologies, Santa Clara, California. 122

The conversion of human iPSCs into myocytes via CRISPR/Cas9

technology
Joseph C. Sanchez, Emilia A. Solomon, Katie L. Davis-Anderson,
Sofiya N. Micheva-Viteva, Rashi S. Iyer, Scott N. Twary. View
Presenter affiliation: Los Alamos National Laboratory, Los Alamos, Poster
New Mexico. 123

Development of a CRISPR/Cas9-based therapy for Hutchinson-

Gilford progeria syndrome
Olaya Santiago-Fernández, Fernando G. Osorio, Víctor Quesada,
Francisco Rodríguez, Sammy Basso, Daniel Maeso, Alicia R. View
Folgueras, José M. P. Freije, Carlos López-Otín. Poster
Presenter affiliation: Universidad de Oviedo, Oviedo, Spain. 124

Manufacture of CRISPR RNPs for clinical use View

Nicole L. Kavanaugh, Samantha Foti, Shawn Shafer. Poster
Presenter affiliation: Aldevron, Madison, Wisconsin. 125

Cleavage-resistant templates for CRISPR-Cas9 editing of SNVs in

human stem cells
William C. Skarnes, Nathaniel Roberts, Nathan Delvaux, Enrica A.
Pellegrino, Justin A. McDonough, Ashley Jacobi, Garrett Rettig. View
Presenter affiliation: Jackson Laboratory for Genomic Medicine, Poster
Farmington, Connecticut. 126

xxvii
A CRISPR/Cas13-based approach for functional study of RNA
modifications View
Huong Ta, Jun Li, Iain Searle. Poster
Presenter affiliation: The University of Adelaide, Adelaide, Australia. 127

CRISPR-Cas9 antimicrobial Resistance reversal potential in

challenging conditions
Thaysa L. Tagliaferri, Natália R. Guimarães, Marcella M. Pereira, Liza
F. Vilela, Hans-Peter Horz, Simone G. Santos, Tiago O. Mendes.
Presenter affiliation: Federal University of Viçosa, Viçosa, Brazil; View
Federal University of Minas Gerais, Belo Horizonte, Brazil; RWTH Poster
Aachen University Hospital, Aachen, Germany. 128

Development and characterization of Pin-point base editor A

modular CRISPR–RNA aptamer-mediated base editing system
Juan C. Collantes, Victor M. Tan, Huiting Xu, Amer Alasadi, Jingjing
Guo, Hanlin Tao, Melany Ruiz-Urigüen, Katarzyna M. Tyc, Tommaso
Selmi, John J. Lambourne, Jennifer A. Harbottle, Jesse Stombaugh,
Jinchuan Xing, Ceri M. Wiggins, Shengkan Jin. View
Presenter affiliation: Robert Wood Johnson Medical School, Poster
Piscataway, New Jersey. 129

Recoding essential genes to reduce resistance to CRISPR-based

gene drives in Drosophila melanogaster
Gerard Terradas, Anna B. Buchman, Omar S. Akbari, Ethan Bier. View
Presenter affiliation: University of California San Diego, La Jolla, Poster
California. 130

Using CRISPR-Cas9 genome editing to introduce naturally

occurring deletional mutations associated with elevated foetal
haemoglobin as an approach for treating sickle cell disease and
-thalassemia
Sarah Topfer, Gabriella Martyn, Kate Quinlan, Merlin Crossley. View
Presenter affiliation: University of New South Whales, Sydney, Poster
Australia. 131

Simple electroporation for efficient CRISPR/Cas9 genome editing

in murine zygotes
Simon E. Tröder, Lena K. Ebert, Linus Butt, Laurens Wachsmuth, View
Bernhard Schermer, Branko Zevnik. Poster
Presenter affiliation: University of Cologne, Cologne, Germany. 132

xxviii
High-throughput off-target detection supports clinical research
Rolf Turk, Garrett Rettig, Gavin Kurgan, Rafet Basar, May Daher,
Jenny Shapiro, Ortal Iancu, Adi Tovin-Recht, Matt McNeill, Mollie View
Schubert, Katy Rezvani, Ayal Hendel, Mark Behlke. Poster
Presenter affiliation: Integrated DNA Technologies, Coralville, Iowa. 133

Targeted Perturb-seq enables genome-scale genetic screens in

single cells
Daniel Schraivogel, Andreas R. Gschwind, Lars Velten, Lars M. View
Steinmetz. Poster
Presenter affiliation: CRG, Barcelona, Spain. 134

Eliminating genome-wide and transcriptome-wide off-target

mutations by base editor
Lijie Wang, Wei Xue, Hongxia Zhang, Runze Gao, Lina Zhou, Yun-Ni
Lei, Jia Wei, Jing Wu, Hanhui Ma, Xingxu Huang, Bei Yang, Hao Yin,
Li Yang, Jia Chen.
Presenter affiliation: ShanghaiTech University, Shanghai, China; View
Chinese Academy of Sciences, Shanghai, China; University of Poster
Chinese Academy of Sciences, Beijing, China. 135

Dissecting epigenetic and chromatin influences on CRISPR/Cas9

genome editing and DNA repair View
Trevor Weiss, Peter Crisp, Nathan Springer, Feng Zhang. Poster
Presenter affiliation: University of Minnesota, Minneapolis, Minnesota. 136

Characterizing and improving the genome-wide specificities of

near-PAMless CRISPR-Cas9 variants
Madelynn N. Whittaker, Russell T. Walton, Kathleen A. Christie,
Benjamin P. Kleinstiver. View
Presenter affiliation: Massachusetts General Hospital, Boston, Poster
Massachusetts. 137

Cytosine base editors with minimized unguided DNA and RNA

off-target events and high on-target activity
Yi Yu, Thomas C. Leete, David A. Born, Lauren Young, Luis A.
Barrera, Seung-Joo Lee, Holly A. Rees, Giuseppe Ciaramella, Nicole View
M. Gaudelli. Poster
Presenter affiliation: Beam Therapeutics, Cambridge, Massachusetts. 138

xxix
Measuring germline variation of human gene essentiality by
genome-wide CRISPR/Cas9 screening in induced pluripotent
stem cells
Yan Zhou, Jing Eugene Kwa, Felicity Allen, Luca Crepaldi, Elin Madli
Peets, Gemma Turner, Verity Goodwin, Rebecca McRae, Katie Urgo,
Jackie Bryant, Kay Clarke, Adam Hunter, Oliver Dovey, Leopold Parts. View
Presenter affiliation: Wellcome Genome Campus, Cambridge, United Poster
Kingdom. 139

Structure and mechanism of two type III CRISPR defence

nucleases
Wenlong Zhu, Stephen A. McMahon, Stuart McQuarrie, Sabine
Gruschow, Shirley Graham, Robert Rambo, Januka S. Athukoralage,
Tracey M. Gloster, Malcolm F. White. View
Presenter affiliation: University of St Andrews, St Andrews, United Poster
Kingdom. 140

TEG-seq A workflow for in cellulo mapping of CRISPR

specificity
Pei-zhong Tang, Bo Ding, Lansha Peng, Vadim Mozhayskiy, Jason View
Potter, Jonathan D. Chesnut. Poster
Presenter affiliation: ThermoFisher, Carlsbad, California. 141

Whole-genome CRISPR knock-in arrayed library creation by

HiUGE
Akiyoshi Uezu, Alan Ma, Richard Kim, Maia Clarkin, Laukik
Nagawekar, Ed Field¸Scott Soderling. View
Presenter affiliation: CasTag Biosciences, Durham, North Carolina; Poster
Duke University Medical School, Durham, North Carolina. 142

Identification of mammalian host factors required for Legionella

pneumophila infection through a functional genomic screen
Devanand D. Bondage, Caroline Esnault, Ryan Dale, Matthias
Machner.
Presenter affiliation: Eunice Kennedy Shriver National Institute of Child View
Health and Human Development, National Institutes of Health, Poster
Bethesda, Maryland. 143

xxx
 AUTHOR INDEX

Abdullahu, Leonora, 77 Bargsten, Katja, 23

Abratte, Christian M., 29 Bari, S.M. Nayeemul, 2
Acree, Christopher, 14 Barnard, Alun R., 76
Adamson, Britt, 34 Barrera, Luis A., 49, 66, 138
Ageely, Eman A., 77 Barry, Kerrie, 41
Ahlskog, Nina, 83 Bartolome, Christopher, 13
Ahmad, Irshad, 110 Basar, Rafet, 133
Akbari, Omar S., 130 Bassik, Michael C., 9
Aklilu, Behailu, 45 Basso, Sammy, 124
Akinci, Ersin, 35 Bauer, Daniel, 118
Al Kawam, Ahmad, 52 Bayurova, Ekaterina, 92
Alarcon, Clara, 78 Beaudoin, Sarah F., 80
Alasadi, Amer, 129 Beeler, Kristina, 114
Alghadban, Samy, 59 Behlke, Mark, 80, 106, 121, 133
Allain, Frédéric H.T., 23 Bensimon, Aaron, 108
Allen, Felicity, 139 Berger, Imre, 27
Alphey, Luke, 53 Berger-Schaffitzel, Christiane, 27
Al-Shayeb, Basem, 17 Beying, Natalja, 39
Altunlu, Engin, 108 Bier, Ethan, 16, 100, 130
Alvarez-Quilon, Alejandro, 32 Biggins, Sue, 11
Amen, Alexandra, 75 Biggs, Daniel, 59
Anbardar, Narges, 101 Billmann, Maximilian, 12, 55, 56
Anderson, Emily M., 85 Bintu, Lacramioara, 9
Anderson, Michelle A., 53 Birchak, Gabriel, 58
Andrews, Brenda, 55, 56 Birk, Alisha, 67, 69
Ang Xin De, Joshua, 53 Bisht, Prakhar, 108
Antony, Jacob, 54 Bisom-Rapp, Ezra, 17
Anzalone, Andrew V., 28 Blaine, Logan J., 36
Aradhana, 9 Blencowe, Benjamin J., 12, 55
Aradhana, Sachdev, 67 Blomme, Jonas, 57
Aratyn-Schaus, Yvonne, 51 Bode, Nicole M., 80
Arbab, Mandana, 31 Bohnuud, Tanggis, 66
Aregger, Michael, 12, 55, 56 Bojorquez-Gomez, Ana, 94
Aryee, Martin J., 46 Bombieri, Nicola, 118
Athukoralage, Januka S., 6, 140 Bondage, Devanand D., 143
Aulicino, Francesco, 27 Boone, Charles, 55, 56
Auradkar, Ankush, 16 Born, David A., 49, 138
Awazu, Akinori, 111 Bosch, Dustin E., 25
Azhar, Mohd, 117 Bosch, Justin A., 58
Bothmer, Anne, 13, 34
Bachhav, Bhagyashree, 115 Bouchareb, Amine, 59
Bachle, Susanna M., 20 Bounds, Lexi R., 47
Balatskyi, Volodymyr V., 119 Braunschweig, Ulrich, 12
Banerjee, Abhimanyu, 9 Brezgin, Sergey, 92
Banfield, Jillian F., 17 Brown, Kevin R., 12, 55, 56

xxxi
Bruhn, Laurakay, 15, 122 Ciaramella, Giuseppe, 49, 51,
Bryant, Jackie, 139 66, 138
Buchman, Anna B., 130 Ciccia, Alberto, 33
Budinich, Krista A., 60 Citorik, Rob, 13
Butler, Corinne, 41 Clark, Karl J., 40
Butt, Linus, 132 Clarke, Kay, 139
Clarkin, Maia, 142
Cafferty, Brian, 51 Cleaver, Stephen, 13
Cameron, Peter, 23 Clelland, Claire D., 67, 69
Campos-Iglesias, Diana, 61 Clement, Kendell, 46, 68
Cancellieri, Samuele, 118 Cléry, Antoine, 23
Cantor, Aaron J., 50 Clow, Patricia A., 63
Canver, Matthew C., 46 Coenen-Stass, Anna M., 83
Cao, Zhendong, 60 Collantes, Juan C., 129
Capin, Julien, 27 Collingwood, Michael A., 80
Carter, Lucas, 11 Conant, David, 114
Carter, Matthew A., 69 Concordet, Jean-Paul, 81
Cassa, Chris, 31 Conklin, Bruce R., 67, 69
Castaneda, Angelica F., 50 Cooper, Robert M., 7
Cavallo, Anna Lina, 30 Corbett, Sybilla, 70
Chaffey, Patrick, 122 Costanzo, Michael, 55, 56
Chakraborty, Debojyoti, 95, 117 Costello, Joseph, 75
Chan, Katherine, 56 Cotta-Ramusino, Cecilia, 13, 34
Chandode, Rakesh, 30 Cowley, Eleanor, 98
Chen, Delai, 51 Crepaldi, Luca, 139
Chen, Ethan, 14 Cress, Brady F., 17
Chen, Fuqiang, 88 Crisp, Peter, 136
Chen, Janice S., 89 Crossley, Merlin, 48, 131
Chen, Jia, 26, 135 Cuella-Martin, Raquel, 33
Chen, Peter J., 62 Cui, Xiaoxia, 71
Chen, Qingzhou, 60 Cunningham, Fiona, 70
Chen, Richelle, 102 Curcuru, Jennifer L., 78
Chen, Xiao, 33 Curiel, David, 103, 107
Cheng, Albert W., 63, 63 Curry, Bo, 15, 122
Cheng, Lo-I, 51
Cheng, Yanhao, 120 Daby, Camden L., 40
Cheng, Yong, 98 Daher, May, 133
Cheng, Zhi, 21 Dale, Ryan, 143
Chesnut, Jonathan D., 101, 141 Damha, Masad J., 77
Chilamkurthy, Ramadevi, 77 Danner, Eric W., 72
Chilcoat, Doane, 78 Davies, Ben, 59
Cho, Tiffany, 32 Davis, Gregory D., 88
Choi, Mihyun, 29 Davis-Anderson, Katie, 73, 123
Chou, Eldon, 85 de la Rosa, Kathrin, 72
Chowdhary, Vivek, 51 de Moraes, Marcos H., 25
Christie, Kathleen A., 8, 65, 137 Decker, Jeremy, 66
Chu, S. Haihua, 66 Dede, Merve, 10
Chulanov, Vladimir, 92 Dellinger, Doug, 15, 122

xxxii
DelRosso, Nicole, 9 Freije, Jose Maria P., 61, 124
Delvaux, Nathan, 126 Freilich, Elizabeth S., 60
Demesa Arevalo, Edgar, 102 Fry, Lewis E., 76
Dewell, Tessa, 69 Fu, Yanfang, 13
Di Palma, Federica, 104 Fuchs, Ryan T., 78
Dickerson Matheny, Sarah, 85 Fuchss, Thomas, 88
Dillingham, Mark, 27
Dilworth, Kimberley, 112 Gagnon, Keith T., 77
Ding, Bo, 141 Gallagher, Danielle N., 37
Ding, Chengfeng, 26 Gallego-Bartolome, Javier, 43
Ding, James, 74 Gantz, Valentino M., 100
Dirupo, Elia, 118 Gao, Pan, 29
Doerfler, Phillip A., 36 Gao, Runze, 135
Dolbee, Alexas, 13 Garachtchenko, Tatiana, 109
Dong, Oliver Xiaoou, 41 Garcia, Sara P., 46
Donohoue, Paul, 23 Gardiner, Jason, 43
Dooley, Shane K., 78 Gasiunas, Giedrius, 78
Doudna, Jennifer A., 17, 75, 89 Gaudelli, Nicole M., 49, 66, 138
Dovey, Oliver, 139 Gehrke, Jason M., 49
Dozio, Vito, 114 Gersbach, Charles A., 47
Duffus, Kate, 74 Ghanam, Amr R., 29
Duong, Phat, 41 Ghanim, Hana Y., 69
Dupuis, Nicholas, 114 Ghoshal, Basudev, 43, 79
Durocher, Daniel, 32 Gier, Rodrigo A., 60
Gierer, John, 45
Ebert, Lena K., 132 Gingras, Anne-Claude, 55
Edwards, Aaron, 49 Girish, Vishruth, 97
Ego, Braeden, 9 Giugno, Rosalba, 118
Ekker, Stephen C., 40 Gjoni, Katie, 69
Esnault, Caroline, 143 Glebe, Dieter, 92
Eugene Kwa, Jing, 139 Glenn, Steve E., 80
Evitt, Niklaus H., 60 Gloster, Tracey M., 6, 140
Golin, Susanne M., 91
Fan, Xiao, 33 Gonatopoulos-Pournatzis,
Farhangmehr, Sha, 12 Thomas, 12, 55
Farmer, Andrew, 109 Gonzalez, Estela, 53
Feliciano, Carissa M., 69 Goodwin, Verity, 139
Fellmann, Christof, 75 Gordeychuk, Ilya, 92
Feng, MIngxia, 24 Graham, Shirley, 6, 140
Feng, Suhua, 79 Grassian, Alexandra, 52
Feng, Yuehan, 114 Gregoire, Francine M., 51
Field, Ed, 142 Gregory, Georgia L., 69
Finkelstein, Ilya J., 89 Grünewald, Julian, 28
Folgueras, Alicia R., 124 Grüschow, Sabine, 6, 140
Fong, Samson H., 94 Gschwind, Andreas R., 134
Ford, Kyle, 94 Guichard, Annabel, 16
Foti, Samantha, 125 Guimarães, Natália R., 128
Fransz, Paul, 39 Guo, Jimmy A., 46

xxxiii
Guo, Jingjing, 129 Hussmann, Jeffrey A., 34
Gut, Michelle, 114
Iancu, Ortal, 133
Ha, Kevin, 12 Ideker, Trey, 94
Haber, James E., 37 Ilsley, Garth, 70
Haerty, Wilfried, 104 Indik, Stanislav, 86
Haeussler, Maximilian, 81 Indikova, Ivana, 86
Hager, Jeff, 94 Ira, Grzegorz, 37
Halder, Viola, 82 Irby, Matthew J., 23
Hanson, Britt, 83 Ireland, Rebekah, 53
Harb, Jerry F., 84 Iyer, Rashi, 73, 123
Harbottle, Jennifer A., 129 Iyer, Sowmya, 46
Hardcastle, Travis, 85 Iyer, Sruja S., 50
Harris, Jennifer, 73
Hart, Traver, 10, 99 Jackson, David, 102
Hartigan, Adam, 66 Jacob, Leni, 52
Hasty, Jeff, 7 Jacobi, Ashley, 87, 126
Hatoum-Aslan, Asma, 2 Jacobs, Thomas, 57
Haugwtiz, Michael, 109 Jacobsen, Steven E., 17, 43, 79
Hawkins, John A., 89 Jain, Rashmi, 41
Hayward, Samuel B., 33 Jana, Sunit, 77
He, Siting, 26 Janto, Abigail N., 37
He, Yanghua, 98 Jasnauskaite, Monika, 78
Hemphill, Kevin, 85 Jason, Potter, 101
Hempstead, Andrew, 20 Jayaram, Hariharan, 50
Hendel, Ayal, 133 Jensik, Philip J., 77
Henrique Ferreira Julio, Alison, Ji, Qingzhou, 88
16 Jia, Gengxiang, 45
Herman, Jake, 11 Jillette, Nathaniel, 63
Hernandez-Pliego, Polinka, 59 Jin, Shengkan, 129
Hess, Gaelen, 9 Jinek, Martin, 23
Hewitt, Alex W., 112 Jividen, Kasey, 98
Hinz, John, 54 Johnson, Marie A., 91
Hiro, Ata, 40 Johnson, Nicole V., 89
Hoeflich, Klaus, 52 Jones Jr, Stephen K., 89
Hofmann, Sidney, 13 Joung, J. Keith, 28, 46, 68
Holden, Kevin, 29 Judge, Luke M., 67, 69
Horng, Joy E., 46 Juettemann, Thomas, 70
Horz, Hans-Peter, 128 Jung, Cheulhee, 89
Hou, Zhenglin, 78
Houben, Andreas, 39 Kadina, Anastasia P., 29
Hsu, FoSheng, 25 Kaduskar, Bhagyashree D., 16
Hsu, Jonathan Y., 28 Kaiser, Rob, 15, 122
Hu, Kuang, 89 Kalajdzic, Predrag, 112
Huang, Jeffrey Y., 84 Kamens, Joanne, 20
Huang, Xingxu, 26, 135 Kan, Shih-Hsin, 84
Huang, Xue, 21 Kantor, Ariel, 90
Hunter, Adam, 139 Katta, Varun, 98

xxxiv
Kavanaugh, Nicole L., 125 Lei, Yun-Ni, 135
Kazlauskas, Darius, 78 Leibowitz, Mitchell L., 36
Keough, Kathleen C., 69 Lenoir, Walter F., 99
Khushwa, Raja babu Singh, 16 Lew, Rachel, 75
Kibbler, Emily, 13 Lewis, Dale W., 91
Kim, Daniel Y., 46 Li, Donghui, 13
Kim, Eiru, 10, 99 Li, Heng, 106
Kim, Richard, 142 Li, Jun, 127
Kleinstiver, Benjamin P., 8, 65, Li, Meng, 1
137 Li, Xueyan, 21
Klompe, Sanne, 14 Li, Yan, 41
Knott, Gavin, 17, 75 Li, Yichao, 98
Kohli, Rahul M., 60 Li, Zheng, 17
Kolmakova, Natalia, 98 Li, Zhiqian, 100
Koonin, Eugene V., 1 Liang, Xiquan, 101
Koppes, Erik A., 91 Licon, Katherine, 94
Kostadima, Myrto, 70 Lidgerwood, Grace E., 112
Kostyushev, Dmitry, 92 Lin, Chun-Han, 23
Kostyusheva, Anastasiya, 92 Lin, Lin, 35
Kotrys, Anna V., 25 Lippman, Zach, 42
Krausz, Sarah L., 93 Lipzen, Anna, 41
Kreisberg, Jason F., 94 Liquori, Alexander J., 49, 66
Krenciute, Giedre, 98 Lisowski, Leszek, 112
Kuenzi, Brent M., 94 Liu, David R., 25, 28, 29, 31, 46,
Kühn, Ralf, 72 62
Kumar, Manoj, 95, 117 Liu, Lei, 102
Kundaje, Anshul, 9 Liu, Liang, 21
Kunii, Atsushi, 96 Liu, Shishi, 120
Kurgan, Gavin, 106, 121, 133 Liu, Wanlu, 43
Liu, Yang, 1
Lakhani, Asad A., 97 Liu, Zhen, 26
Lam, Daisy, 66 Llambi, Fabien, 52
Lam, Dieter K., 49 Long, Xiaochun, 29
Lam, Kin Chung, 28 Lopez-Otin, Carlos, 61, 124
Lamberth, Jacob T., 88 Lorincz, Reka, 103
Lambourne, John J., 129 Lu, Chao, 33
Lan, Yemin, 60 Lung, Genesis, 51
Lawson, Keith, 55 Lunstad, Ben, 15, 122
Lazzarotto, Cicera, 29, 98 Ly, Lana C., 48
Lebedin, Mikhail, 72 Lyu, Qing, 29
Lee, GaHyun, 98
Lee, Han B., 40 Ma, Alan, 142
Lee, Hyunho, 46 Ma, Hanhui, 135
Lee, John, 94 Ma, Jian, 98
Lee, Lily, 109 Mabuchi, Megumu, 78
Lee, Seung-Joo, 49, 138 Machner, Matthias, 143
Lee, So Jung, 38 MacLaren, Robert E., 76, 90,
Leete, Thomas C., 138 105, 116

xxxv
Madli Peets, Elin, 139 Mootha, Vamsi K., 25
Maeso, Daniel, 124 Morales, Elvin P., 84
Maggiorella, Laurence, 108 Morell, Montse, 109
Mahé, Florence, 108 Morgado, Micaela, 99
Makarova, Kira S., 1 Mougous, Joseph D., 25
Maksimova, Elena, 85 Mozhayskiy, Vadim, 141
Mali, Prashant, 94 Mueller, Christian, 51
Malinin, Nikolay, 98 Mukund, Aditya, 9
Malzahn, Aimee A., 120 Munroe, Rob J., 29
Man, Angela L., 104 Munson, Brenton P., 94
Mancini, Andrew, 75 Murray, Ryan, 49
Maragh, Samantha, 98 Musunuru, Kiran, 46
Marshall, Jeff, 66 Myers, Chad, 12, 55, 56
Martin, Joel, 41 Myerson, Joel, 15
Martin, Paul, 74 Myronova, Anna, 119
Martyn, Gabriella, 48, 131 Mzalouat, Samira, 108
Mashimo, Tomoji, 96
Matuszek, Zaneta, 31 Naeem, Muhammad, 110
Maures, Travis, 114 Nagawekar, Laukik, 142
Maxwell, Karen, 3 Nakade, Shota, 111
Mayer Gross, Maren, 85 Nakamae, Kazuki, 111
McCaffrey, Ryan, 15, 122 Nakamura, Shiho, 96
McClements, Michelle E., 76, 90, Nazuka, Ichiro, 111
105, 116 Nevard, Katherine, 53
McDonough, Justin A., 126 Nevin, Zachary S., 69
McKinney, Andrew, 75 Newby, Gregory A., 29, 46
McLaughlin, Megan, 10, 99 Nguyen Tran, Minh Thuan, 112
McMahon, Stephen A., 140 Nicholls, Robert D., 91
McNeill, Matthew, 106, 121, 133 Nitsch, Roberto, 30
McQuarrie, Stuart, 6, 140 Nureki, Osamu, 22
McRae, Rebecca, 139
Mehta, Tarang K., 104 Ogawa, Yuki, 113
Mei, Yu, 45 Oki, Jennifer, 114
Mendes, Tiago O., 128 Olins, Jenny, 66
Mendonca, Samir A., 107 Olivieri, Michele, 32
Mepani, Rina J., 50 O'Reilly, Daniel, 77
Mestiri, Imen, 108 Origel Marmolejo, Carlos A., 115
Meysami, Parisa, 27 Orozco, Gisela, 74
Miano, Joseph M., 29 Osorio, Fernando G., 124
Miao, Zhuang, 62
Micheva-Viteva, Sofiya, 73, 123 Pacesa, Martin, 23
Moffat, Jason, 12, 55, 56 Packer, Michael S., 49, 51, 66
Mohd Khalid, Mohd Khairul Paddison, Patrick, 11
Nizam, 112 Pan, Changtian, 120
Moineau, Sylvain, 4 Papathanasiou, Stamatis, 36
Mok, Beverly, 25, 31 Pape, Tillmann, 24
Moll, Theodore, 45 Papikian, Ashot, 43
Montoya, Guillermo, 24 Park, Jesslyn, 75

xxxvi
Parts, Leopold, 139 Rapvisankar, Purnima, 34
Patibandla, Sahiti D., 115 Rees, Holly A., 49, 138
Pattison, Scott, 114 Ren, Qiurong, 120
Paulraj, Sushmitha, 78 Ren, Shawn, 75
Pausch, Patrick, 17 Rettig, Garrett, 87, 121, 126, 133
Pebay, Alice, 112 Rezvani, Katy, 133
Peddle, Caroline F., 105, 116 Rinaldi, Conrad, 49
Pellegrino, Enrica A., 126 Robb, Brett, 78
Pellman, David, 36 Roberts, Nathan, 87, 126
Pelosse, Martin, 27 Roberts, Thomas C., 83
Peng, Lansha, 141 Rocha, Cristina, 30
Pereira, Marcella M., 128 Rodríguez, Francisco, 124
Perez-Bermejo, Juan A., 69 Roenspies, Michelle, 39
Perkett, Matthew, 85 Ronald, Pamela C., 41
Perrimon, Norbert, 58 Ronda, Carlotta, 14
Petersen, Christopher, 98 Ross, Catherine, 56
Peterson, S. Brook, 25 Rothstein, Rodney, 38
Petit, Aurélien, 108 Roy, Subhadeep, 15
Petri, Karl, 28, 46 Rubens, Jacob, 13
Pham, Nhung, 37 Ruiz-Urigüen, Melany, 129
Phutela, Rhythm, 117 Ryan, Daniel, 15, 122
Picard, Colette L., 79 Rybarski, James R., 89
Pinello, Luca, 28, 46, 68, 118
Piven, Oksana O., 119 Sachdev, Aradhana R., 69
Potter, Jason, 141 Sakamoto, Naoaki, 111
Preece, Chris, 59 Sakuma, Tetsushi, 96, 111
Press, William H., 89 Salomon, Wes, 13
Puchta, Holger, 39 Sanchez, Joseph C., 123
Pyhtila, Brook, 20 Sanchez, Kyle, 94
Santiago-Fernández, Olaya, 124
Qi, Jun, 60 Santos, Simone G., 128
Qi, Qian, 98 Sasaki, Kanae E., 46
Qi, Stanley, 19 Scaduto, Christine, 97
Qi, Yiping, 120 Schermer, Bernhard, 132
Que, Qiudeng, 120 Schildkraut, Ezra, 78
Quesada, Víctor, 124 Schimenti, John C., 29
Quinlan, Kate, 48, 131 Schmidt, Carla, 39
Quinn, Thomas P., 109 Schmutz, Jeremy, 41
Schraivogel, Daniel, 134
Rabadan, Raul, 33 Schubert, Mollie, 121, 133
Radey, Matthew C., 25 Searle, Iain, 127
Raguram, Aditya, 25 Segatori, Laura, 115
Rahman, Mahfuzur, 56 Selmi, Tommaso, 129
Ramadoss, Gokul N., 69 Sentmanat, Monica F., 71
Rambo, Robert, 140 Shackleford, Lewis, 53
Ranganathan, Sridhar, 101 Shafer, Shawn, 125
Rasband, Matthew, 113 Shah, Aalok, 51
Rath, Phalguni, 59 Shah, Hina, 46

xxxvii
Shah, Manan, 48 Takenaga, Mitsumasa, 111
Shapiro, Jenny, 133 Tam, Janet M., 79
Shapiro, Rebecca, 82 Tambe, Akshay, 50
Sharma, Namrata, 117 Tan, Victor M., 129
Shcherbakova, Inna, 13 Tang, Pei-zhong, 141
She, Qunxin, 24 Tang, Xu, 120
Sheltzer, Jason, 97 Tao, Hanlin, 129
Shen, Anne, 118 Taussig, David, 15, 122
Shen, John Paul, 94 Terradas, Gerard, 130
Shen, Max W., 28, 31, 35 Thakker, Suhani, 15, 122
Shen, Yufeng, 33 Tian, Li, 41
Sherwood, Richard, 35 Toelzer, Christine, 27
Shi, Junwei, 60 Tong, Amy, 56
Shmakov, Serge A., 1 Topfer, Sarah, 131
Siksnys, Virginijus, 78 Tovin-Recht, Adi, 133
Simone, Brandon W., 40 Townsend, Justin, 122
Sinha, Dipanjali, 117 Tröder, Simon E., 132
Skarnes, William C., 126 Tsai, Annie M., 37
Skopelitis, Tara, 102 Tsai, Shengdar, 29, 98
Slaymaker, Ian, 49, 66 Tsang, Jennifer, 20
Smith, Sarah, 51 Tsuchida, Connor A., 17
So, Po-Lin So, 69 Turk, Rolf, 87, 133
Soczek, Katarzyna, 75 Turner, Gemma, 139
Soderling, Scott, 142 Twary, Scott, 73, 123
Sofos, Nicholas, 24 Tyc, Katarzyna M., 129
Sola-Esteves, Noris M., 49 Tycko, Josh, 9
Solomon, Emilia A., 123
Soppa, Lena, 92 Uezu, Akiyoshi, 142
Spees, Kaitlyn, 9 Urbaitis, Tomas, 78
Springer, Nathan, 136 Urgo, Katie, 139
Stabb, Hannah N., 93 Usaj, Matej, 56
Steinberg, Barrett, 13
Steinfeld, Israel, 15, 122 Vahl, Christoph, 88
Steinmetz, Lars M., 134 Vakulskas, Christopher A., 80
Stella, Stefano, 24 van Brabant Smith, Anja, 85
Stengel, Katharina, 23 Van, Mike, 9
Stenler, Sofia, 83 Varga, Éva, 93
Sternberg, Samuel, 14 Velten, Lars, 134
Stitilyte, Migle, 78 Vemuri, Mohan c., 101
Stombaugh, Jesse, 85, 129 Venclovas, Ceslovas, 78
Stout, Elizabeth S., 48 Vermeulen, Annaleen, 85
Strezoska, Žaklina, 85 Vilela, Liza F., 128
Sullivan, Tim, 13 Vo, Phuc Leo, 14
Sun, Lili, 36 Vong, Brandon, 79
Sun, Wei, 21 Voytas, Dan, 44

Ta, Huong, 127 Wachsmuth, Laurens, 132

Tagliaferri, Thaysa L., 128 Walker, Jacqueline K., 112

xxxviii
Walker, John A., 29 Yang, Lu, 48
Walton, Russell T., 8, 65, 137 Yang, Yang, 98
Wang, Harris, 14 Yao, David, 9
Wang, Jiaheng, 120 Yao, Yu, 36
Wang, Jiuyu, 21 Ye, Chaoyang, 52
Wang, Lijie, 135 Yen, Jonathan S., 49
Wang, Qi, 112 Yin, Desuo, 120
Wang, Raymond Y., 84 Yin, Hao, 135
Wang, Xiao, 26 Yoshimi, Kazuto, 96
Wang, Xiao, 46 Young, Joshua K., 78
Wang, Yanli, 21 Young, Lauren, 49, 138
Wang, Ying, 26 Yu, Shu, 41
Wang, Yu, 106 Yu, Wenxia, 26
Ward, Henry, 12, 56 Yu, Yi, 138
Watry, Hannah L., 67, 69
Wei, Jia, 135 Zeng, Jun, 25
Weiss, Mitchell J., 36 Zenke, Frank T., 88
Weiss, Trevor, 136 Zerbino, Daniel R., 70
Weissman, Jonathan S., 34 Zevnik, Branko, 132
Welker, Ervin, 93 Zhang, Cheng-Zhong, 36
Weng, Zhigang, 52 Zhang, Feng, 18
White, Malcolm F., 6, 140 Zhang, Feng, 136
White, Michael, 71 Zhang, Hongxia, 135
Whittaker, Madelynn N., 8, 65, Zhang, Nan, 41
137 Zhang, Ruochi, 98
Wiedenheft, Blake, 5 Zhang, Wei, 29
Wienert, Beeke, 48, 69 Zhang, Yingxiao, 120
Wiggins, Ceri M., 129 Zhang, Yong, 120
Wilson, Christopher, 31, 62 Zhao, Junfei, 33
Wolf, Yuri I., 1 Zhou, Lina, 135
Wood, Matthew J., 83 Zhou, Yan, 139
Woodley, Jessica, 121 Zhu, Jacqueline J., 63, 64
Wu, Jing, 135 Zhu, Wenlong, 140
Wu, Qingyu, 102 Zieger, Marina, 51
Wyrick, John, 54

Xing, Jinchuan, 129

Xu, Albert, 34
Xu, Huiting, 129
Xue, Wei, 135

Yamamoto, Takashi, 96, 111

Yan, Bo, 51
Yan, Jun, 34
Yang, Alexander L., 115
Yang, Bei, 26, 135
Yang, Jing, 21
Yang, Li, 26, 135

xxxix
GROWING DIVERSITY OF UNIQUE CRISPR-CAS-RELATED
SYSTEMS AND CAS1 HOMOLOGS

Kira S Makarova1, Yuri I Wolf1, Serge A Shmakov1, Yang Liu2, Meng Li2,
Eugene V Koonin1
1
National Institutes of Health, National Center for Biotechnology
Information, National Library of Medicine, Bethesda, MD, 2Shenzhen
University, Shenzhen Key Laboratory of Marine Microbiome Engineering,
Institute for Advanced Study, Guangdong, China

The principal function of archaeal and bacterial CRISPR-Cas systems is

antivirus adaptive immunity. However, recent genome analyses identified a
variety of derived CRISPR-Cas variants at least some of which appear to
perform different functions. I will review the status of current knowledge
about such systems, both that lack adaptation module and those that include
derived variants of Cas1, the integrase involved in CRISPR adaptation as
well as casposon transposition. The later include recently described systems
found in archaeal metagenomes of the Asgard superphylum. Notably, the
diversity of Cas1 in Asgard archaea alone is greater than that detected so far
among the rest of archaea and bacteria. The Asgard CRISPR-Cas
derivatives also encode distinct forms of Cas4, Cas5 and Cas7 proteins,
and/or additional nucleases. Some of these systems are predicted to perform
defense functions, but possibly, not programmable ones, whereas others are
likely to represent previously unknown mobile genetic elements.

1
CRISPR-ASSISTED ENGINEERING OF BACTERIAL VIRUSES TO
COMBAT ANTIBIOTIC-RESISTANT PATHOGENS

S.M. Nayeemul Bari, Asma Hatoum-Aslan

The University of Alabama, Dept. of Biological Sciences, Tuscaloosa, AL

The rise in antibiotic resistant bacterial pathogens, coupled with the sharp
decline of new antibiotics in the development pipeline, have sparked a
renewed interest in the use of bacterial viruses (phages) to control drug-
resistant infections. While there have been spectacular recent successes,
many challenges remain before the therapeutic use of phages can become a
routine practice. For example, phages are replete with genes of unknown
function, and current therapeutic strategies utilize a mixture of wild-caught
phages that are poorly-characterized and thus have the potential to cause
adverse side-effects through the release of toxins and the spread of
virulence factors on mobile genetic elements. Therefore, a more rational
approach to phage therapy is needed, which employs well-characterized
phages that are genetically predefined. However, therapeutic phages are
intractable by most genetic techniques due to their swift and destructive
replication cycle. This underscores the pressing need to develop robust tools
for phage genome engineering. Here, we present strategies to genetically
engineer therapeutic phages using a Type III-A CRISPR-Cas system native
to Staphylococcus epidermidis. Given that Type III CRISPR-Cas systems
are distributed in diverse bacteria, it is anticipated that similar approaches
can be broadly applied to enable the systematic study of uncharacterized
phage genes and the rational design of phage-based antimicrobials that
target diverse bacterial pathogens.

2
MULTIPLE MECHANISMS OF INHIBITION OF CRISPR-CAS9

Karen Maxwell

University of Toronto, Dept. of Biochemistry, Toronto, Canada

CRISPR-Cas systems provide bacteria with an adaptive immune system that

protects them from invasion by phages, plasmids and other mobile genetic
elements. These systems are widespread in bacteria and archaea. In
response to this defence, phages have evolved protein inhibitors of these
systems, known as anti-CRISPRs. We are characterizing anti-CRISPR
proteins that inhibit the CRISPR-Cas9 enzyme commonly used for genome
editing purposes. Using a combination of structural and biochemical
analyses, we have identified a variety of mechanisms of activity among this
diverse group of inhibitors. Several of these anti-CRISPRs show activity
against distantly related Cas9 proteins, and different anti-CRISPRs are able
to inhibit the same Cas9 functional domain through different means. These
studies provide insight into the role of anti-CRISPRs in the diversification
and evolution of CRISPR-Cas systems.

3
THE UPS AND DOWNS OF CRISPR-CAS SYSTEMS IN
STREPTOCOCCUS THERMOPHILUS

Sylvain Moineau

Université Laval, Félix d'Hérelle Reference Center for Bacterial Viruses,

Département de Biochimie, de Microbiologie, et de Bio-Informatique,
Québec City, Canada

Streptococcus thermophilus (ST) is an industrially relevant Gram-positive

bacterium added to milk to manufacture high-quality yogurt and several
cheeses worldwide. It is widely documented that dairy manufacturing
facilities are constantly at risk of milk fermentation failures due to the
evolving population of virulent phages, which may inactivate the added ST
cells, leading to lower quality products. Thus, constant phage monitoring
and stringent phage control measures are indispensable. One of the key
measures is to use phage-resistant ST cultures. CRISPR-Cas systems are by
far the most important phage defense mechanisms in ST and CRISPR-
phage-resistant ST strains are now used globally. Virulent streptococcal
phages can overcome CRISPR-Cas resistance through a variety of means,
including nucleotide mutations, deletions, modular exchanges, and the
production of anti-CRISPR proteins (ACR). ST has also evolved various
means to block these ACR-containing phages. Taken altogether, ST and its
phages have been a fantastic model to study and teach CRISPR-Cas
systems. This presentation will provide an overview of the on-going battles
between phages and ST as well as the use of the ST CRISPR-Cas systems
for genome editing purposes to better understand phage biology.

4
PHASE-DEPENDENT CRISPR EVOLUTION

Blake Wiedenheft

Montana State University, Immunology & Infectious Diseases, Bozeman,

MT

CRISPR-associated proteins (Cas1 and Cas2) integrate foreign DNA at the

“leader” end of CRISPR loci. Some leader sequences contain a binding site
for IHF (Integration Host Factor). A heterodimer of IHF protein binds to
and kinks the leader of Type I-E CRISPRs, thereby recruiting an upstream
sequence motif that helps dock Cas1-2 onto the first repeat of the CRISPR
locus. To determine the prevalence of this IHF-dependent mechanism of
CRISPR expansion, we analyzed 1,173 I-E and I-F CRISPR leaders.
Results from these experiments reveal a phased distribution of IHF binding
sites and upstream sequence motifs. In vitro adaptation assays reveal that
phase of regulatory elements, rather than distance from the CRISPR locus,
controls spacer acquisition.

5
KILLING THE MESSENGER: THE HOW AND WHY OF TYPE III
CRISPR DEACTIVATION

Januka S Athukoralage, Stuart McQuarrie, Sabine Grüschow, Shirley

Graham, Tracey M Gloster, Malcolm F White

Biomedical Sciences Research Complex, University of St Andrews, School

of Biology, St Andrews, United Kingdom

Type III CRISPR systems detect foreign RNA and generate cyclic
oligoadenylate (cOA) molecules that act as a second messenger to signal
infection within prokaryotes. cOA activate nucleases that degrade the
nucleic acid of both invader and host. This can lead to dormancy or cell
death; to avoid this, cells require mechanisms to remove cOA from the cell
once a viral infection has been defeated. Enzymes specialised for this task
are known as ring nucleases. Four distinct families of ring nucleases have
been characterised to date. These include CRISPR ring nuclease 1 (Crn1)
which are only found the crenarchaea, certain bacterial cOA activated
nucleases that act as self-limiting enzymes by slowly degrading their own
cOA activators, anti-CRSPR viral ring nucleases (AcrIII-1) of the DUF1874
family used by viruses to subvert type III immunity and the homologous
DUF1874 family Crn2 enzymes possessed by prokaryotes.

Here, we demonstrate that the widespread CRISPR associated protein Csx3,

previously described as an RNA deadenylase, is a ring nuclease that rapidly
degrades the cyclic tetra-adenylate (cA4) antiviral signal. The enzyme has
an unusual cooperative reaction mechanism involving an active site that
spans the interface between two dimers, sandwiching the cA4 substrate. The
widespread occurrence of cOA degrading enzymes in prokaryotes, as well
as use by viruses, highlight the crucial role of ring nucleases in regulating
cOA levels to benefit either host or parasite. To gain insight into this
process, we combine our knowledge of cOA activated ribonucleases and
host and viral ring nucleases and present a comprehensive kinetic model of
a well-studied type III CRISPR system. This model allows us to examine
the dynamic interplay between cOA production, ribonuclease activation and
its control by ring nucleases, which govern cyclic nucleotide levels and
determine infection outcomes during virus-host conflict. Accordingly, ring
nucleases are promising candidates for use in disarming the type III immune
response of pathogenic bacteria targeted by phage therapies.

6
CRISPR VS NATURAL COMPETENCE: ALTRUISTIC GROUP
DEFENSE MORE THAN INDIVIDUAL IMMUNE SYSTEM

Robert M Cooper1,2, Jeff Hasty1,2,3,4

1
UC San Diego, BioCircuits Institute, La Jolla, CA, 2UC San Diego, San
Diego Center for Systems Biology, La Jolla, CA, 3UC San Diego,
Molecular Biology, La Jolla, CA, 4UC San Diego, Bioengineering, La Jolla,
CA

CRISPR-Cas systems seem to present a difficult evolutionary tradeoff:

CRISPR-Cas defends against phages and parasitic DNA, but does it also
prevent cells from acquiring new, potentially helpful genes? Genomic
analyses of this conundrum have arrived at often contradictory conclusions.
In some cases, CRISPR-Cas systems do reduce the spread of antibiotic
resistance genes, but on evolutionary time scales, CRISPR-Cas does not
significantly hinder horizontal gene transfer (HGT). Experimental studies
pitting CRISPR against HGT have mainly focused on phages, conjugation,
or artificial transformation. However, less work has been done with the
natural competence route for HGT, a major driver of evolution and
antibiotic resistance. Here, we use the bacterium Acinetobacter baylyi,
which combines high natural competence with a functional Type I-F
CRISPR-Cas system, to experimentally probe the interactions between
CRISPR and natural competence. Surprisingly, we find that most spacers
throughout the endogenous 90-spacer CRISPR array allow targeted DNA to
enter the cell and integrate into its genome. However, following integration
of a targeted protospacer, the newly autoimmune cells kill themselves in a
form of programmed cell death. The rate of CRISPR-induced death is
usually slow enough that self-targeting cells can divide and often form
colonies, albeit with reduced growth rate. Thus in A. baylyi, CRISPR
appears to block natural competence more through altruistic group defense
than as an individual immune system, and it allows for bet hedging by
allowing targeted protospacers time to mutate. In addition to informing the
CRISPR vs HGT conundrum, this dynamic has implications for
applications using CRISPR for targeted antimicrobials or to block
dissemination of antibiotic resistance genes.

7
APPLICATIONS OF MINIMAL-PAM CRISPR-CAS ENZYMES

Kathleen A Christie1,2,3, Russell T Walton1,2, Madelynn N Whittaker1,2,

Benjamin P Kleinstiver1,2,3
1
Massachusetts General Hospital, Center for Genomic Medicine, Boston,
MA, 2Massachusetts General Hospital, Department of Pathology, Boston,
MA, 3Harvard Medical School, Department of Pathology, Boston, MA

Advances in genome editing technologies have led to exciting innovations

in biomedical research and clinical applications to treat human disease.
Despite the vast potential of CRISPR technologies, certain properties of
CRISPR-Cas enzymes remain suboptimal for modern-day laboratory or
therapeutic implementations. To reduce or eliminate these constraints, we
utilize protein engineering strategies to improve the intrinsic properties of
CRISPR nucleases. We recently expanded the targeting range of SpCas9 via
molecular engineering to alter PAM preference, leading to the development
of two new enzymes named SpG and SpRY (capable of targeting NGN and
NRN>NYN PAMs, respectively). These variants function efficiently as
nucleases, C-to-T base editors (CBEs), and A-to-G base editors (ABEs),
collectively offering unprecedented access to the genome to generate
previously impossible edits. In ongoing work, we are expanding the potency
and utility of these enzymes for a variety of applications, including the
development of BEs with enhanced activities, to improve their safety, and
for their use in molecular biology. We are also expanding upon the high-
throughput methods that we developed to engineer and characterize these
proteins, and are now applying these systems to understand other important
properties of CRISPR-Cas enzymes. Together, our results provide insight
into important properties of CRISPR nucleases, while offering optimized
technologies for research and the treatment of genetic diseases.

8
HIGH-THROUGHPUT DISCOVERY AND CHARACTERIZATION OF
HUMAN TRANSCRIPTIONAL REPRESSOR AND ACTIVATOR
DOMAINS

Josh Tycko1, Nicole DelRosso2, Gaelen Hess1, Aradhana 1, Abhimanyu

Banerjee3, Aditya Mukund2, Mike Van4, Braeden Ego1, David Yao1,
Kaitlyn Spees1, Anshul Kundaje1,5, Michael C Bassik#1, Lacramioara
Bintu#6
1
Stanford University, Genetics, Stanford, CA, 2Stanford University,
Biophysics, Stanford, CA, 3Stanford University, Physics, Stanford, CA,
4
Stanford University, Biology, Stanford, CA, 5Stanford University,
Computer Science, Stanford, CA, 6Stanford University, Bioengineering,
Stanford, CA

# Co-corresponding authors

Thousands of proteins localize to the human nucleus, but we lack a

complete accounting of which contain effector domains that regulate
messenger RNA transcription. To systematically discover and characterize
effector domains in cells, we developed a high-throughput pooled assay
where libraries of 80 amino acid domains are fused to a DNA-binding
domain and their effects upon recruitment to a reporter gene are read out by
sequencing.

Using this approach, we quantified gene silencing and epigenetic memory

after recruitment for >5,000 nuclear protein domains. We discover a
relationship between repressor domain strength and evolutionary history for
the complete family of >300 KRAB domains.

Our domain screening approach is also compatible with deep mutational

scans, which we used to identify enhanced variants of the ZNF10 KRAB
effector used in CRISPRi.

To search for unannotated effectors, we tiled 238 nuclear proteins with a

library of >10,000 sequences and discovered efficient repressors as short as
10 amino acids, for example, in the non-canonical polycomb protein MGA.

Finally, we discovered new activator domains in nuclear proteins including

a divergent ancestral KRAB variant.

Together, we demonstrate a scalable approach to systematically measure

transcriptional effector activity in human cells, discover novel effectors,
establish a resource for interpreting the transcriptional regulatory roles of
nuclear proteins, and define compact transcriptional effector domains for
application in synthetic transcription perturbation technologies.

9
MULTIPLEX ENCAS12A SCREENS SHOW FUNCTIONAL
BUFFERING BY PARALOGS IS SYSTEMATICALLY ABSENT FROM
GENOME-WIDE CRISPR/CAS9 KNOCKOUT SCREENS

Merve Dede1,2, Megan McLaughlin1,2, Eiru Kim1, Traver Hart1,3

1
The University of Texas MD Anderson Cancer Center, Department of
Bioinformatics and Computational Biology, Houston, TX, 2The University
of Texas MD Anderson Cancer Center, Graduate School of Biomedical
Sciences, Houston, TX, 3The University of Texas MD Anderson Cancer
Center, Department of Cancer Biology, Houston, TX

Monogenic pooled library CRISPR knockout screens across hundreds of

cell lines identified thousands of essential genes, whose disruption leads to
fitness defects. These large-scale efforts by the Cancer Dependency Map
have been critical for identifying candidate cancer targets. However, the
number of essential genes detected from these screens are very low
compared to the number of constitutively expressed genes in a cell, raising
the question of why we observe so few essential genes. Through a
systematic analysis of monogenic knockout screen data, we observed that
half of all constitutively expressed genes are never observed as essential in
any CRISPR screen. Notably, these never-essentials are highly enriched for
paralogs, suggesting a major blind spot due to functional redundancy,
masking detection of a substantial number of genes.
Recent studies investigated paralog dependencies in monogenic CRISPR
knockout screens, revealing differential effects of paralogs on cellular
fitness. A study showed paralogs are less likely to be essential than
singletons, and integrated expression and mutation data to identify
paralog synthetic lethals1. However, exclusively computational approaches
cannot identify paralog buffering where neither gene suffers loss of
function. Multiplex gene perturbation methods are required to address this
question.
We investigated paralog buffering among constitutively expressed genes
through systematic dual-gene knockout screening by testing an
algorithmically defined set of ~400 candidate paralog pairs, using enCas12a
multiplex knockout system in three cell lines. We observed 24 synthetic
lethal paralog pairs which have escaped detection by monogenic knockout
screens at stringent thresholds. 19 of 24 (79%) synthetic lethal interactions
were present in at least two out of three cell lines and 14 of 24 (58%) were
present in all three cell lines, including alternate subunits of stable protein
complexes and functionally redundant enzymes. Together these
observations strongly suggest that paralogs represent a targetable set of
genetic dependencies that are systematically under-represented among
essential genes due to genetic buffering in monogenic CRISPR-based
functional genomics approaches.

1. De Kegel,B.& Ryan, C.J. Paralog buffering contributes to the variable essentiality

of genes in cancer cell lines. PLoS genetics,15(10).

10
FUNCTIONAL ANNOTATION OF GENE DOMAINS IN LIVING
CELLS

Jake Herman1,2, Lucas Carter1, Sue Biggins2, Patrick Paddison1

1
Fred Hutchinson Cancer Research Center, Human Biology Division,
Seattle, WA, 2Fred Hutchinson Cancer Research Center, Basic Sciences
Division, Seattle, WA

A critical knowledge gap for the human genome has arisen from our
inability to resolve important functional domains and motifs within protein
coding genes at the large scale. Historically, large scale annotation of
protein domains and motifs relied on homology based-inference by
searching against the current 5494 conserved protein family (Pfam)
domains documented in the human genome (e.g., methyltransferase-like
domain). This approach is ineffective for the ~45% of the proteome that is
devoid of Pfam domains, and still requires validation for the remaining
genes. Closing this knowledge gap is critical for both basic and disease-
focused biomedical research, where years, if not decades, can be spent
dissecting gene functions. Here we leverage the mutagenic properties of
CRISPR-Cas9 by saturating sgRNAs across the coding sequence of a gene
to identify essential domains and motifs. A panel of diploid and aneuploid
cells suggest critical, phenotypically constrained regions do not tolerate in-
frame CRISPR-Cas9 indels. As a result, each gene produces a unique
mutational signature, with constrained regions scoring as phenotypic
"peaks". As a test case, we performed tiling mutagenesis to resolve the
domain structure for 48 well characterized kinetochore-associated genes and
identified approximately 160 functional regions, of which nearly 1/4 have
not been previously described. Novel regions were found in a diverse set of
mitotic factors including: CENPK, KNL1, SKA3, Shugoshin/SGO1,
Mad1/MAD1L1, and ch-TOG/CKAP5. We will present preliminary data on
functional domains for ch-TOG, responsible for its spindle organizing
function, and for Mad1, key for kinetochore localization and spindle
assembly checkpoint function. This powerful genetic approach allows rapid
and inexpensive dissection of essential protein activities expanding our
understanding of genic structure for application to both basic science and
disease-focused questions.

11
GENETIC INTERACTION MAPPING AND EXON-RESOLUTION
FUNCTIONAL GENOMICS WITH A HYBRID CAS9–CAS12A
PLATFORM

Thomas Gonatopoulos-Pournatzis1,2, Michael Aregger1,2, Kevin R Brown1, Sha

Farhangmehr1, Ulrich Braunschweig1, Henry N Ward3, Kevin Ha1, Max
Billmann3, Chad Myers3, Ben Blencowe1, Jason Moffat1
1
University of Toronto, Donnelly Centre, Toronto, Canada, 2National cancer
Institute (NCI/NIH), RNA Biology Laboratory, Frederick, MD, 3University of
Minnesota, Computer Science and Engineering, Minneapolis, MN

Efficient and systematic identification of the functions of genomic segments

and the comprehensive mapping of genetic interactions in mammalian cells
represent major challenges in biomedical research. To address these, we have
developed a CRISPR-based screening platform for combinatorial genetic
perturbations that employs co-expression of Cas9 and Cas12a nucleases and
libraries of hybrid guide (hg)RNAs, which are generated from fusion constructs
comprising Cas9 and Cas12a gRNAs. We named this system CHyMErA (Cas
Hybrid for Multiplexed Editing and Screening Applications). Through iterative
rounds of pooled hgRNA library construction and screening, and by employing
a deep learning framework, we optimized hgRNA designs in both the human
and mouse genomes. Application of CHyMErA to assess the phenotypic roles
of paralogous gene pairs reveals that ~25% of paralogs display non-additive
fitness phenotypes when targeted in combination. These data reveal extensive
genetic interactions between paralogs, and demonstrate that CHyMErA offers a
tool for uncovering previously masked phenotypes due to genetic redundancy.

In addition, we have applied CHyMErA to assess phenotypic roles of individual

splice variants by directing Cas9 and Cas12a to exon-flanking sites. To assess
CHyMErA’s efficacy for exon deletion, we targeted frame-altering exons
predicted to result in gene inactivation and examined the impact on cell fitness.
As expected, hgRNAs targeting frame-altering exons in essential genes
displayed a highly significant enrichment for effects on cell growth compared to
exons residing in non-essential genes. We next applied CHyMErA to
systematically assess the phenotypic impact of deleting ~2,000 frame preserving
alternative exons, located in transcripts expressed in a panel of human cell lines
and belonging to functionally diverse genes with a range of fitness profiles.
These screens detected over 100 alternative exons whose deletion impacts cell
growth. A few of these exons have previously described roles in cell
proliferation. However, the majority have unexpected roles in cell fitness and
are represented by a range of functional categories such as cell signalling and
gene regulation. In summary, CHyMErA represents an effective system for the
combinatorial perturbation of genetic elements in mammalian cells.
Importantly, this system opens the door to systematically interrogating the
functions of exons belonging to diverse alternative splicing regulatory networks
that are amenable to high-throughput phenotyping assays.

12
WRITING LARGE DNA SEQUENCES INTO THE GENOME WITH
NOVEL ENZYMATIC MACHINERY

Barrett Steinberg1, Yanfang Fu1, Donghui Li1, Tim Sullivan1, Emily

Kibbler1, Alexas Dolbee1, Sidney Hofmann1, Christopher Bartolome1, Inna
Shcherbakova1, Anne Bothmer1, Wes Salomon 1, Rob Citorik1,2, Stephen
Cleaver1, Jacob Rubens1,2, Cecilia Cotta-Ramusino1
1
Tessera Therapeutics, Cambridge, MA, 2Flagship Pioneering, Flagship
Pioneering, Cambridge, MA

The ability to introduce long DNA sequence into genomes with high
specificity and efficiency would provide a critical complement to existing
gene-editing approaches. Canonically, DNA integration after a nuclease-
mediated double-stranded break relies upon DNA damage response
pathways, which are variably active across cell types and are biased towards
repair with small insertions or deletions. Naturally occurring mobile genetic
element enzymes, such as transposases, retrotransposases, and
recombinases, afford a rich reservoir of biochemical activity that in
principle can be exploited to integrate DNA independently of DNA damage
repair pathways. These highly abundant, and functionally diverse elements
vary in the structure of their nucleic acid substrate, the biochemical
mechanism of substrate integration, and the efficiency and specificity with
which they integrate their substrate. Here we have identified, prioritized,
and tested hundreds of previously uncharacterized mobile genetic elements
for their ability to mediate site-specific integration of genes into the human
genome. We first conducted a systematic in silico analysis of tens of
thousands of naturally occurring mobile genetic elements from across the
tree of life and prioritized species for screening based upon their proteomic
and nucleic acid signatures. We then established a high throughput
screening strategy to evaluate the activity of these enzymes in human cells.
We identified several novel enzymes that enable site-specific genomic
integration in diverse cell types, including primary cells. These enzymes
and our approach have the potential to fundamentally expand the
possibilities for genome engineering.

13
CRISPR RNA-GUIDED INTEGRASES FOR HIGH-EFFICIENCY AND
MULTIPLEXED BACTERIAL GENOME ENGINEERING

Phuc Leo Vo1, Carlotta Ronda2, Sanne Klompe3, Ethan Chen4, Christopher
Acree3, Harris Wang2,5, Samuel Sternberg3
1
Columbia University Irving Medical Center, Pharmacology, New York,
NY, 2Columbia University Irving Medical Center, Systems Biology, New
York, NY, 3Columbia University, Biochemistry and Molecular Biophysics,
New York, NY, 4Columbia University, Biological Sciences, New York,
NY, 5Columbia University Irving Medical Center, Pathology and Cell
Biology, New York, NY

Tn7-like transposons are pervasive mobile genetic elements in bacteria that

mobilize using heteromeric transposase complexes comprising distinct
targeting modules. We recently described a Tn7-like transposon from
Vibrio cholerae that employs a Type I-F CRISPR–Cas system for RNA-
guided transposition, in which Cascade directly recruits transposition
proteins to integrate donor DNA downstream of genomic target sites
complementary to CRISPR RNA. However, the requirement for multiple
expression vectors and low overall integration efficiencies, particularly for
large genetic payloads, hindered the practical utility of the transposon. Here,
we present a significantly improved INTEGRATE (insertion of
transposable elements by guide RNA-assisted targeting) system for
targeted, multiplexed, and marker-free DNA integration of up to 10
kilobases at ~100% efficiency. Using multi-spacer CRISPR arrays, we
achieved simultaneous multiplex insertions in three genomic loci, and facile
multi-loci deletions when combining orthogonal integrases and
recombinases. Finally, we demonstrated robust function in other
biomedically- and industrially-relevant bacteria, and developed an
accessible computational algorithm for guide RNA design. This work
establishes INTEGRATE as a versatile and portable tool that enables
multiplex and kilobase-scale genome engineering.

14
IMPROVING THE CHEMICAL SYNTHESIS AND PERFORMANCE OF
EXTENDED-LENGTH GUIDE RNAs FOR GENOME EDITING AND
CONTROL OF GENE EXPRESSION

Laurakay Bruhn1, Bo Curry1, Rob Kaiser1, Ben Lunstad2, Ryan McCaffrey1,

Joel Myerson1, Subhadeep Roy2, Daniel Ryan1, Israel Steinfeld1, David
Taussig1, Suhani Thakker1, Doug Dellinger2
1
Agilent Technologies, Santa Clara, CA, 2Agilent Technologies, Boulder,
CO

The landscape of CRISPR-based technologies for genome editing and

control of gene expression is expanding rapidly and many of these
technologies utilize extended-length guide RNAs that are challenging to
produce by traditional RNA synthesis chemistries. Two examples include
(i) the SAM CRISPR-activation system (Konerman, et. al., Nature, 2014)
which employs guide RNAs around 160-nt long containing two MS2 RNA
aptamers appended to stem-loops in the guide RNA to recruit
transcriptional activation domains, and (ii) Prime Editing (Anzalone et. al.,
Nature, 2019) which employs extensions of the guide RNA to direct the
replacement or insertion of new DNA sequences into the genome. Using a
novel RNA synthesis chemistry (Dellinger et. al. JACS, 2011) we find it
straightforward to chemically synthesize long RNA oligos in the size range
currently utilized for these applications. We are continuing to further
develop synthesis and purification methodologies to enable effective
synthesis of even longer RNA oligos. Chemical synthesis of RNAs affords
multiple advantages including (i) increased efficacy, (ii) robust and scalable
production of highly pure sgRNAs for biotechnological and therapeutic
applications, and (iii) greater flexibility in the sgRNA design including the
ability to incorporate chemical modifications site-specifically to enhance
performance. We are studying the impact of various types of chemical
modifications in 163mer guide RNAs designed for the SAM CRISPR-
activation system aimed at enhancing their stability in cells while
maintaining their guide RNA functionality. Experimental results using qRT-
PCR to directly measure the half-lives of these modified guide RNAs
transfected into mammalian cells without Cas9 protein, as well as other
cell-based assays to monitor the activity of the extended-length modified
guide RNAs complexed with Cas9 protein, will be presented. These studies
elucidate the types and positions of chemical modifications which do not
interfere with the activity of extended-length guide RNAs but can improve
their performance and utility, for example, by increasing the duration of
their activity when delivered into cells or in vivo.

15
STUDYING THE PHYSIOLOGICAL EFFECTS OF MUTATIONS
CONFERRING INSECTICIDE RESISTANCE IN DROSOPHILA
MELANOGASTER AND THE APPLICATION OF ALLELIC DRIVE FOR
REVERTING THESE MUTATIONS IN FLY POPULATIONS

Bhagyashree D Kaduskar1,2, Raja babu Singh Khushwa1,2, Ankush

Auradkar2,3, Alison Henrique Ferreira Julio3,4, Annabel Guichard2,3, Ethan
Bier2,3
1
Tata Institute for Genetics and Society (TIGS-India), Bangalore, India,
2
Tata Institute for Genetics and Society (TIGS-UCSD), San Diego, CA,
3
University of California, Section of Cell and Developmental Biology, San
Diego, CA, 4Instituto de Ciências Biomédicas (ICB), Universidade Federal
do Rio de Janeiro, Rio de Janeiro, Brazil

Insecticides currently in use primarily target two proteins critical for

neuronal function, the VGSC (Voltage Gated Sodium Channel) and Ace1
(Acetylcholine esterase 1). Insects acquire resistance to these insecticides,
mainly via two mechanisms: by selecting mutations affecting the
insecticide-sensitive site (target-site resistance) and by up-regulating
insecticide-metabolizing enzymes (metabolic resistance). A recurring
target-site mutation identified in various pests such as housefly and
mosquitoes, is kdr (knockdown resistance) -affecting the vgsc gene- which
confers resistance to both pyrethroids and DDT. kdr, thus poses a major
global threat to the continued use of insecticides as a means for vector
control. Despite consistent efforts to analyze functional features of the kdr
mutations in the surrogate xenopus oocyte system, it has been challenging
to link these findings with corresponding physiological effects in insects.
Drosophila melanogaster, with its many advances in genome editing, is an
attractive model for studying the physiological outcomes of these
mutations, as well as for designing strategies to reverse such mutations. In
this study, we have generated common kdr mutations identified in field-
caught mosquitoes (Anopheles gambiae and Aedes aegypti) in Drosophila
using CRISPR/Cas9 editing. Here we analyze the physiological effects of
these mutations in flies. We discovered differential sensitivities to
pyrethroids (Permethrin and Deltamethrin) versus DDT of these mutants.
Importantly, we show successful application of the allelic-drive technology,
wherein a gene drive platform inserted at one location in the genome also
biases the inheritance of a preferred (wt, sensitive in this case) allelic
variant at a second genomic site, to revert the resistant kdr mutations to its
susceptible wild type counterpart in caged populations.
The successful application of the allelic-drive systems opens up numerous
possibilities including targeted reversal of wild insecticide-resistant
populations to susceptible ones, particularly in cases where resistance is
conferred by well-identified point mutations.

16
CRISPR-Cas FROM HUGE PHAGES IS A HYPERCOMPACT
GENOME EDITOR

Patrick Pausch1,2, Basem Al-Shayeb1,3, Ezra Bisom-Rapp4, Connor A

Tsuchida1,5, Zheng Li6, Brady F Cress1,2, Gavin J Knott1,2, Steven E
Jacobsen6,7, Jillian F Banfield1,8, Jennifer A Doudna1,2,7,9
1
University of California, Innovative Genomics Institute, Berkeley, CA,
2
University of California, Department of Molecular and Cell Biology,
Berkeley, CA, 3University of California, Department of Plant and Microbial
Biology, Berkeley, CA, 4University of California, College of Natural
Resources, Berkeley, CA, 5University of California, University of
California, Berkeley - University of California, San Francisco Graduate
Program in Bioengineering, Berkeley, CA, 6University of California,
Department of Molecular, Cellular and Developmental Biology, Los
Angeles, CA, 7Howard Hughes Medical Institute, Investigator, Berkeley,
CA, 8University of California, Department of Earth and Planetary Sciences,
Berkeley, CA, 9University of California, Department of Chemistry,
Berkeley, CA

CRISPR-Cas systems are found widely in prokaryotes where they provide

adaptive immunity against virus infection and plasmid transformation. We
describe a minimal functional CRISPR-Cas system, comprising a single
~70-80 kilodalton protein, Cas (type V-J), and a CRISPR array, encoded
exclusively in the genomes of huge bacteriophages. Cas employs a single
active site for both CRISPR RNA (crRNA) processing and crRNA-guided
DNA cutting to target foreign nucleic acids. This hypercompact system is
active in vitro and in bacterial, human and plant cells with expanded target
recognition capabilities relative to other CRISPR-Cas proteins. Useful for
genome editing and DNA detection but with a molecular weight half that of
Cas9 and Cas12a genome-editing enzymes, Cas offers advantages for
cellular delivery that expand the genome editing toolbox.

17
HARNESSING NATURE’S DIVERSITY FOR GENE EDITING AND
BEYOND

Feng Zhang1,2,3
1
Howard Hughes Medical Institute, Chevy Chase, MD, 2Broad Institute of
MIT and Harvard, Cambridge, MA, 3Massachusetts Institute of Technology,
McGovern Institute for Brain Research, Dept. of Brain & Cognitive
Sciences, and Dept of Biological Engineering, Cambridge, MA

Precision genome editing, which can be used to alter specific DNA

sequences, is a powerful tool for understanding the molecular circuitry
underlying cellular processes. Over the past several years, we have
harnessed microbial CRISPR-Cas systems for use as platforms for a range
of genome manipulations, including single and multiplex gene knockout,
gene activation, and large-scale screening applications. In addition, we have
discovered and characterized several novel CRISPR systems, including the
RNA-targeting CRISPR-Cas13 systems, which we have developed for
transcriptome editing and modulation. We also leveraged the unique
properties of Cas13 to create a highly sensitive diagnostic platform for
detection of nucleic acids, called SHERLOCK. Recently, we discovered a
CRISPR-associated transposase (CAST), which we showed performs RNA-
guided transposition. We engineered CAST for precision gene insertion in
bacteria, achieving unidirectional integration of cargoes of DNA up to 10
kb at targeted genomic loci. We are continuing to refine and extend
CRISPR-based technologies as well as explore microbial diversity to find
new enzymes and systems that can be adapted for use as molecular biology
tools and novel therapeutics.

18
DEVELOPMENT OF CRISPR AS AN ANTIVIRAL STRATEGY TO
COMBAT SARS-COV-2 AND RNA VIRUSES

Stanley Qi

Stanford University, Stanford, CA

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has

highlighted the need for antiviral approaches that can be rapidly developed
and deployed to target RNA viruses. We explored the RNA-guided
endoribonuclease activity of the CRISPR-Cas13d system for viral inhibition
against SARS-CoV-2 and other viruses. The resulting method, PAC-MAN
(Prophylactic Antiviral CRISPR in huMAN cells), can effectively degrade
RNA from SARS-CoV-2 sequences, live influenza A virus (IAV) and other
live coronaviruses in human lung epithelial cells. By targeting conserved
viral regions, our bioinformatic analysis showed the strategy allows using a
small number of crRNAs to target the majority of RNA virus families
including coronaviruses. With a safe and effective system for respiratory
tract delivery, we will test its potential as prophylaxis and/or treatment to
target different RNA viruses.

19
CRISPR TOOLS IN THE TIME OF COVID-19 RESEARCH - TRENDS
FROM ADDGENE

Susanna M Bachle, Jennifer Tsang, Brook Pyhtila, Joanne Kamens, Andrew

Hempstead

Addgene, the nonprofit plasmid repository, Watertown, MA

In the beginning of this year, most of the world came to a standstill due to
the COVID-19 pandemic. This included scientific research on most topics,
with the exception of research on coronavirus and COVID-19. Scientists all
over the world came together to tackle this ongoing challenge by
developing new tools to study the virus, inventing diagnostics and assessing
treatments for the disease. One interesting trend was the development and
focusing of various CRISPR tools on COVID-19 research. Open sharing
through reagent repositories such as Addgene allows timely access and the
opportunity to improve and adapt existing tools.

Addgene has been instrumental in disseminating CRISPR diagnostic tools

for SARS-CoV-2 detection and related research globally. From March to
May 2020, just two months after Addgene started to build a COVID-19
plasmids collection, ~4,500 COVID-19 plasmid requests were received
from 50 countries. These included 81 requests from 25 countries for
CRISPR plasmids to be used in COVID-19 research. Thus far, we identified
11 CRISPR plasmids in our collection with published information
describing a use in SARS-CoV-2 detection. Since late 2012 when CRISPR
tool development began, Addgene has distributed over 180,000 CRISPR
reagents to 4,200 institutions in 87 different countries. With this widespread
sharing and adoption of CRISPR tools in previous years, we predict that
once research labs reopen, many will utilize these tools to study
coronaviruses and will continue to build new diagnostic tools using openly
shared reagents from Addgene’s repository.

The COVID-19 pandemic has demonstrated once again that CRISPR tools
are versatile and easily adaptable to many systems. The flexibility of
CRISPR tools combined with the rapid dissemination through centralized
repositories and other open sharing measures has contributed to the quick
development of CRISPR detection methods during the COVID-19
pandemic. In line with the power of many CRISPR tools, the broad
application of CRISPR-based detection tools is about to transform many
areas of disease detection and diagnostics. We plan to present distribution
statistics and application examples of CRISPR-based tools used in COVID-
19 and other disease detection and research.

20
DNA CLEAVAGE BY CLASS 2 CRISPR-CAS SYSTEMS

Wei Sun1, Zhi Cheng1,2, Xue Huang1,2, Jing Yang 1, Liang Liu1, Jiuyu
Wang1, Xueyan Li1,2, Yanli Wang1,2
1
Chinese Academy of Sciences, Key Laboratory of RNA Biology, Institute
of Biophysics, Beijing, China, 2University of Chinese Academy of
Sciences, Beijing, China

CRISPR-Cas systems are broadly grouped into two classes, and class 2
systems require only a single protein to target foreign DNA or RNA. Class
2 CRISPR-Cas systems are further divided into type II, V and VI. Type II
CRISPR-Cas9 has been widely used as genome editing tool. We reported
the structural analyses of Cas9 in multiple functional conformations,
including pre-catalytic state, catalytically activated state, and post-clavage
state. These results provide an improved framework for understanding the
mechanistic basis for Cas9 function and genome editing. Type V CRISPR-
Cas systems are further divided into subtypes A-K. We determined the
crystal structures of Cas12i2-crRNA-DNA ternary and Cas12i2-crRNA
binary complexes. We also found that the complementary ssDNA binding
activates the catalytic pocket of Cas12i2, enabling it to cleave the ssDNA
non-specially and in trans. Together, our studies of Cas9 and Cas12i2
provides new insights into the molecular mechanisms of this CRISPR
effectors.

21
STRUCTURE-GUIDED MINI AND PAM-FLEX CAS9

Osamu Nureki

The University of Tokyo, Graduate School of Science, Dept. of Biological

Sciences, Tokyo, Japan

The CRISPR-associated endonuclease Cas9 can be targeted to specific

genomic loci by single guide RNAs (sgRNAs). We have solved the crystal
structures of Cas proteins, from 7 species, complexed with sgRNA and its
target DNA at atomic resolutions. These high-resolution structures
combined with functional analyses revealed the generality and diversity of
molecular mechanism of RNA-guided DNA targeting by Cas nucleases, and
uncovered the distinct mechanisms of PAM recognition. On the basis of the
structures, we succeeded in changing the specificity of PAM recognition,
which paves the way for rational design of new, versatile genome-editing
technologies. We have generated single guanine recognizing Cas9 variants
and PMA-flex Cas9 variants, which would be very useful for base editing
and gene modulation using dCas9 as well as widening target space for
genome editing.

22
STRUCTURAL INSIGHTS INTO THE OFF-TARGET ACTIVITY OF
CAS9

Martin Pacesa1, Chun-Han Lin2, Antoine Cléry3, Katja Bargsten1, Matthew

J Irby2, Katharina Stengel2, Frédéric H.T. Allain3, Peter Cameron2, Paul
Donohoue2, Martin Jinek1
1
University of Zurich, Dept. of Biochemistry, Zurich, Switzerland, 2Caribou
Biosciences, Berkeley, CA, 3ETH Zurich, Institute of Molecular Biology
and Biophysics, Zurich, Switzerland

Target DNA specificity of the CRISPR-associated nuclease Cas9 from

Streptococcus pyogenes is determined by complementarity of a 20-
nucleotide segment in its guide RNA. This makes the system easily
programmable for a variety of genomic targets and has been extensively
utilized for genome editing in vivo. However, undesired binding and
cleavage of partially complementary targets (off-targets) creates safety
concerns for its use in clinical and agricultural applications. Currently, we
lack structural and mechanistic understanding of Cas9 off-target DNA
interactions, and how these permit off-target cleavage. Using a
crystallographic approach, we determined multiple high-resolution
structures of Cas9-sgRNA complexes bound to validated off-target DNA
substrates. We observe that preservation of proper base stacking, base
skipping, and the formation of non-canonical base pairs promotes duplex
formation with mismatched targets. This is highly dependent on the type of
mismatched nucleobase pair and the position within the guide-target DNA
strand heteroduplex. These results explain mismatch tolerance in Cas9
binding and might contribute to improved rational design of guide RNAs
and off-target prediction algorithms.

23
STRUCTURES OF THE CMR- COMPLEX REVEAL THE
REGULATION OF THE IMMUNITY MECHANISM OF TYPE III-B
CRISPR-CAS

Nicholas Sofos1, MIngxia Feng1,2, Stefano Stella1, Tillmann Pape1, Qunxin

She2, Guillermo Montoya1
1
Structural Molecular Biology Group, Novo Nordisk Foundation Center fro
Protein Research. University of Copenhagen, Protein Structure & Function,
Copenhagen, Denmark, 2State Key Laboratory of Microbial Technology,
Shandong University, Qingdao, China

Cmr- is a Type III-B CRISPR-Cas complex that upon target RNA

recognition unleashes a multifaceted immune response against invading
genetic elements, including ssDNA cleavage, cyclic oligoadenylate
synthesis, and also a unique UA-specific ssRNA hydrolysis by the Cmr2
subunit. Here, we present the structure-function relationship of Cmr-
unveiling how binding of the target RNA regulates the Cmr2 activities.
CryoEM analysis revealed the unique subunit architecture of Cmr- and
captured the complex in different conformational stages of the immune
response, including the non-cognate and cognate target-RNA bound
complexes. The binding of the target RNA induces a conformational change
of Cmr2, which together with the complementation between the 5´-tag in
the crRNA and the 3´-antitag of the target RNA, activate different
configurations in a unique loop of the Cmr3 subunit, which acts as an
allosteric sensor signaling the self vs. non-self recognition. These findings
highlight the diverse defense strategies of Type III complexes, resulting in
novel tactics of fine-tuning the immune response against invading MGEs.

24
CRISPR-FREE MITOCHONDRIAL BASE EDITING ENABLED BY AN
INTERBACTERIAL CYTIDINE DEAMINASE TOXIN

Beverly Y Mok, Marcos H de Moraes, Jun Zeng, Dustin E Bosch, Anna V

Kotrys, Aditya Raguram, FoSheng Hsu, Matthew C Radey, S. Brook
Peterson, Vamsi K Mootha, Joseph D Mougous, David R Liu

Broad Institute of Harvard and MIT / Howard Hughes Medical Institute,

Cambridge, MA

Bacterial toxins represent a vast reservoir of biochemical diversity that can

be repurposed for biomedical applications. Such proteins include a group of
predicted interbacterial toxins of the deaminase superfamily, members of
which have found application in gene-editing techniques. Because
previously described cytidine deaminases operate on single-stranded nucleic
acids, their use in base editing requires the unwinding of double-stranded
DNA (dsDNA)—for example by a CRISPR–Cas9 system. Base editing
within mitochondrial DNA (mtDNA), however, has thus far been hindered
by challenges associated with the delivery of guide RNA into the
mitochondria. As a consequence, manipulation of mtDNA to date has been
limited to the targeted destruction of the mitochondrial genome by designer
nucleases. Here we describe an interbacterial toxin, which we name DddA,
that catalyzes the deamination of cytidines within dsDNA. We engineered
split-DddA halves that are non-toxic and inactive until brought together on
target DNA by adjacently bound programmable DNA-binding proteins.
Fusions of the split-DddA halves, transcription activator-like effector array
proteins, and a uracil glycosylase inhibitor resulted in RNA-free DddA-
derived cytosine base editors (DdCBEs) that catalyze C•G-to-T•A
conversions in human mtDNA with high target specificity and product
purity. We used DdCBEs to model a disease-associated mtDNA mutation in
human cells, resulting in changes in respiration rates and oxidative
phosphorylation. CRISPR-free DdCBEs enable the precise manipulation of
mtDNA, rather than the elimination of mtDNA copies that results from its
cleavage by targeted nucleases, with broad implications for the study and
potential treatment of mitochondrial disorders.

25
CAS12A BASE EDITORS INDUCE EFFICIENT AND SPECIFIC
EDITING WITH LOW DNA DAMAGE RESPONSE

Xiao Wang1, Chengfeng Ding1, Wenxia Yu1, Ying Wang2, Siting He3, Bei
Yang4, Xingxu Huang1, Zhen Liu3, Li Yang2, Jia Chen1
1
ShanghaiTech University, School of Life Science and Technology,
Shanghai, China, 2Chinese Academy of Science, CAS Key Laboratory of
Computational Biology, CAS-MPG Partner Institute for Computational
Biology, Shanghai, China, 3Chinese Academy of Science, Institute of
Neuroscience, CAS Key Laboratory of Primate Neurobiology, Shanghai,
China, 4ShanghaiTech University, Shanghai Institute for Advanced
Immunochemical Studies, Shanghai, China

The advent of base editors (BEs) holds a great potential in correcting

pathogenic-related point mutations to treat relevant diseases. However,
Cas9 nickase (nCas9) derived BEs lead to DNA double-strand breaks,
which can trigger unwanted DNA damage response (DDR). Here, we
showed that the original version of catalytically-dead-Cas12a (dCas12a)
conjugated BEs induced a basal level of DNA breaks and minimally
activated DDR proteins including H2AX, ATM, ATR and p53. By fusing
dCas12a with engineered human APOBEC3A, we further developed the
BEACON (Base Editing induced by human APOBEC3A and Cas12a
without DNA break) system to achieve enhanced editing efficiency and
specificity. Efficient C-to-T editing was achieved by BEACON in
mammalian cells at levels comparable to AncBE4max, with only low levels
of DDR and minimal RNA off-target mutations. Importantly, BEACON
induced in vivo base editing in mouse embryos and targeted C-to-T
conversions were detected in F0 mice.

26
BACULOVIRUS VECTORED HIGHLY EFFICIENT LARGE CARGO
DNA DOCKING AND PRIME EDITING IN GENOMES

Francesco Aulicino1,2, Martin Pelosse1, Christine Toelzer1, Julien Capin1,

Parisa Meysami1, Christiane Berger-Schaffitzel1,2, Mark Dillingham1,2, Imre
Berger1,2,3
1
University of Bristol, School of Biochemistry, Bristol, United Kingdom,
2
BrisSynBio, Biological Sciences, Bristol, United Kingdom, 3Max Planck-
Bristol, Centre for Minimal Biology, Bristol, United Kingdom

The restricted heterologous DNA packaging capacity of currently used

adeno associated virus (AAV) and lentivirus (LV) severely limit the
efficacy and safety of CRISPR-based precise genome editing and
engineering interventions. In marked contrast, baculovirus possesses a very
large heterologous DNA cargo capacity, far exceeding AAV and LV.
Baculoviral vectors can efficiently transduce a large variety of mammalian
cell types and do not replicate or integrate in a mammalian host, resulting in
a favourable safety profile. Moreover, due to their widespread use as a
heterologous protein expression system, tool-kits are readily available to
facilitate heterologous DNA cargo insertion into the baculovirus.
To unlock baculovirus for state-of-the-art synthetic biology applications and
accelerate the design-build-test-learn (DBTL) cycle, we introduce and
validate MultiMate, a hybrid DNA assembly method for efficient error-free
assembly of up to 25 customized DNA modules in a single baculovirus.
MultiMate is tailormade for generating and delivering all-in-one baculoviral
vectors for CRISPR-based precise genome intervention in human cells.
We generated homology-independent targeted integration (HITI) all-in-one
baculoviral vectors for C-terminal tagging of Actin in human cells, with
efficiencies surpassing homologous directed repair (HDR) and achieving up
to 30% correct genomic insertion of DNA cargos. We progressively
increased the size of the DNA payload beyond what is feasible with AAV
or LV, and demonstrate efficient baculovirus vectored CRISPR-based DNA
docking with base pair precision irrespective of cargo size.
In addition, we applied MultiMate for baculovirus vectored prime editing in
four distinct human cell lines, achieving highly efficient and cleavage-free
trinucleotide insertion with no detectable in-del occurrence.

27
PRIMEDESIGN SOFTWARE FOR RAPID AND SIMPLIFIED DESIGN
OF PRIME EDITING GUIDE RNAs

Jonathan Y Hsu1,2,3,4, Andrew V Anzalone5,6,7, Julian Grünewald2,3,4, Kin

Chung Lam2,3,4, Max W Shen5,6,7,8, Karl Petri2,3,4, David R Liu5,6,7, J. Keith
Joung2,3,4, Luca Pinello2,3,4
1
Massachusetts Institute of Technology, Department of Biological
Engineering, Cambridge, MA, 2Massachusetts General Hospital, Molecular
Pathology Unit, Charlestown, MA, 3Massachusetts General Hospital, Center
for Cancer Research and Center for Computational and Integrative Biology,
Charlestown, MA, 4Harvard Medical School, Department of Pathology,
Boston, MA, 5Broad Institute, Merkin Institute of Transformative
Technologies in Healthcare, Cambridge, MA, 6Harvard University,
Department of Chemistry and Chemical Biology, Cambridge, MA,
7
Harvard University, Howard Hughes Medical Institute, Cambridge, MA,
8
Massachusetts Institute of Technology, Computational and Systems
Biology Program, Cambridge, MA

Prime editing (PE) is a new class of mammalian cell genome editing

technology that enables unprecedented precision in the installation of
specific substitutions, insertions, and deletions into the genome, offering
greater versatility than CRISPR nucleases and base editors. However,
design of the required guide RNAs for prime editors is more complex than
for standard CRISPR-based nucleases or base editors. Here we describe
PrimeDesign, a user-friendly, end-to-end web application
(http://primedesign.pinellolab.org/) and command-line tool
(https://github.com/pinellolab/PrimeDesign) for the design of PE
experiments. PrimeDesign can be used for single and combination editing
applications, as well as genome-wide and saturation mutagenesis screens.
Using PrimeDesign, we also constructed PrimeVar, a comprehensive and
searchable database for prime editing guide RNA (pegRNA) and nicking
sgRNA (ngRNA) combinations to install or correct >68,500 pathogenic
human genetic variants from the ClinVar database.

28
COMPARATIVE ANALYSIS OF CONVENTIONAL CRISPR-CAS9
EDITING AND PRIME EDITING OF A REGULATORY ELEMENT IN
MICE

Pan Gao*1, Qing Lyu*1, Amr R Ghanam*1, Cicera R Lazzarotto2, Gregory A

Newby3, Wei Zhang1, Mihyun Choi4, Kevin Holden5, John A Walker5,
Anastasia P Kadina5, Rob J Munroe6, Christian M Abratte6, John C Schimenti6,
David R Liu3, Shengdar Q Tsai2, Xiaochun Long1,4, Joseph M Miano1
1
Medical College of Georgia at Augusta University, Vascular Biology Center,
Augusta, GA, 2St Jude Children’s Research Hospital, Hematology, Memphis,
TN, 3Broad Institute of MIT and Harvard, Chemistry, Cambridge, MA, 4Albany
Medical College, Physiology, Albany, NY, 5Synthego, Redwood City, CA,
6
Cornell University, Biomedical Sciences, Ithaca, NY
*Contributed equally

Most sequence variation associated with disease occurs in genomic ‘dark

matter’ where millions of transcription factor binding sites (TFBS) and
thousands of short and long non-coding genes reside. Precise substitutions
within such noncoding sequences are achievable in mice using 3-component
CRISPR (Cas9, sgRNA, and repair template). The recent reporting of a 2-
component prime editing (PE) system, comprising Cas9 nickase fused to an
engineered MMLV reverse transcriptase and a prime editing guide RNA
(pegRNA), has further simplified precision editing. Here, we report results of a
comparison between CRISPR-Cas9 and PE wherein the sgRNA and pegRNA
carry the same protospacer sequence targeting a TFBS called a CArG box in the
promoter of the mouse Tspan2 gene. 3-component CRISPR targeting of the
Tspan2 CArG box (3-bp substitution) resulted in 20/37 (54%) pups with editing
detected by PCR. MiSeq analysis of 11 of the CRISPR-Cas9 founders identified
6/11 (55%) with most sequencing reads showing correct 3-bp editing of the
CArG box (range, 62.7%-95.1%). The remaining 5/11 (45%) showed >50%
local indels, with an average of only 20.45% correct alleles (range, 1.7%-
42.7%). 2-component PE of the same Tspan2 CArG box (1-bp substitution)
yielded 12/47 (26%) pups with editing detected by PCR. Strikingly, sequence
analysis showed 11/12 (92%) founders exhibited perfect 1-bp editing of the
CArG box with no evidence of local indels or other undesired editing
byproducts. CHANGE-seq performed with Cas9 nuclease nominated a greater
number of predicted off-targets associated with a standard length sgRNA than
pegRNA. Precision editing of the Tspan2 CArG box resulted in loss of Tspan2
expression in aorta and bladder, but no change in brain where expression of
Tspan2 is highest. These findings demonstrate cell type-specific loss in target
gene expression upon subtle editing of a single TFBS suggesting probable
existence of TFBS sequence variants in humans that result in strong expression
changes. PE-mediated precision editing enables future modeling or correction
of disease-associated TFBS variants in mice. *Authors contributed equally.

29
HIGH THROUGHPUT PROTEOME ANALYSIS OF gRNA-
INDEPENDENT OFF-TARGETING OF BASE EDITORS

Cristina Rocha1, Rakesh Chandode1, Anna Lina Cavallo2, Roberto Nitsch1

1
AstraZeneca R&D, Clinical Pharmacology and Safety Sciences - Gene
Therapy, Gothenburg, Sweden, 2AstraZeneca R&D, Discovery Sciences -
Discovery Biology, Gothenburg, Sweden

In most genome editing applications using CRISPR/Cas9, the Cas9

nuclease induces a double-stranded DNA break (DSB) which may result in
mis-repair events with stochastic nucleotide insertions and deletions (indels)
at the target site. Recently, Base Editors were presented as novel and safer
gene editors lacking the ability to induce DSB. Nevertheless, their use in
therapy is still hampered by a reported gRNA-independent Off-Target
editing effect at RNA level. Base editing is a multistep process where the
deamination of the target nucleotide generates an intermediate "alien" base
subsequently replaced with the intended nucleotide by the hijacked cell-
endogenous machinery. Off-Target editing of mRNA has been
demonstrated and although transient, the edited base will remain mutated
until RNA degradation. Consequently, proteins translated from the mutated
mRNA could result different from their WT counterparts (truncated,
misfolded or non-functioning). Our study relies on a high-throughput mass-
spec analysis of post edited cells. Our method compares the proteome of
WT unedited cells with the one edited by Base Editors under the canonical
conditions needed to edit the DNA. We identified the most recurrent protein
mutations and run them through a bioinformatic analysis using the immune
epitope prediction database (NetMHC) to determine whether the new
peptides presented a different immunogenic profile. This work represents
the first estimation of the risk of using Base Editors in vivo and will help
designing novel editors with reduced random deamination.

30
DETERMINANTS OF BASE EDITING OUTCOMES FROM TARGET
LIBRARY ANALYSIS AND MACHINE LEARNING

Mandana Arbab, Max W Shen, Beverly Mok, Chris Wilson, Zaneta

Matuszek, Chris Cassa, David R Liu

Broad Institute of MIT and Harvard, CBTS, Cambridge, MA

While base editors are widely used to enable targeted point mutations in
DNA, the factors that determine base editing outcomes are not well
understood, impeding the optimal choice and use of base editors from
among many reported variants. We characterized sequence-activity
relationships of 11 cytosine and adenine base editors (CBEs and ABEs) on
38,538 genomically-integrated targets in mammalian cells and used the
resulting outcomes to develop BE-Hive, a machine learning model that
accurately predicts base editing genotypic outcomes (R 0.9) and
efficiency (R 0.7). We corrected 3,388 disease-associated SNVs with
90% precision, consistent with prediction (R = 0.78–0.92), including 675
alleles with bystander nucleotides within the canonical base editing activity
window that BE-Hive correctly predicted would not be substantially edited.
We discovered sequence determinants of previously unpredictable CBE-
mediated transversions, and used these determinants to correct via C•G-to-
G•C and C•G-to-A•T editing 174 SNVs with 90% precision among edited
amino acid sequences. We discovered additional base editing outcomes
such as efficient edits of some nucleotides outside each editor’s activity
window that were not predicted by inspection, but that could be accurately
captured and predicted by BE-Hive. Finally, we established a new role for
deaminase domains in resolving U•G intermediates in CBEs, and
engineered novel CBE variants that modulate this process. These
discoveries deepen our understanding of base editors, enable their use at
previously intractable targets, and provide new base editors with improved
editing capabilities.

31
USING CRISPR-BASED GENETIC SCREENS TO STUDY DNA
REPAIR

Michele Olivieri, Alejandro Alvarez-Quilon, Tiffany Cho, Daniel Durocher

The Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital,

Toronto, Canada

The orchestration of DNA repair is of fundamental importance to the

maintenance of genomic integrity and tumor suppression. DNA repair also
profoundly influences gene editing reactions, be it via programmable
nucleases or base editing modalities. DNA damage is detected in the
context of the varied chromatin landscape, its presence must be
communicated throughout the cell to alter many ongoing processes, and the
machinery that will mend the lesion must be recruited to the damage site.
To bring new insights into the organization of the DNA damage response,
we use genome-scale genetic screens to map drug-gene and gene-gene
interactions. My presentation will focus on the chemogenomics of the DNA
damage response, which revealed some surprising new insights into DNA
repair processes, including the identification of a previously unaccounted
non-homologous end-joining factor, ERCC6L2.

32
LARGE-SCALE PHENOTYPIC CHARACTERIZATION OF GENETIC
VARIANTS OF THE DNA DAMAGE RESPONSE USING CRISPR-
DEPENDENT BASE EDITING

Raquel Cuella-Martin1, Samuel B Hayward*1, Xiao Fan*2,3, Xiao Chen1,

Junfei Zhao2,3,4, Raul Rabadan2,3,4, Chao Lu1, Yufeng Shen2,3, Alberto
Ciccia1
1
Columbia University Irving Medical Center, Dept. of Genetics &
Development, Herbert Irving Comprehensive Cancer Center, New York,
NY, 2Clumbia University Irving Medical Center, Dept. of Systems Biology,
New York, NY, 3Columbia University Irving Medical Center, Dept. of
Biomedical Informatics, New York, NY, 4Columbia University Irving
Medical Center, Program for Mathematical Genomics, New York, NY

CRISPR-dependent base editing has shown great promise for modeling and
correcting nucleotide variants of interest. However, its use on a high-
throughput scale remains to be established. In this talk, I will present our
recent work demonstrating that cytosine base editors enable large-scale
phenotypic characterization of nucleotide variants on a large-scale. In
particular, I will describe our mutational tiling studies on 86 genes of the
DNA damage response (DDR), which led to the identification of >2,000
sgRNAs generating variants that affect cellular fitness in response to DNA
damage. Among those variants, I will describe loss-of-function mutations
categorized as of uncertain significance in cancer patients, mutations with
separation-of-function patterns, and mutations with treatment-specific
phenotypes. In addition, I will outline the use of base editing screens to
identify protein motifs, post-translational modification sites and non-coding
elements of functional relevance. This work demonstrates that base editing
screens are powerful approaches to characterize human genetic variants and
define their impact on human disease.

*Authors contributed equally

33
UNDERSTANDING MECHANISMS OF GENOME EDITING WITH
HIGH-RESOLUTION FUNCTIONAL GENOMICS

Jeffrey A Hussmann1,2,3, Purnima Ravisankar4,5, Jun Yan4,5, Albert Xu1,3,

Anne Bothmer6,7, Cecilia Cotta-Ramusino6,7, Jonathan S Weissman1,3, Britt
Adamson4,5
1
UCSF, Department of Cellular and Molecular Pharmacology, San
Francisco, CA, 2UCSF, Department of Microbiology and Immunology, San
Francisco, CA, 3UCSF, Howard Hughes Medical Institute, San Francsico,
CA, 4Princeton University, Lewis-Sigler Institute for Integrative Genomics,
Princeton, NJ, 5Princeton University, Department of Molecular Biology,
Princeton, NJ, 6Editas Medicine, Cambridge, MA, 7Tessera Therapeutics,
Cambridge, MA

The ability to efficiently introduce precise changes to genomic sequence is

of broad interest for both basic research and therapeutic purposes. Widely
used genome engineering techniques introduce targeted damage to genomic
loci with CRISPR-Cas nucleases and rely on cellular DNA repair pathways
to carry out the installation of desired sequence changes. Despite intense
interest, the organization of the repair pathways engaged by these processes
and the exact molecular mechanisms by which different mutational
outcomes are produced remain incompletely understood. To address these
gaps, we developed a high throughput, high information content screening
approach for simultaneously measuring the effects of thousands of genetic
perturbations on the distribution of mutations introduced at targeted double
strand breaks. We have applied this screening platform to profile editing
outcomes after knockdown of ~500 individual repair genes and ~1000 pairs
of genes across a range of different editing strategies. Identifying
hierarchical structure in repair outcome changes across many different
perturbations allows us to perform data-driven inference of the organization
of classical and alternative end-joining pathways. We characterize the
complex interplay between local sequence content and DNA repair factors
in dictating mutational outcomes, produce insights into the mechanisms of
homology directed repair by single-stranded and linear double-stranded
donors, and identify factors involved in promoting and suppressing the
capture of non-homologous genomic sequence at double strand break sites.
The systematic maps of DNA repair processes we have produced will guide
efforts to optimize editing efficiency and precision across diverse genetic
backgrounds and editing modalities.

34
HIGH-THROUGHPUT APPROACHES TO CONTROLLING CAS9 AND
DCAS9 OUTCOMES

Lin Lin2, Ersin Akinci1, Max Shen3, Richard Sherwood1

1
Brigham and Women's Hospital and Harvard Medical School, Medicine,
Boston, MA, 2Hubrecht institute, NA, Utrecht, Netherlands, 3Broad
Institute, NA, Cambridge, MA

Repair outcomes of Cas9-nuclease-induced double-strand breaks at a given

target site have recently been shown to be predictable. Controlling these
outcomes is a major challenge toward precision gene editing. We have
devised a platform to attach 12,000 peptides derived from DNA repair
proteins to Cas9 and sensitively measure their influence on Cas9 repair
outcome distribution. Using this approach, we have identified peptides
capable of altering the repair outcome distribution of template-free and
HDR-templated Cas9-nuclease editing.

dCas9-based transcriptional modulation has relied on a small set of peptide

attachments to activate or repress gene expression. Using a similar
quantitative measurement platform, we have assessed the effects of 18,000
human peptides on dCas9-based transcriptional modulation, identifying
human peptides that enable stronger transcriptional activation than existing
dCas9 activation platforms. We will discuss these novel platforms and the
new CRISPR tools they have yielded.

35
MITOTIC ERRORS AND MASSIVE CHROMOSOME
REARRANGEMENT ARE ON-TARGET CONSEQUENCES OF
GENOME EDITING

Mitchell L Leibowitz*1,2, Stamatis Papathanasiou*1,2, Phillip A Doerfler^3,

Logan J Blaine^2, Lili Sun4, Yu Yao3, Cheng-Zhong Zhang5,6, Mitchell J
Weiss3, David Pellman1,2,7
1
Harvard Medical School, Department of Cell Biology, Boston, MA, 2Dana-
Farber Cancer Institute, Department of Pediatric Oncology, Boston, MA,
3
St. Jude Children's Research Hospital, Department of Hematology,
Memphis, TN, 4Dana-Farber Cancer Institute, Single-Cell Sequencing
Program, Boston, MA, 5Harvard Medical School, Department of
Biomedical Informatics, Boston, MA, 6Dana-Farber Cancer Institute,
Department of Data Sciences, Boston, MA, 7Howard Hughes Medical
Institute, Chevy Chase, MD

Genome editing has transformed biomedical research and has significant

therapeutic potential for genetic disease and cancer. Currently, the most
practicable approach for genome editing relies on the generation of DNA
double strand breaks by the programable Cas9 nuclease from bacterial
CRISPR systems (clustered regularly interspaced short palindromic
repeats). Despite its promise, toxicities for Cas9 genome editing are
incompletely understood. Here we report that a catastrophic mutational
process called chromothripsis is a previously unappreciated consequence of
Cas9 genome editing. Chromothripsis is extensive chromosome
rearrangement restricted to one or a few chromosomes that causes human
congenital disease and cancer. Using model cell systems and a gene therapy
protocol in clinical trials (NCT03745287) we show that Cas9 DNA breaks
generate abnormal nuclear structures, micronuclei, that trigger
chromothripsis. Chromothripsis is an on-target toxicity that may be
addressable by cell manipulation protocols and screening but cannot be
circumvented by optimization of Cas9 cutting.

36
A RAD51-INDEPENDENT PATHWAY PROMOTES SINGLE-STRAND
TEMPLATE REPAIR IN GENE EDITING

Danielle N Gallagher1, Nhung Pham2, Annie M Tsai1, Abigail N Janto1,

Grzegorz Ira2, James E Haber2
1
Brandeis University, Biology, Waltham, MA, 2Baylor College of
Medicine, Molecular and Human Genetics, Houston, TX

The Rad51/RecA family of recombinases perform a critical function in

typical repair of double-strand breaks (DSBs): strand invasion of a resected
DSB end into a homologous double-stranded DNA (dsDNA) template
sequence to facilitate repair. However, repair of a DSB using single
stranded DNA (ssDNA) as a template, a common method of CRISPR/Cas9-
mediated gene editing, is Rad51-independent. We have analyzed the genetic
requirements for these Rad51-independent events in Saccharomyces
cerevisiae in two different assays. Gene editing events were carried out
either by creating a DSB with the site-specific HO endonuclease and
repairing the DSB with 80-nt single-stranded oligonucleotides (ssODNs) or
by using a bacterial retron system that produces ssDNA templates in vivo to
repair a Cas9-mediated DSB . We show that single strand template repair
(SSTR), is dependent on Rad52, Rad59, Srs2 and the Mre11-Rad50-Xrs2
(MRX) complex, but not Rad51, Rad54 or Rad55. Srs2 acts to prevent
overloading of Rad51 on the ssDNA filament, whereas Rad59 appears to
alleviate the inhibition of Rad51 on Rad52’s strand annealing activity, as
deletion of RAD51 suppresses both the srs2 and rad59 phenotypes. In
contrast, gene targeting using an 80-bp dsDNA template of the same
sequence is Rad51-dependent. We also examined SSTR events in which the
ssODN carried several mismatches. In the absence of the mismatch repair
protein Msh2, we found that the fate of edits templated by the ssODN are
very different at the 3’ end, which can anneal directly to the resected DSB
end, compared to the 5’ end. We also find that DNA polymerase Pol ’s 3’
to 5’ proofreading activity frequently excises edits encoded close to the 3’
end of the template. We further report that SSTR is accompanied by a 600-
fold increase in mutations in a region adjacent to the sequences directly
undergoing repair. These DNA polymerase -dependent mutations may
compromise the accuracy of gene editing.

37
COMPARISON OF I-SCEI AND SPCAS9 DOUBLE-STRAND BREAK
END TETHERING IN VIVO

So Jung Lee, Rodney Rothstein

Columbia Univ Med Ctr, Genetics and Development, New York, NY

Meganucleases such as HO and I-SceI are frequently used to generate site-

specific double-strand breaks (DSBs) to study repair processes. It is unclear
how much of this knowledge can be applied to SpCas9 DSBs. For example,
it is well-established that in WT cells, repair proteins are recruited to
meganuclease-induced DSBs, and that DSB ends are mostly tethered.
However, ends become untethered in the absence of the Mre11-Rad50-Xrs2
(MRX) complex.
There is in vitro evidence that, unlike HO and I-SceI, SpCas9 stays bound to
both ends of a DSB after cleavage and blocks access of repair proteins.
However, the effect of Cas9 on DSB end tethering and repair protein
recruitment in vivo has not been studied. We developed a system in S.
cerevisiae to visualize both ends of an inducible DSB as well as the Rad52
repair protein by fluorescence microscopy in vivo. We compared I-SceI and
SpCas9 cleavages in this system by examining Rad52 recruitment and DSB
end tethering.
We observe that WT cells recruit Rad52 to both I-SceI and Cas9-induced
DSB ends with similar efficiencies, showing that Cas9 does not block
access of repair proteins in vivo. In the absence of the MRX complex
(xrs2 ), cells recruit Rad52 less efficiently than WT. Interestingly, Rad52
recruitment is lower at Cas9 DSBs compared to I-SceI DSBs in xrs2 cells.
Perhaps the end processing deficiency in xrs2 cells leads to sensitivity to
the type of DSB; Cas9 DSBs have blunt ends while I-SceI DSBs have 5’
overhangs.
Next, we assessed DSB end tethering. In WT cells, both I-SceI and Cas9
DSB ends are mostly tethered. In xrs2 cells, however, most DSBs
generated by either nuclease become untethered, providing evidence that
Cas9 does not remain bound to DSB ends in vivo. If Cas9 were bound to
DSB ends, tethering by Cas9 would have suppressed the untethering
phenotype of xrs2 .
Interestingly, the majority of untethered ends after cutting with I-SceI or
Cas9 nuclease do not recruit Rad52 foci to either end in xrs2 , again
reflecting a deficiency in end processing. A smaller group of untethered
cells have a single Rad52 focus on one end of the DSB. Surprisingly,
recruitment of Rad52 to either end is not random. Rad52 foci form more
often on the telomere-proximal end compared to the centromere-proximal
end for both I-SceI and Cas9 DSBs in xrs2 . This bias in Rad52 recruitment
was not observed at the untethered ends of WT cells.
In conclusion, we find that Cas9-induced DSB ends efficiently recruit
Rad52 and become untethered in the absence of MRX, suggesting that Cas9
does not bind the two ends of a DSB in vivo. Additionally, in the absence of
MRX, Rad52 is recruited asymmetrically to the untethered ends, where the
telomere-proximal end is preferred.
38
CRISPR/CAS-MEDIATED CHROMOSOME ENGINEERING IN
ARABIDOPSIS THALIANA

Michelle Roenspies1, Carla Schmidt1, Natalja Beying1, Andreas Houben2,

Paul Fransz3, Holger Puchta1
1
Karlsruhe Institute of Technology, Botanical Institute, Karlsruhe,
Germany, 2Leibniz Institute of Plant Genetics and Crop Plant Research,
Breeding Research, Seeland, Germany, 3University of Amsterdam, Plant
Development and (Epi)Genetics, Amsterdam, Netherlands

Ever advancing genome editing tools are in the process of revolutionizing

plant breeding. The current state of this technology enables many
applications which aim to improve plant productivity or other desirable
traits. Up until a few years ago, the main focus of researchers was to simply
knock-in or knock-out single genes or to induce single base changes, but
constant improvements of genome editing tools have lead researches to
tackle more ambitious projects. A long-standing aim was the establishment
of efficient genome engineering tools with the prospect of being able to
shape chromosomes based on our needs and to even mimic genome
evolution, both with great potential to impact plant breeding. Recently, we
were able to show for the first time that the CRISPR/Cas system can be
utilized for the induction of heritable chromosomal rearrangements, such as
inversions and translocations, in the Mb-range in plants. The application of
this technique has the potential to transform plant breeding as, for example,
inversions typically present obstacles to the breeding process due to absent
recombination between inverted regions. On the other hand, the targeted
induction of chromosomal translocations provides access to fix or break
genetic linkages between traits on different chromosomes, a current
limitation to the breeding process. By combining an egg-cell specific
promoter and the Cas9 orthologue from Staphylococcus aureus we were
able to reverse the well-known 1,1 Mb heterochromatic knob (hk4S) on
chromosome 4 in Arabidopsis, which is present in only some of its
accessions, such as Columbia-0 (Col-0). It has been shown that in case of
crossing Col-0 with a knob-less accession such as Landsberg erecta (Ler-1),
meiotic recombination is completely suppressed within the inverted region.
By crossing Col-0, harbouring the reversed knob, with Ler-1, we were able
to target meiotic crossovers into a region that was previously inaccessible
for genetic exchange. Using the same strategy, we were able to reach
another milestone towards efficient genome engineering: the induction of
reciprocal heritable translocations in the Mbp range between heterologous
chromosomes in A. thaliana, reaching frequencies of up to 2.5 % for
individual lines. Using molecular and cytological analysis, it could be
confirmed that the chromosome-arm exchanges between chromosomes 1
and 2 and between chromosomes 1 and 5 were conservative and reciprocal.

39
PRECISE INTRODUCTION OF SMALL GENETIC CHANGES
WITHOUT BYSTANDER MUTATIONS IN VITRO AND IN VIVO BY
SCARLESS GENE EDITING

Brandon W Simone1, Ata Hiro2, Han B Lee1, Camden L Daby1, Stephen C

Ekker1,2, Karl J Clark1,2
1
Mayo Clinic, Biochemistry and Molecular Biology, Rochester, MN, 2Mayo
Clinic, Clinical and Translational Sciences, Rochester, MN

Introducing small genetic changes to study specific alleles or reverting

clinically relevant mutations to normal has been an area of interest in
precision genomics for several years. Estimates suggest nearly 90% of
known human pathogenic mutations are caused by small genetic variations,
and the ability to correct these errors is highly sought after. Several
technologies have been developed to address the issue, but each have their
limitations. One of the most common ways to correct these small sequence
variations is by providing a single stranded DNA (ssDNA) donor containing
the desired alteration, typically flanked by variable regions of homology
from ~30-100bp in length for correction by the homology directed repair
(HDR) pathway. ssDNA donors coupled with a CRISPR-Cas9 mediated
Double-stranded breaks (DSB) is one of the most streamlined approaches to
introduce such small changes. However, ssDNA-mediated gene editing
often results in undesired indel mutations at the insertion junction as a result
of the DNA repair by HDR. This imprecision represents a major technical
bottleneck that can preclude the use of this method for high resolution
genome engineering applications. We herein report Scarless Gene Editing
(SGE) as a genome engineering technology that results in precise insertion
of small genetic changes without bystander indel mutations introduced by
classical ssDNA-mediated HDR. We show that SGE operates in diverse
systems from in vitro HEK293T cells and human induced pluripotent stem
cells (iPSCs) and somatic and germline changes in vivo in zebrafish. Data is
consistent with SGE inducing precise edits using a conserved mechanism
that is independent of the canonical HDR DNA repair pathway. SGE
represents a new approach for precise gene editing that enables more rapid
and precise gene editing from vertebrate models in vivo to human primary
cells in vitro.

40
TARGETED INSERTION OF A LARGE DNA FRAGMENT USING
CRISPR-CAS9 IN THE MODEL PLANT RICE

Oliver Xiaoou Dong, Shu Yu, Rashmi Jain, Phat Duong, Nan Zhang,
Corinne Butler, Yan Li, Anna Lipzen, Joel Martin, Kerrie Barry, Jeremy
Schmutz, Li Tian, Pamela C Ronald

University of California, Davis, College of Biological Sciences, Davis, CA

Targeted gene insertion at a pre-determined genomic safe harbor in plants

provides a desirable alternative to random transgene insertion obtained
through conventional methods. Most cases of targeted gene insertion in
plants reported to date either relied on the presence of a selectable marker
gene in the inserted DNA or occurred at low frequency (< 2.2 %) with
relatively small DNA fragments (< 1.8 kb). We demonstrated the use of an
optimized CRISPR-Cas9-based method to achieve targeted gene insertion
of a 5.2 kb DNA fragment at newly identified genomic safe harbors in rice.
We obtained a marker-free rice plant with high carotenoid content in the
seeds and no detectable penalty in morphology or yield. Whole genome
sequencing revealed the absence of detectable off-target mutations by Cas9
in this plant. This study offers a promising strategy for genetic improvement
of rice and other crops.

41
DISSECTING QUANTITATIVE TRAIT VARIATION BY GENOME
EDITING.

Zach Lippman

Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

Genome editing is being lauded as a revolutionary technology that will

invigorate plant breeding. However, crop domestication and improvement is
founded on genetic complexity that goes well beyond single gene mutations
with dramatic phenotypic effects, which have dominanted plant genome
editing thus far. To advance to the next stage, for both agricultural
application and to address fundamental questions, we are dissecting the
mechanisms and principles underlying quantitative trait variation. Our early
focus was on epistasis, which revealed that interacting genes are highly
dose-sensitive systems and can exploited to create continuums of
quantitative variation. More recently we have applied genome editing to
investigate the genetic architectures of cis-regulatory regions that control
transcriptional and phenotypic outputs from developmental genes. We have
found that gene promoters are also highly dose-sensitive and can serve as
tunable control regions, which can be manipulated to create novel alleles
and quantitative variation that goes beyond what nature has provided. I will
present case studies that shed light on if and to what extent rules can be
established to achieve predicable outcomes from genome editing.

42
CRISPR-MEDIATED TARGETING OF EPIGENETIC
MODIFICATIONS IN PLANTS.

Steven E Jacobsen, Javier Gallego-Bartolome, Ashot Papikian, Basudev

Ghoshal, Jason Gardiner, Wanlu Liu

HHMI/University of California, Los Angeles, Dept. of Molecular, Cell and

Developmental Biology, Los Angeles, CA

Our laboratory uses genetics, genomics, and biochemical approaches to

study cytosine DNA methylation and gene silencing in the model plant
Arabidopsis thaliana. DNA methylation in plants is stably inherited from
generation to generation due the activity of four interlinked DNA
methylation pathways which form a complex self-reinforcing loop to
maintain methylation in three cytosine sequence contexts, CG, CHG and
CHH. The initiation of DNA methylation requires the DNA
methyltransferase DRM2 (homolog of mammalian Dnmt3) which is guided
by 24 nucleotide small RNAs and longer non-coding RNAs in a pathway
called RNA-directed DNA methylation. We are developing CRISPR-based
approaches to target components of this pathway to initiate methylation and
silencing at gene promoters. We have demonstrated that once initiated, this
methylation and silencing can be maintained in subsequent plant
generations after the removal of the CRISPR tools. We have also developed
CRISPR systems for removing DNA methylation from genes, and shown
that this unmethylated state can also be heritable in the absence of the
demethyating transgene. Finally, we are developing novel systems for gene
activation and gene silencing based on the CRISPR SunTag system.
Progress in the development and use of these tools will be presented.

43
OVERCOMING BOTTLENECKS IN EDITING PLANT GENOMES

Dan Voytas

University of Minnesota, Department of Genetics, Cell Biology &

Development, Center for Genome Engineering and Center for Precision
Plant Genomic, St. Paul, MN

Plant gene editing is usually carried out by delivering reagents such as Cas9
and sgRNAs to explants in culture. Edited cells are then induced to
differentiate into whole plants by exposure to various hormones. Creating
edited plants through tissue culture is often inefficient, requires
considerable time, only works with limited species and genotypes and
causes unintended changes to the genome and epigenome. We report
methods to generate gene edited dicotyledonous plants through de novo
meristem induction. Developmental regulators and gene editing reagents are
delivered to somatic cells on whole plants. Meristems are induced that
produce shoots with targeted DNA modifications, and gene edits are
transmitted to the next generation. The de novo induction of gene edited
meristems sidesteps the need for tissue culture, promising to overcome a
bottleneck in plant gene-editing.

44
PRECISE GENE EDITING IN CROPS

Yu Mei, John Gierer, Theodore Moll, Gengxiang Jia, Behailu Aklilu

KWS, Gateway Research Center, St Louis, MO

Genome editing (GE) technologies, such as CRISPR/Cas-based systems are

powerful tools for agriculture. GE shows great potential in both functional
validation of genes, and crop improvement through molecular breeding.
Precise gene edits, and sequence modifications e.g. gene insertions or
replacements after double-strand DNA breaks via homology targeted gene
directed repair pathway (HDR) are very much desired. Nevertheless, low
efficiency of HDR-mediated sequence modifications is a major roadblock to
success, since non-homologous end joining (NHEJ) is the dominant repair
pathway in plants. KWS is a world leading plant breeding company, and
our GE team seeks to improve the efficiency of HDR in important crop
species. We have established an efficient and fast assay for targeted
integration in a regenerable cell culture system. Together with the use of our
regeneration booster proteins, we have been able to detect targeted
integration in both model, and elite corn lines. We further identified and
screened multiple potential plant HDR boosters and found significant
improvement on efficiency of targeted integration with the best conditions.
These findings enable us to produce gene edited plants with precise targeted
integration in an efficient and cost-effective way.

45
ONE-SEQ: A UNIVERSAL PLATFORM FOR DEFINING OFF-TARGET
PROFILES OF CRISPR GENE EDITORS WITH UNSURPASSED
SENSITIVITY

Karl Petri1, Daniel Y Kim1, Kanae E Sasaki1, Matthew C Canver1, Xiao

Wang2, Hyunho Lee1, Hina Shah1, Joy E Horng1, Kendell Clement1,
Sowmya Iyer1, Sara P Garcia1, Jimmy A Guo1, Gregory A Newby3, Luca
Pinello1, David R Liu3, Martin J Aryee1, Kiran Musunuru2, J Keith Joung*1
1
Massachusetts General Hospital, Molecular Pathology Unit, Charlestown,
MA, 2Perelman School of Medicine at the University of Pennsylvania,
Cardiovascular Institute, Philadelphia, PA, 3Harvard University and Howard
Hughes Medical Institute, Department of Chemistry and Chemical Biology,
Cambridge, MA

Defining off-target profiles of gene-editing nucleases and CRISPR base

editors remains an important challenge for use of these technologies,
therapeutic or otherwise. Here we describe OligoNucleotide Enrichment
and sequencing (ONE-seq), a novel in vitro method that leverages
customizable, high-throughput DNA synthesis technology instead of
purified genomic DNA (gDNA) from individual genomes to profile gene
editor off-target sites. We show that ONE-seq matches or exceeds the
sensitivity of existing methods for identifying bona fide CRISPR-Cas9 off-
target sites in cultured human cells and in vivo in a liver-humanized mouse
model. In addition, ONE-seq outperforms existing best-in-class methods for
defining off-target sites of CRISPR-Cas12a nucleases, cytosine base editors
(CBEs), and adenine base editors (ABEs), unveiling previously
undescribed bona fide off-target sites for all these editors in human cells.
Collectively, our results demonstrate the unsurpassed sensitivity and
flexibility of ONE-seq for defining off-target effects of various CRISPR-
based gene editors.

*To whom correspondence should be addressed

46
PERTURBING GENE REGULATORY NETWORKS DYSREGULATED
IN CHD8-RELATED AUTISM USING CRISPR-BASED
TRANSCRIPTION FACTORS.

Lexi R Bounds1,2, Charles A Gersbach1,2,3

1
Duke University, Department of Biomedical Engineering, Durham, NC, 2Duke
University, Center for Advanced Genomic Technologies, Durham, NC, 3Duke
University Medical Center, Department of Surgery, Durham, NC

119 rare variants in the gene encoding Chromodomain-helicase-DNA-binding

protein 8 (CHD8) have been identified that result in ASD-related phenotypes1.
These heterozygous, loss-of-function mutations share clinical phenotypes including
macrocephaly and development delay. CHD8 is known to regulate the Wnt signaling
pathway through interactions with -catenin, and functions as a transcriptional
repressor by remodeling chromatin structure. Previous work established that CHD8
regulates the expression of many ASD-related genes and transcriptional networks
specific to neurodevelopment in human induced pluripotent stem cell (hiPSC)-
derived neural progenitor cells (hNPCs), human midfetal cortex, and embryonic
mouse cortex2-4. However, the specific gene regulatory networks (GRNs)
dysregulated by CHD8 haploinsufficiency still remain unclear.

We hypothesized targeted modulation of CHD8 using nuclease-dead Cas9 (dCas9)-

effector fusions could clarify the underlying GRNs that are disrupted in CHD8-
related ASD. hNPCs were transduced with lentiviral vectors containing either the
repressive fusion, dCas9KRAB, or the activating fusion, VP64dCas9VP64, and a single
guide RNA targeting the promoter of CHD8 or a non-targeting control. Changes in
mRNA expression were assayed via RT-qPCR and RNA-seq four days post-
transduction. We show 4-fold increase and 50% reduction in mRNA levels of CHD8
when targeted with VP64dCas9VP64 or dCas9KRAB, respectively.

Transcriptome sequencing revealed 2,184 and 1,465 differentially expressed genes

(DEGs) upon activation or repression of CHD8, respectively (q < 0.05). Further, the
specificity of dCas9-effector fusions is demonstrated as CHD8 is the most
upregulated- or downregulated-gene, even though it is known to regulate many other
genes. The DEGs upon modulation of CHD8 were enriched for ASD-risk genes1 (p
< 0.001). Likewise, gene ontology biological process and pathway analysis revealed
that DEGs upregulated upon CHD8 repression were enriched in neurodevelopment
pathways, including “axon guidance”, “neuron projection development”, and
“positive regulation of neuron differentiation” (q < 0.05). In contrast, DEGs
downregulated upon CHD8 activation were enriched in cell cycle, chromatin
organization, and biosynthesis processes (q < 0.05), suggesting that the loss of
CHD8 expression has distinct consequences.

In summary, we demonstrated the use of dCas9-based epigenome editors to dissect

the GRNs underlying ASD-risk genes. We are currently conducting high-throughput
CRISPR-Cas9-based epigenomic regulatory element screening (CERES)5 to identify
distal regulatory elements that control CHD8 expression.
[1] Abrahams, B. S. et al. Mol Autism (2013).
[2] Cotney, J. et al. Nat Communications (2015).
[3] Durak, O. et al. Nat Neuroscience (2016).
[4] Wang, P. et al. Mol Autism (2017).
[5] Klann, T. S. et al. Nat Biotechnology (2017).

47
MODELLING MUTATIONS AND VARIANTS THAT ALTER GAMMA
GLOBIN EXPRESSION USING GENOME EDITING IN ERYTHROID
CELLS

Gabriella E Martyn, Beeke Wienert, Elizabeth S Stout, Lu Yang, Manan

Shah, Lana C Ly, Merlin Crossley, Kate G Quinlan

UNSW Sydney, School of Biotechnology and Biomolecular Sciences,

Sydney, Australia

-haemoglobinopathies, such as sickle cell anaemia and -thalassemia, arise

from mutations in the adult -globin gene that disrupt the normal function
of haemoglobin. Reactivation of the developmentally silenced foetal
globin gene in in adult red blood cells is an attractive therapeutic option for
these genetic diseases as it leads to elevated foetal haemoglobin and
alleviates the symptoms of -haemoglobinopathies.

Hereditary Persistence of Foetal Hemoglobin (HPFH) is a rare benign

condition in which individuals express globin throughout adulthood. Co-
inheritance of one of these beneficial HPFH mutations reduces the severity
of symptoms in an individual with a -haemoglobinopathy mutation.
Making globin promoter HPFH mutations in established erythroid cell
lines using CRISPR/Cas9 has allowed us to define how these mutations lead
to upregulation of the normally silenced foetal globin and uncover the
transcription factors involved. Two clusters of the mutations, at ~200bp and
~115bp upstream from the transcription start site disrupt the binding of the
major globin repressor transcription factors ZBTB7A and BCL11A, thus
leading to globin upregulation in adult cells. Conversely, single base pair
mutations at 198bp, 175bp and 113bp upstream of the transcription start site
create de novo binding sites for well-known erythroid activating
transcription factors KLF1, TAL1 and GATA-1, respectively, to upregulate
globin.

We have also used both CRISPR/Cas9 and base editing to model genetic
disease modifiers identified by GWAS studies as linked to elevated foetal
haemoglobin and reduced -haemoglobinopathy disease severity. These
studies have allowed us to identify a number of disease modifier variants
that are causative of foetal globin upregulation and to define the
mechanism by which they act.

Our work provides insights into both the utility of genome editing with
naturally occurring beneficial mutations and disease modifiers to treat sickle
cell anaemia and -thalassemia and the fundamental mechanistic knowledge
required for clinical translation of this approach.

48
DIRECTED EVOLUTION OF ADENINE BASE EDITORS WITH
INCREASED ACTIVITY AND THERAPEUTIC APPLICATION

Nicole M Gaudelli, Dieter K Lam, Holly A Rees, Noris M Sola-Esteves,

Luis A Barrera, David A Born, Aaron Edwards, Jason M Gehrke, Seung-
Joo Lee, Alexander J Liquori, Ryan Murray, Michael S Packer, Conrad
Rinaldi, Ian M Slaymaker, Jonathan S Yen, Lauren E Young, Giuseppe
Ciaramella

Beam Therapeutics, Cambridge, MA

The foundational adenine base editors (e.g. ABE7.10) enable programmable

C•G to T•A point mutations, but editing efficiencies can be low at some
challenging loci in primary human cells. We recently employed directed
evolution strategies to further evolve ABE7.10, using a chemically-
synthesized library of adenosine deaminase variants, to create next-
generation adenine base editors (ABE8s). At NGG PAM sites, ABE8s
result in ~1.5x higher editing at protospacer positions A5-A7 and ~3.2x
higher editing at positions A3-A4 and A8-A10 compared with ABE7.10.
Non-NGG PAM variants have a ~4.2-fold overall higher on-target editing
efficiency than ABE7.10. In human CD34+ cells, ABE8 can recreate a
natural allele at the promoter of the -globin genes HBG1 and HBG2, with
up to 60% efficiency, causing persistence of fetal hemoglobin. In primary
human T cells, ABE8s achieve 98-99% target modification which is
maintained when multiplexed across three loci. Delivered as mRNA,
ABE8s induce no significant levels of sgRNA-independent off-target
adenine deamination in genomic DNA and very low levels of adenine
deamination in cellular mRNA.

49
A POOLED SCREEN TO ENGINEER AND OPTIMIZE CAS RNP
VARIANTS FOR CELL-TARGETED DELIVERY

Akshay Tambe, Rina J Mepani, Aaron J Cantor, Sruja S Iyer, Angelica F

Castaneda, Hariharan Jayaram

Spotlight Therapeutics, NA, Hayward, CA

Editing genomes with CRISPR-Cas proteins has the potential to

revolutionize the way human genetic diseases are treated. Although
remarkable progress has been made using these nucleases to modify a
genome in a defined manner, delivering Cas / guide RNA complexes to
selected cells remains a significant challenge.

To address this, we developed a high-throughput screen to identify Cas

fusion proteins that can selectively internalize and localize to the nucleus of
primary cells. This workflow does not require engineering of reporters into
the target cells, thereby enabling the use of primary cells and a wide variety
of cell types. First, we developed a streamlined and scalable technique to
prepare large libraries of RNA-barcoded Cas9 complexes, which pairs
unique sequence identifiers on the guide RNA with a library of Cas9 protein
variants. Then we identified cell-internalizing variants by co-incubating the
RNA-barcoded protein complexes with cells of interest, isolating the
nuclear subcompartment, and sequencing the barcodes within that fraction.
Quantitation of the internalized barcodes allows us to measure the uptake
dynamics of multiple Cas9 variants simultaneously.

To demonstrate feasibility, we prepared a library of approximately five

thousand Cas9 variants, each bearing a unique cell penetrating peptide and
its identifying guide RNA barcode. We then used our high-throughput
sequencing assay to quantitatively rank each cell penetrating peptide’s
capacity to deliver the Cas9 payload to the nucleus of primary mouse
fibroblasts. As an internal validation of the method, the library included
sequences that have been previously shown to improve Cas9 internalization,
and as expected, the screen recovered the positive control peptides. We also
identified a number of previously uncharacterized peptides that enhance
Cas9 internalization. Global analysis of the chemical properties of the
peptide library revealed chemical signatures that substantially improve Cas9
internalization.

Taken together, our methods serve as a proof-of-principle for a pooled

screen for CRISPR-Cas delivery. It can be easily expanded to investigate
larger and more diverse libraries of Cas variants, as well as other cell types
of interest. Additionally, our approach to preparing libraries of barcoded
CRISPR-Cas proteins is likely to be widely applicable to other protein
engineering problems in the field.

50
TRANSLATING ADENINE BASE EDITOR TECHNOLOGY INTO A
POTENTIAL TREATMENT FOR LUNG AND LIVER
MANIFESTATIONS OF ALPHA-1 ANTITRYPSIN DEFICIENCY

Michael S Packer1, Vivek Chowdhary2, Genesis Lung1, Lo-I Cheng1,

Yvonne Aratyn-Schaus1, Sarah Smith1, Aalok Shah1, Delai Chen1, Marina
Zieger2, Brian Cafferty1, Bo Yan1, Christian Mueller2, Giuseppe
Ciaramella1, Francine M Gregoire1
1
Beam Therapeutics, Liver, Cambridge, MA, 2University of Massachusetts
Medical School, Horae Gene Therapy Center, Worcester, MA

Alpha-1 Antitrypsin Deficiency (A1AD) is a rare monogenic liver and lung

disease caused by mutations within the SERPINA1 gene. The most
common genetic variant associated with clinical disease is the PiZ allele
containing a single G>A transition mutation. Alpha-1 antitryspin (A1AT)
expressed from the PiZ allele harbors the E342K amino acid substitution
responsible for protein misfolding and formation of toxic aggregates within
hepatocytes. Due to aberrant protein folding, insufficient A1AT is secreted
into circulation where it serves as an inhibitor of neutrophil elastase. A1AT
deficiency leaves elastin-rich tissue in the lungs vulnerable to damage that
ultimately presents as emphysema. In principle, adenine base editors
(ABEs) can catalyze the chemistry required to precisely correct the disease-
causing PiZ mutation. However, generating an ABE that efficiently corrects
the PiZ mutation required modification of the ABE Cas9 domain to permit
acceptance of a non-canonical protospacer-adjacent motif (PAM) as well as
mutation of the TadA deaminase to increase enzymatic activity. Chemical
modification of the gRNA led to further increases in base editing
efficiencies. In patient-derived cells, the most prevalent base editing product
was correction of the PiZ allele to a wild-type gene (PiM), but we also
observed editing of neighboring bystander adenines. In vitro biochemical
assays suggest that both precise correction to wild-type as well as correction
linked to a D341G bystander edit would be beneficial editing outcomes.

Using lipid nanoparticle (LNP) delivery technology, we assessed our base

editing approach in the NSG-PiZ transgenic mouse model. In total liver
genomic DNA, we observed considerable levels of beneficial alleles that
increase with the passage of time after treatment. This increase would be
consistent with a proliferative advantage in gene-corrected hepatocytes that
no longer express the toxic PiZ protein. Consistent with this observation,
liver pathology was markedly improved as measured by a reduction in PAS-
D stained PiZ globules. Consequently, treated animals also exhibited an
increase in serum A1AT and serum elastase inhibitory capacity. Using
mass-spectrometry, this increase in serum A1AT was shown to be
constituted by wild-type and D341G A1AT proteins. These results indicate
that base editing has the potential to address both lung and liver disease in
A1AT deficiency.

51
ASSESSING THE IMPORTANCE OF THE KINASE DOMAIN
THROUGH KINOME-FOCUSED CRISPR SCREENING

Ahmad Al Kawam, Alexandra Grassian, Fabien Llambi, Leni Jacob,

Zhigang Weng, Klaus Hoeflich, Chaoyang Ye

Blueprint Medicines, Biology, Cambridge, MA

The identification of new therapeutic targets is critical in the fight against

cancer. Over the past few decades, the inhibition of kinase cancer drivers
has dramatically changed the cancer treatment landscape. Kinases play an
important role in modulating the cellular signaling cascades that are often
altered in cancer. This role is mainly carried out by the kinase domain(s)
which control catalytic activity through binding ATP. Consequently, small
molecule inhibition of catalytic activity can disrupt cancer signaling and
halt tumor growth.

Although examples of kinase drivers in cancer development are well

established, there are still numerous understudied kinases with limited
information about their biological function. Therefore, we designed a
comprehensive kinase-focused CRISPR library specifically aimed at
interrogating the contribution of kinase catalytic activity. Our library
contains a high density of guides targeting the kinase domain, allowing for
quick assessment of the importance of the active site. Our library aims to
create opportunities for identifying novel therapeutic targets and for further
understanding the cancer signaling mechanisms.

We utilized our newly designed library in a high throughput, kinome-wide

CRISPR pooled screen applied to multiple cellular models. CRISPR Pooled
Screening is a powerful tool for conducting large-scale studies involving
hundreds or thousands of genes. However, due to a general preference to
increase throughput, most screening efforts include a limited number of
guides per gene. This increases chances of detecting false positives and
reduces the screen’s power to differentiate between the activity of different
gene domains. In our library, however, we used a minimum of 24 guides
per gene strategically distributed both inside and outside the catalytic
domains of each kinase. Additionally, our library allows us to rule out likely
false-positive hits due to genomic copy number amplifications through the
addition of guides targeting the nearest neighbor gene.

As a reference, we used the Broad Institute’s Achilles Avana CRISPR

Screen. Our library was able to reproduce 100% of Avana’s significant
kinase hits. Furthermore, we identified novel hits that did not show
dependency in Avana. These hits produced a phenotype only when their
kinase domain was targeted and did not show any phenotype for the guides
outside the kinase domain. Most importantly, our screen highlighted the
value of domain-centric CRISPR libraries and how they could be used for
identifying kinase targets that have so far eluded previous screens.
52
UNDERSTANDING HOMOLOGY-DIRECTED REPAIR IN AEDES
AEGYPTI GERMLINE ENGINEERING

Joshua Ang Xin De, Katherine Nevard, Rebekah Ireland, Lewis

Shackleford, Estela Gonzalez, Michelle A Anderson, Luke Alphey

The Pirbright Institute, Arthropod Genetics, Pirbright, United Kingdom

The increasing prevalence of insecticide resistance and ongoing global

disease burden has encouraged new efforts in mosquito control. Genetic-
based strategies, some of which require site-specific CRISPR/Cas-9 genome
engineering of the mosquito germline have great potential as species-
specific mosquito control strategies. Despite recent successes in Aedes
aegypti and other mosquito species, little is known of the conversion tract or
any other factors that influence the integrity and efficiency of homology-
directed repair (HDR) events in the germline. Plasmid constructs containing
a fluorescent marker with perfect, recoded, or perfect, but with a small
deletion, homology arms were injected with Cas9 protein and in-vitro
transcribed sgRNAs into Ae. aegypti embryos of the Liverpool strain.
Single-stranded, biotinylated linearised double-stranded, and/or biotinylated
single-stranded versions of two constructs were also assessed. Biotinylated
donors were co-injected with mRNA coding for Cas9 fused with a
monomeric streptavidin. Injection survivors were subsequently outcrossed
with wild-type and the next generation screened for fluorescence indicating
an HDR event into the germline of the injected individual. PCR and
sequencing was used to confirm the site of transgene integration and
characterise the conversion tract of insertion events. The plasmid construct
with perfect homology arms gave the highest integration rate (4.8%), and
recoding of just 1.2% of the sequence decreased the integration rate to
1.63% (Binomial test, p = 0.02). A reduction in integration rate was also
observed when a 170 bp-long sequence homologous to the region around
the double stranded break was deleted from the homology arm (Binomial
test, p = 0.01). On the other hand, the various donor types did not cause any
significant effects (Binomial test, p > 0.1) to the integration rates when
compared to the corresponding plasmid versions. Just over half of the
positive G1s obtained with a plasmid donor were results of perfect HDR
events while the other donor types generated integrations which could not
be fully characterised by PCR, indicating they have likely not integrated
into the intended target site. Sequencing of the transgene flanking region in
65 individuals has revealed all conversion tracts to be unidirectional and
span within 220 bp proximal to the break, except in one individual where
bidirectional conversion of up to 725 bp proximal to the break was
observed. This suggests that HDR in the germline is usually initiated by
strand invasion on only one side of the break. The HDR mechanism in the
Ae. aegypti germline is highly sensitive to donor type and homology arm
heterology. Gene conversion resulting from HDR events is almost always
unidirectional and spans within 220 bp proximal to the double strand break.

53
DNA BINDING BY CAS9 INTERFERES WITH BASE EXCISION
REPAIR EFFICIENCY

Jacob Antony, John Wyrick, John Hinz

Washington State University, School of Molecular Biosciences, Pullman,

WA

DNA cleavage by the Cas9 endonuclease requires RNA-directed binding to

specific genomic targets. Genome editing with the CRISPR/Cas system is a
well characterized process of targeted mutagenesis due to the generation of
double strand breaks by Cas9, however DNA targeting and inadvertent off-
target binding by Cas9 throughout the genome can lead to unwanted
sporadic mutagenesis. We aim to explore the mechanisms critical to
mutagenesis resulting from Cas9 binding. Using catalytically inactive dead
Cas9 (dCas9), we address whether the inhibition of DNA repair processes,
such as base excision repair (BER), contribute to mutagenesis from DNA
targeting in CRISPR/Cas systems. We propose that dCas9 limits repair
protein accessibility to DNA lesions thus inhibiting repair activity and
contributing towards mutagenesis at both target and off-target binding sites.
We found that dCas9 targeting is inherently mutagenic in yeast, particularly
in a repair-deficient background. Our data indicates that cytosine
deamination on the DNA strand opposing the guide RNA-targeted strand
within the genomic target region is an important source of mutagenesis
during genome editing with the CRISPR/Cas9 system. Analysis of
mutations in the CAN1 gene upon dCas9 targeting in ung1 yeast mutants
using mismatch-containing guide RNA further implicate a role for BER in
off-target mutagenesis during genome editing. Using an in-vitro uracil DNA
glycosylase (UDG) activity assay, we show that uracil lesions close to the
protospacer adjacent motif (PAM) site are inhibited by dCas9 binding.
Taken together these data suggest that inhibition of uracil DNA glycosylase
(UDG) likely contributes to mutagenesis mediated by Cas9 binding. Our
results suggest that different DNA glycosylases responsible for recognizing
a variety of DNA lesions, as well as subsequent enzymatic steps of base
excision repair, might be impaired during genome editing. Our study
provides novel insights into the mechanism driving mutagenesis during
genome editing and expands the applications of the CRISPR toolbox.

54
SYSTEMATIC MAPPING OF GENETIC INTERACTIONS FOR
METABOLIC GENES USING CRISPR-CAS SCREENS

Michael Aregger1,4, Keith Lawson1, Maximilian Billmann2, Michael Costanzo1,

Thomas Gonatopoulos-Pournatzis1,4, Kevin R Brown1, Anne-Claude Gingras3,
Benjamin J Blencowe1, Chad L Myers2, Brenda J Andrews1, Charles Boone1,
Jason Moffat1
1
University of Toronto, Donnelly Centre, Toronto, Canada, 2University of
Minnesota, Computer Science and Engineering, Minneapolis, MN, 3University
of Toronto, LTRI, Toronto, Canada, 4National Cancer Institute (NCI/NIH),
RNA Biology Laboratory, Frederick, MD

A common characteristic of cancer is the reprogramming of cellular metabolism

in order to fuel growth and division. It is thus not surprising that several
metabolic pathways have emerged as therapeutic targets for cancer. However,
because cancer cells are intrinsically buffered to combat metabolic stress, it is
important to understand how cells may adapt to the loss of specific metabolic
pathways. Towards this, we use pooled genome-wide CRISPR screens to
systematically map genetic interactions (GIs) in co-isogenic human HAP1 cells
carrying a loss-of-function mutation of a single metabolic gene of interest. By
comparing fitness profiles between WT and query-mutant cells we identified
negative (e.g. synthetic lethals) and positive (e.g. suppressors) GIs.

We initially aimed to map GIs for the fatty acid synthase (FASN), whose
product catalyses the formation of long-chain fatty acids. FASN-mutant cells
show a strong dependence on lipid uptake that is reflected in negative GIs with
genes involved in the LDL receptor pathway, vesicle trafficking and protein
glycosylation. Further support for these functional relationships is derived from
additional GI screens in query cell lines deficient for other genes involved in
lipid metabolism, including LDLR, SREBF1, SREBF2 and ACACA. We
uncovered abundant buffering and crosstalk between metabolic pathways that
highlight the major principles of metabolic plasticity and pinpointing to genetic
vulnerabilities. Our GI profiles also identify a role for the previously
uncharacterized gene C12orf49 (LUR1) in regulation of exogenous lipid uptake
through modulation of SREBF2 signalling in response to lipid starvation.

As a complementary approach to study GIs in co-isogenic query lines, we have

developed CHyMErA, a novel multi-targeting CRISPR-based screening system
that employs co-expression of Cas9 and Cas12a nucleases and libraries of
hybrid guide RNAs. This new platform allows to concurrently perturb gene
pairs and its application across a panel of cell lines revealed wide-spread GIs
between metabolic genes.

Overall, our GI profiles for de novo fatty acid synthesis and related lipid-uptake
genes provide a resource for studying metabolic rewiring and disease
phenotypes linked to lipid metabolism. We also demonstrate the power of
systematic GI profiling using query mutants in a co-isogenic cell line, an
approach that can be applied to other bioprocesses and expanded to begin
generating more comprehensive GI maps for human genes.

55
GENOME-WIDE IDENTIFICATION OF QUANTITATIVE GENETIC
INTERACTIONS IN HUMAN CELLS USING CRISPR/CAS9 SCREENS

Maximilian Billmann1, Mahfuzur Rahman1, Amy Tong3, Katherine Chan3,

Michael Costanzo3, Henry Ward2, Michael Aregger3, Matej Usaj3,
Catherine Ross3, Kevin Brown3, Brenda Andrews3,4, Charles Boone3,4,
Jason Moffat3,4, Chad Myers1,2
1
University of Minnesota-Twin Cities, Dept. Computer Science and
Engineering, Minneapolis, MN, 2University of Minnesota-Twin Cities,
Graduate Program in Bioinformatics and Computational Biology,
Minneapolis, MN, 3University of Toronto, The Donnelly Centre and
Banting and Best Department of Medical Research, Toronto, Canada,
4
University of Toronto, Dept. Molecular Genetics, Toronto, Canada

A major focus of systems biology and genomic medicine is to link genotype

to phenotype, yet, we remain far from accurately predicting disease states
from genome sequence. Genetic interaction networks in model organisms
have shed light on this problem, highlighting how combinations of genome
variants can impact phenotypes. The disruptive CRISPR-based genome
editing technology enables this combinatorial mutation approach in human
cells. We systematically map genome-wide genetic interactions using
CRISPR/Cas9 in human cells. We performed a large number of genome-
wide screens with specific, loss-of-function mutation in HAP1 cells along
with more than 20 screens in wildtype HAP1 cells to be used as a basis for
robust scoring of genetic interactions. We developed a computational
pipeline to identify quantitative genetic interactions qGI from these data.
The qGI pipeline corrects unwanted effects such as surprising frequent
interactions in wt HAP1 screens. Moreover, we applied a novel framework
to explore reproducibility of qGI scores. We believe that our observations
generalize to other differential CRISPR screening platforms. In summary,
we developed a computational pipeline that will guide the generation of a
genome-wide reference genetic interaction network in human cells.

56
INCREASING GENOME EDITING EFFICIENCY WITH HEAT
TREATMENT IN ARABIDOPSIS THALIANA

Jonas Blomme1,2,3, Thomas Jacobs1,2

1
Ghent University, Department of Plant Biotechnology and Bioinformatics,
Ghent, Belgium, 2Ghent University, VIB Center for Plant Systems Biology,
Ghent, Belgium, 3Ghent University, Department of Biology, Ghent,
Belgium

CRISPR/Cas is a genome editing tool that has greatly improved targeted

mutagenesis during the last years thanks to its straightforward and
inexpensive design. However, the efficiency of the system varies depending
on the species and/or method of delivery. Some reports show that applying
heat treatment to plants and some other species can increase genome editing
efficiency. Unfortunately, the described methods often require dedicated
growth chambers or climate rooms not available for all research labs. Our
results confirm that heat but no other biotic or abiotic stress increases
genome editing efficiency in Arabidopsis thaliana. In this study we
evaluated the effect of three heat-shock treatments (37°C) on CRISPR/Cas
activity in Arabidopsis. We use common equipment available in any
molecular lab to induce heat stress. We report an 1,21-8,1x increase in
genome editing efficiency upon heat treatment for standard Cas9 (nine
targets) and Cas12a (three targets) applications, but also for cytidine base
editors (two targets). Our data also suggests that the generation of large
deletions using two gRNAs nor homology-directed repair is promoted after
heat treatment. We investigate the inheritability of the generated mutations,
and postulate that heat stress induces the formation of mosaic mutations that
can be transmitted to the following generations.

57
PRECISE GENOME ENGINEERING IN DROSOPHILA USING PRIME
EDITING

Justin A Bosch1, Gabriel Birchak1, Norbert Perrimon1,2

1
Blavatnick Institute, Harvard Medical School, Department of Genetics,
Boston, MA, 2Howard Hughes Medical Institute, Chevy Chase, MD

Precise genome editing is a valuable tool to study gene function in model

organisms. Prime editing, a precise editing system developed in human
cells, does not require double strand breaks or DNA template and has low
off-target effects. Here, we adapted prime editing for the model organism
Drosophila melanogaster by expressing components in transfected S2R+
cells and in transgenic flies. Using this system, we installed premature stop
codons in three classical visible marker genes, ebony, white, and forked.
Sequence analysis of genomic target sites revealed that editing efficiency
was similar to published results in human cells. Furthermore, edited flies
had mutant phenotypes that were consistent with loss of gene function by
premature protein truncation. Our results suggest that prime editing is a
useful system in Drosophila to study gene function, such as installing point
mutations, deletions, or epitope tags.

58
REDUCING CAS9 HALF-LIFE LEADS TO EFFICIENCY
ENHANCEMENT AND REDUCES MOSAICISM

Amine Bouchareb, Samy Alghadban, Phalguni Rath, Polinka Hernandez-

Pliego, Daniel Biggs, Chris Preece, Ben Davies

Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford,

United Kingdom

The high efficiency of CRISPR/Cas9 system in generating gene-edited

animals have raised the possibility that this technology could enable mouse
mutants to be generated and phenotyped without the need to breed lines of
mutant mice. Since the majority of mice used in research are involved in
breeding procedures, this technology has the potential to impact the animal
cost of biomedical research significantly. One significant disadvantage of
the technology which precludes this possibility, however, is the frequent
generation of genetic mosaics, due to the persistence of the Cas9 nuclease
after the first embryonic cleavage.
In an attempt to reduce mosaicism, we have built and tested a Cas9 fused to
a mutant FKBP12-derived destabilizing domain (DD-Cas9) and a Cas9
fused to the N-terminal domain of the replication-licensing factor Geminin
(Gem-Cas9). The latter is of interest, as it would be selectively degraded by
the anaphase-promoting complex during cell division. Furthermore, this
variant has previously been shown to improve homology directed repair
(HDR) by concentrating activity of the nuclease to the S/G2 phase.
First, we confirmed the efficiency of our Cas9 variants in the context of
gene editing. Using a mouse ES cell line harboring a Traffic Light Reporter
system, we observed that the Geminin domain fusion led to an enhancement
in the absolute rate of HDR.
Using targeted deep sequencing we assessed the level of mosaicism after
the introduction of these Cas9 variants as ribonucleoprotein into mouse
zygotes. Data from mouse blastocysts across different gene loci showed that
Gem-Cas9 resulted in a significant decrease in the level of mosaicism
compared to unmodified Cas9. We next targeted the Tyrosinase gene which
leads to absence of pigmentation i.e. albinism in homozygous mutant mice.
Phenotypic observations supported by targeted deep sequencing suggest a
decrease in the level of mosaicism for Gem-Cas9 generated mice.
Since high and prolonged activity of Cas9 is associated with increased off-
targets, four gRNA that are known to have a high off-Target cleavage
activity, were selected to assess simultaneously on-target and off-target
efficiency in human iPS and mouse ES cells. Our preliminary sequencing
data suggest an improved on-target efficiency along with a lower or
comparable off-target activity for Gem-Cas9 compared to unmodified Cas9.
With a high efficiency, comparable or reduced level of off-target
mutagenesis and a reduced level of mosaicism, Gem-Cas9 could be a
promising alternative to WT-Cas9 for genetically modified animal
generation and research applications.

59
HIGH-PERFORMANCE CRISPR-CAS12A GENOME EDITING FOR
COMBINATORIAL GENETIC SCREENING.
Rodrigo A Gier1,2, Krista A Budinich*1,2, Niklaus H Evitt1,2, Zhendong Cao1,2,
Elizabeth S Freilich1,2, Qingzhou Chen1,2, Jun Qi3, Yemin Lan2, Rahul M Kohli1,2,
Junwei Shi1,2
1Perelman School of Medicine, Univ of Penn, Philadelphia, PA, 2Epigenetics

Institute, Perelman School of Medicine, Univ of Penn, Philadelphia, PA, 3Dana-

Farber Cancer Institute, Harvard Medical School, Boston, MA

CRISPR-based genetic screening has revolutionized cancer drug target discovery.

Combinatorial genetic screening, a high-throughput strategy to perturb multiple
genes simultaneously, promises to elucidate therapeutically tractable synthetic
sick/lethal interactions in diverse diseases. Here we present a simple, robust
CRISPR-Cas12a-based approach for combinatorial genetic screening in cancer cells.
Currently, Cas9-based methods are efficient in single-gene knockout screening but
greatly underperform in combinatorial genetic screening, primarily due to the
complicated cloning and high recombination frequency of the dual CRISPR-Cas9
RNA (sgRNA) expression vector. Cas12a (Cpf1) has the potential to overcome these
limitations via intrinsic RNase activity that allows it to process its own crRNA array,
enabling multi-gene editing from a single RNA transcript. Though Cas12’s RNA
processing activity makes it ideal for multiplex targeting, Cas12a-based
combinatorial genetic screening remains incompatible with negative selection
screens due to intrinsically low Cas12a editing efficiency in mammalian cells.

To elevate the Cas12a editing efficiency, we (1) modified the nuclear localization
signal, (2) added a second CRISPR RNA (crRNA) direct repeat, and (3)
implemented the enhanced AsCas12a E174R/S542R mutations. Combining these
elements improved editing efficiency ~32-64-fold in mammalian cells. We term this
optimized AsCas12a system opAsCas12a.

To increase sensitivity and achieve efficient functional knockout in negative

selection screens, we established crRNA library design principles by targeting pan-
essential genes and known cancer dependencies with tiled crRNA. We found that
crRNA that cleave functional domains better generate loss-of-function alleles,
increasing the signal-to-noise ratio in pooled genetic screens. We benchmarked the
robustness of domain-targeted opAsCas12a against SpCas9 in a single-gene
knockout screen to verify known leukemia dependencies, where our opAsCas12a
system performed similarly to SpCas9.

Lastly, we demonstrated the performance of opAsCas12a in combinatorial genetic

screening by conducting double knockout screening to reveal synthetic sick/lethal
epigenetic interactions in leukemia cells. We selected 21 epigenetic domains from
single-crRNA knockout screens in leukemia. Through one-step T4 ligation, we
created a dual-crRNA library with 8,281 combinations and minimal crRNA cassette
recombination (~0.3%). This pairwise screen identified three novel synthetic sick
interactions in leukemia: Brd9&Jmjd6, Kat6a&Jmjd6, and Brpf1&Jmjd6. We
validated two of these synthetic sick interactions through competition-based
proliferation assays, chemical inhibition, and RNA-seq. Collectively, these data
demonstrate the utility and power of our opAsCas12a method in combinatorial
genetic screens for therapeutic target discovery.

60
BASE-EDITING MEDIATED TARGETING OF PROGERIN
ACCUMULATION

Diana Campos-Iglesias1,2, Jose Maria P Freije1,2, Carlos Lopez-Otin1,2

1
Universidad de Oviedo, Departamento de Bioquímica y Biología
Molecular, Facultad de Medicina, Instituto Universitario de Oncología
(IUOPA), Oviedo, Spain, 2Centro de Investigación Biomédica en Red de
Cáncer, (CIBERONC), Madrid, Spain

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare genetic disorder,

characterized by accelerated aging beginning in childhood. The underlying
molecular cause is a de novo point mutation in exon 11 of LMNA gene,
which causes the activation of a cryptic splice donor site, leading to the
accumulation of a mutant form of lamin A called progerin. Modifications in
the splicing of this gene are also present in healthy cells and therefore, a
small amount of this toxic protein is also produced during normal aging.
Over the last decades, numerous potential treatment options have been
explored through different approaches. In this sense, CRISPR-Cas-based
genome editing has emerged as a promising strategy in the study and
treatment of genetic diseases like HGPS. Base editors have the potential to
directly insert a desired point mutation in the genome, without needing
double-strand breaks or donor DNA templates.

Here, we explore the use of CRISPR-Cas base editors efficacy for the
prevention of the aberrant splicing that leads to progerin accumulation.
Thus, the use of this technology allowed us to specifically target the cryptic
splicing site in human cells with high efficiency. These findings suggest
that CRISPR-Cas-based genome editing tools could drive major advances in
the discovery of new treatments for pathogenic conditions in humans,
including progeria.

61
PROGRAMMABLE M6A MODIFICATION OF CELLULAR RNAs
WITH A CAS13-DIRECTED METHYLTRANSFERASE

Christopher Wilson1,2,3, Peter J Chen1,2,3, Zhuang Miao1,2,3, David R Liu1,2,3

1
Harvard University, Chemistry and Chemical Biology, Cambridge, MA,
2
Broad Institute of Harvard and MIT, Merkin Institute of Transformative
Technologies in Healthcare, Cambridge, MA, 3Harvard University, Howard
Hughes Medical Institute, Cambridge, MA

N6-methyladenosine (m6A) is the most widespread internal mRNA

modification in humans. Despite recent progress in understanding the
biological roles of m6A, the inability to install m6A site-specifically in
individual transcripts has hampered efforts to elucidate causal relationships
between the presence of a specific m6A and phenotypic outcomes. Here we
demonstrate that nucleus-localized dCas13 fusions with a truncated
METTL3 methyltransferase domain and cytoplasm-localized fusions with a
modified METTL3:METTL14 methyltransferase complex can direct site-
specific m6A incorporation in distinct cellular compartments, with the
former fusion protein having particularly low off-target activity.
Independent cellular assays across multiple sites confirm that this targeted
RNA methylation (TRM) system mediates efficient m6A installation in
endogenous RNA transcripts with high specificity. Finally, we show that
TRM can induce m6A-mediated changes to transcript abundance and
alternative splicing. These findings establish TRM as a tool for targeted
epitranscriptome engineering to help reveal the effect of individual m6A
modifications and dissect their functional roles.

62
CRISPR-MEDIATED MULTIPLEXED LIVE CELL IMAGING OF
NONREPETITIVE GENOMIC LOCI

Patricia A Clow1, Nathaniel Jillette1, Jacqueline J Zhu1, Albert W

Cheng1,2,3,4
1
The Jackson Laboratory, Genomic Medicine, Farmington, CT, 2The
Jackson Laboratory, Cancer Center, Bar Harbor, ME, 3University of
Connecticut Health Center, Genetics and Genome Sciences, Farmington,
CT, 4University of Connecticut Health Center, Institute for Systems
Genomics, Farmington, CT

Three-dimensional (3D) structures of the genome are dynamic,

heterogeneous and functionally important. Live cell imaging has become
the leading method for chromatin dynamics tracking. However, existing
CRISPR- and TALE-based genomic labeling techniques have been
hampered by laborious protocols and low signal-to-noise ratios (SNRs), and
are thus 20 mostly applicable to repetitive sequences. Here, we report a
versatile CRISPR/Casilio-based imaging method, with an enhanced SNR,
that allows for one nonrepetitive genomic locus to be labeled using a single
sgRNA. We constructed Casilio dual-color probes to visualize the dynamic
interactions of cohesin-bound elements in single live cells. By forming a
binary sequence of multiple Casilio probes (PISCES) across a continuous
stretch of DNA, we track the dynamic 3D 25 folding of a 74kb genomic
region over time. This method offers unprecedented resolution and
scalability for delineating the dynamic 4D nucleome.

63
JACKIE: ENUMERATION OF ALL SINGLE- AND MULTI-COPY
CRISPR BINDING SITES IN A GENOME

Jacqueline J Zhu1, Albert W Cheng1,2,3,4

1
The Jackson Laboratory, Genomic Medicine, Farmington, CT, 2The
Jackson Laboratory, Cancer Center, Bar Harbor, ME, 3University of
Connecticut Health Center, Genetics and Genome Sciences, Farmington,
CT, 4University of Connecticut Health Center, Institute for Systems
Genomics, Farmington, CT

CRISPR-based methods for genome, epigenome editing and imaging have

provided powerful tools to interrogate the functions of the genome. The
design of guide RNA (gRNA) is a vital step of CRISPR experiments. We
report here the implementation of JACKIE (Jackie and Albert’s CRISPR K-
mer Instances Enumerator), a pipeline for enumerating all potential single
and multi-copy CRISPR sites in the genome. We demonstrate the
application of JACKIE to identify locus-specific repetitive sequences for
CRISPR/Casilio-based genomic labeling.

Website: http://cheng.bio/JACKIE

64
ENHANCED PRECISION OF BASE EDITING WITH NEAR-PAMLESS
CRISPR-CAS9 VARIANTS

Kathleen A Christie1,2,3, Russell T Walton1,2, Madelynn N Whittaker1,2,

Benjamin P Kleinstiver1,2,3
1
Massachusetts General Hospital, Center for Genomic Medicine, Boston,
MA, 2Massachusetts General Hospital, Department of Pathology, Boston,
MA, 3Harvard Medical School, Department of Pathology, Boston, MA

Base editors (BEs) are a promising genome editing platform that enable
programmable and heritable DNA base substitutions in living cells. They
consist of catalytically attenuated CRISPR enzymes fused to deaminase
domains that generate C-to-T or A-to-G substitutions. However, one major
limitation of BEs that diminishes their utility is their dependence on the
availability of protospacer adjacent motifs (PAMs) required for Cas9
binding. The restriction of PAM recognition is magnified by the necessity
to place the target base within the editing window of the deaminase domain.
One method to relieve this limitation would be to relax PAM recognition,
greatly expanding the scope of C or A bases amenable for base editing
technologies.

To overcome the limitation of PAM recognition, we engineered SpCas9

variants with relaxed PAM preferences. We generated SpG, a potent
SpCas9 variant capable of uniformly targeting across NGN PAMs. To
further reduce PAM preference we then generated a near-PAMless Cas9
variant, named SpRY, which is capable of efficiently targeting sites with
NRN PAMs and can more weakly target sites with NYN PAMs. We then
demonstrated that SpG and SpRY function as potent nucleases and adenine
and cytosine BEs (CBEs and ABEs, respectively). As a proof-of-concept to
demonstrate the potential of SpG and SpRY BEs for generating previously
inaccessible point mutations, we utilized SpG- and SpRY-CBEs to
introduce several genetic variants associated with protecting individuals
from developing disease. While these variants are inaccessible to wild-type
and previously engineered CBEs, we demonstrate SpG and SpRY CBEs can
be utilized to achieve efficient C>T on-target editing, while avoiding
unwanted bystander edits. These results establish that the enhanced
targeting resolution of SpG and SpRY can improve the utility of BEs, work
that we are expanding for other BE applications. Furthermore, we are
investigating additional applications of SpG and SpRY for many other
genome editing applications that require high resolution targeting.

65
A BASE EDITING APPROACH TO ELIMINATE SICKLE
HEMOGLOBIN

S. Haihua Chu, Daisy Lam, Jenny Olins, Alex Liquori, Jeff Marshall,
Jeremy Decker, Tanggis Bohnuud, Luis Barrera, Ian Slaymaker, Michael
Packer, Nicole Gaudelli, Adam Hartigan, Giuseppe Ciaramella

Beam Therapeutics, Beam Tx, Cambridge, MA

Sickle cell disease (SCD) is a monogenic disorder that alters the structure
and function of oxygen-carrying hemoglobin in red blood cells and is
caused by the substitution of a single amino acid (E6V) on the human beta
hemoglobin subunit. Gene therapy approaches to treat SCD have focused on
either expression of an engineered anti-sickling -globin or upregulation of
fetal hemoglobin and rely on lentiviral integration or double strand DNA
breaks to elicit their function. In contrast, adenine base editors (ABEs) have
been shown to precisely convert A-T to G-C base pairs without inducing
double strand DNA breaks, allowing for correction of the single nucleotide
mutation responsible for SCD.
Our studies have identified ABE variants that efficiently recognize and edit
the sickle mutation, converting the valine to an alanine (E6A). This
conversion generates a variant form of -globin, Hb G-Makassar, which
occurs naturally in human genetics: homozygous individuals are
asymptomatic with normal hematologic parameters and have no evidence of
hemoglobin polymerization or sickling of red blood cells. In in vitro studies,
an alanine substitution at this position did not contribute to polymerization
of deoxygenated hemoglobin.
Through ex vivo delivery of guide RNA and mRNA encoding our ABE
variants, we successfully edited human CD34+ cells both from sickle trait
individuals and homozygous SCD (HbSS) patients. Subsequent in vitro
erythroid differentiation (IVED) of edited CD34+ cells confirmed that
Makassar editing was retained throughout erythropoiesis. In CD34+ cells
isolated from HbSS patients, >75% bulk Makassar editing could be
achieved in IVED cells. At these high Makassar editing levels, nearly 75%
of cells were bi-allelically edited, with fewer than 3% unedited, as
determined by BFU-E analysis.
We additionally developed an ultra-high-performance liquid
chromatography (UPLC) method (confirmed by liquid chromatography
mass spectrometry (LC-MS)) that resolves sickle globin (HbS) from Hb G-
Makassar globin in IVED cells. The level of Makassar globin correlated
closely with the percent Makassar editing, with >80% conversion to Hb G-
Makassar which corresponded to <15% HbS. At this high level of
conversion, in vitro sickling under hypoxia was markedly reduced.
Altogether, our Hb G-Makassar direct editing strategy can convert a
pathogenic single nucleotide mutation to a benign, non-sickling, naturally
occurring variant, demonstrating that this next generation gene editing
strategy may be a promising new modality for treating patients with SCD.

66
C9ORF72 FRONTOTEMPORAL DEMENTIA (FTD) AND
AMYOTROPHIC LATERAL SCLEROSIS (ALS): USING PATIENT
CELLS AND CRISPR TO REVEAL THERAPEUTIC APPROACHES

Claire D Clelland1,2, Sachdev Aradhana1, Alisha Birk1, Hannah L Watry1,3,

Luke M Judge1,4, Bruce R Conklin1,3,5
1
Data Sciences and Biotechnology, Gladstone Institutes, San Francisco, CA,
2
Dept. of Neurology, UCSF, San Francisco, CA, 3Innovative Genomics
Institute, UC Berkeley, San Francisco, CA, 4Dept. of Pediatrics, UCSF, San
Francisco, CA, 5Dept. of Medicine, UCSF, San Francisco, CA

Expansion of the GGGGCC hexanucleotide repeat in the C9orf72 gene is

the most frequent known genetic cause of frontotemporal dementia (FTD)
and amyotrophic lateral sclerosis (ALS) (termed C9-FTD/ALS). Although
the cellular dysfunction caused by this disease is multifactorial, targeting
the gene itself by CRISPR/Cas9 editing could be curative. The most
pressing challenge to implementing this technology effectively is
identifying edits that neutralize the C9orf72 mutation without introducing
unintended cellular dysfunction. We have taken three approaches to editing
the C9orf72 gene in human iPSC cells: (1) bi-allelic excision of non-coding
DNA harboring the repeat expansion region, (2) allele-specific excision of
the mutant allele containing the repeat expansion, (3) regulatory region
disruption to selectively silence the C9orf72 repeat expansion. We have
additionally engineered our cell lines with inducible neuron-specific
transcription factors that generate cortical or motor neurons (cell types
relevant to C9FTD/ALS) in a high-throughput and cost effective manner.
By studying the effects of these genetic manipulations in induced neurons
we not only interrogate gene editing approaches, but also advance our
understanding of the normal regulation of the C9orf72 gene.

67
STANDARDIZED QUANTIFICATION OF PRIME EDITING
EXPERIMENTS

Kendell Clement, J. Keith Joung, Luca Pinello

Massachusetts General Hospital, Molecular Pathology Unit, Center for

Computational and Integrative Biology, Center for Cancer Research,
Boston, MA

Prime editing is a highly versatile genome editing technology that

significantly expands the ability to create precise changes to DNA.
However, the technology is in its infancy and no standardized analysis
strategy exists to quantify the efficiency and purity of editing products. This
problem is compounded in that unintended editing outcomes (e.g. scaffold
incorporation) are not accurately quantified using analysis approaches that
have been used for other genome editing technologies. To address this
point, we have extended our tool CRISPResso2 to rapidly and reproducibly
characterize the editing outcomes of prime editors. Inputs for CRISPResso2
prime editing analysis is minimal, requiring only the prime editing guide
RNA (pegRNA) spacer sequence (which specifies the initial nick location)
and pegRNA extension sequence (which encodes the desired edit). Based
on these two input parameters, CRISPresso2 quantifies unintended edits
that could result at the nicking site (with a narrow window), as well as at the
3’ flap ligation site (with a wide window). Optionally, users may 1)
customize analysis by modifying each window size, 2) specify a nicking
sgRNA (ngRNA) sequence targeting the non-edited strand to quantify
mutations at this site, or 3) specify the pegRNA scaffold sequence to
quantify the rate of unintended scaffold incorporation. These extended
capabilities make it possible to analyze simple or complex prime editing
experiments with high reproducibility. We anticipate that our improvements
will increase reproducibility and accuracy, and create a standard for the
analysis of prime editing experiments moving forward.

68
DOMINANT NEGATIVE DISEASE, IDEAL MODELS FOR ALLELE-
SPECIFIC THERAPEUTIC EDITING

Bruce R Conklin1,2,3, Katie Gjoni1, Gokul N Ramadoss1,4, Juan A Perez-

Bermejo1, Alisha M Birk1, Carissa M Feliciano1,6, Aradhana R Sachdev1,
Georgia L Gregory1,7, Hana Y Ghanim1, Hannah L Watry1,2, Kathleen C
Keough1, Matthew A Carter1, Po-Lin So So1, Tessa Dewell1, Beeke
Wienert1,2, Zachary S Nevin1, Claire D Clelland1,5, Luke M Judge1,6
1
Gladstone Institutes, San Francisco, CA, 2Innovative Genomics Institute,
Berkeley, CA, 3UCSF, Medicine & Ophthalmology, San Francisco, CA,
4
UCSF, Biomedical Sciences Program, San Francisco, CA, 5UCSF,
Neurology, San Francisco, CA, 6UCSF, Pediatrics, San Francisco, CA,
7
UCSF, Surgery, San Francisco, CA

Precise in vivo genome editing will provide a path to treat of a large number
of currently incurable genetic diseases. To define this path, we need proof-
of-concept edits that will have highly efficacious, phenotypic changes that
can be measured accurately in the patient. We are focused to dominant
negative (DN) diseases because the selective deletion of the DN allele could
provide a complete cure of the diseased cell. DN diseases were once
thought be exceedingly rare, but we have found that in specific tissues such
as motor neurons or retina, DN diseases are remarkably common. We have
used a set of 50 DN disease genes to identify a DN signature, that could
greatly increase the discovery of DN disease genes in the future.
Additionally, we are investigating induced pluripotent stem cells (iPSCs)
from >40 patients that each have targetable DN disease mutations that can
be modeled in iPSC-derived cell types related to macular degeneration
(BEST1), cardiomyopathy (TNNT2), motor neuron disease (MFN2, NEFL,
HSPB1 and C9orf72). To facilitate allele-specific guide RNA design, we
developed AlleleAnalyzer, an open source software package.
AlleleAnalyzer identifies guide RNAs that target common alleles in cis with
the disease mutation for allele-specific inactivation. Additionally, we use
DISCOVER-seq to identify off-target editing from these allele-specific
guides and to analyze their repair outcomes in diverse cell types. A major
remaining challenge is to achieve temporal control of Cas9 nuclease
expression. Precisely timed Cas9 expression would maximize on-target
editing, limit off-target DNA damage and minimize the risk of triggering an
immune-response to the bacterially derived Cas9. We provide evidence that
patient-specific iPSC-derived cell models provide a powerful platform for
evaluating therapeutic editing DN disease in multiple tissue types.

69
THE EMBL-EBI GENOME TARGETING CATALOGUE

Sybilla Corbett, Thomas Juettemann, Myrto Kostadima, Garth Ilsley, Fiona

Cunningham, Daniel R Zerbino

European Molecular Biology Laboratory, European Bioinformatics

Institute, Hinxton, United Kingdom

Techniques derived from the discovery of the CRISPR/Cas9 system are

revolutionizing biomedical research. New laboratory protocols are
undergoing rapid experimentation and modification, including a broad
range of techniques from gene editing through non-homologous end joining
and homology directed repair, to transcriptional activation and repression,
as well as imaging experiments. As a consequence, there has been a rapid
increase in published experiments using CRISPR-related proteins.
Combined with the ability to carry out genome-wide screens, this has
resulted in a high volume of generated data.

We have launched the EMBL-EBI Genome Targeting Catalogue, to capture

this wealth of information in a systematic fashion, enabling its visibility,
discoverability and re-usability. It is a manually curated collection of
published experiments using CRISPR/Cas enzymes. By including data from
genome-wide pooled screens and single gene experiments we expect to
provide an integrated resource that will aid research and facilitate new
discoveries. The database currently contains over 350 curated papers on 47
species covering a wide range of targeting experiments, giving experimental
parameters such a guide RNA sequences and links to raw results such as
read counts. We are now exploring avenues for author submission of large
datasets.

70
CREATION OF FLOXED ALLELES BY ELECTROPORATING TWO
CRISPR RNPs AND TWO ssODNs

Monica F Sentmanat1, Michael White2, Xiaoxia Cui1

1
Washington University in St. Louis, Genetics, St Louis, MO, 2Washington
University in St. Louis, Pathology & Immunology, St Louis, MO

Floxing is one of more popular type of mouse models desired for

biomedical research. One floxing method uses two CRISPR gRNAs
designed to flank the sequnce to be floxed. Paired with a single stranded
oligonucleotide donor (ssODN), each gRNA complexed with Cas9 protein
(RNP) mediates the insertion of a loxP site. The 2 RNPs/2 ssODNs method
is the most flexible to design, quickest and cheapest to obtain reagents.
However, some labs experienced difficulties to generate floxed models
using this method. Several reasons can contribute to the challenge. Non-
homologous end joining (NHEJ) events at individual cut site and deletion
between target sites compete with the formation of floxed allele.
Additionally, the two independent insertions of loxP site also have to
happen in the same chromosome to result in a floxed allele.
We report here that we have had good success in the past three year with
floxing projects. Out of the 50 projects we designed, 21 are complete, and
another 20 models are pending F1 data. All the models were created by
electroporating two RNPs wtih two ssODNs. Both RNPs and ssODNs are
validated in Neuro 2A cells for efficient loxP insertion before
electroporating embryos. Livebirths are genotyped by using NGS to identify
potential founders with loxP insertion in both target sites. More recently, we
developed an in vitro Cre assay to test whether the inserted loxP sites are in
the same allele, where genomic DNA of a potential founder is incubated
with recombinant Cre protein, followed by a PCR reaction specifically
amplifying an excision product. Animals in positive Cre assay are bred for
F1’s. In projects where no livebirths have two loxP sites, or are positive in
Cre assay, we choose a male animal with loxP insertion in one target site
and a likely in-phase indel at the other as sperm donor and perform IVF on
wild type oocytes to obtain fertilized eggs for a second round targeting to
insert the remaining loxP only into the specific indel. The resulting animals
with two loxP sites are guaranteed to be in phase. The timelines to reach
confirmation of animals with a floxed allele by one-step or two-step
targeting are both around 20 weeks. We conclude that electroporating
embryos with two-gRNA and two-ssODN, when complemented with a
potential second round targeting, is a valid, simple and reasonably efficient
method for introducing floxed alleles.

71
A HOMOLOGY INDEPENDENT SEQUENCE REPLACEMENT
STRATEGY IN HUMAN CELLS USING A CRISPR NUCLEASE

Eric W Danner, Mikhail Lebedin, Kathrin de la Rosa, Ralf Kühn

Max Delbrück Center for Molecular Medicine, Helmholtz Association,

Berlin, Germany

Precision genomic alterations largely rely on Homology Directed Repair

(HDR), but targeting without homology using the Non-Homologous End
Joining (NHEJ) pathway has gained attention as a promising alternative.
Previous studies demonstrated precise insertions formed by the ligation of
donor DNA into a targeted genomic double strand break in both dividing
and non-dividing cells. In this work we extend this idea and use NHEJ
repair to replace genomic segments with donor sequences; we name this
method ‘Replace’ editing (Rational end-joining protocol delivering a
targeted sequence exchange). Using CRISPR/Cas9 we create two genomic
breaks and ligate a donor sequence in-between. This exchange of a genomic
for a donor sequence uses neither microhomology nor homology arms. We
target four loci and show successful exchange of exons in 16% to 54% of
cells. Using linear amplification methods and deep sequencing pipelines we
quantify the diversity of outcomes following Replace editing and profile
mutations formed at the ligated interfaces. The ability to replace exons or
other genomic sequences in cells not efficiently modified by HDR holds
promise for both basic research and medicine.

72
CRISPR-CAS9 REPROGRAMMING OF HUMAN STEM CELLS INTO
MOTOR NEURONS

Katie Davis-Anderson1, Sofiya Micheva-Viteva1, Jennifer Harris1, Scott

Twary1, Rashi Iyer2
1
Los Alamos National Laboratory, Bioscience Division, Los Alamos, NM,
2
Los Alamos National Laboratory, Analytics, Intelligence, and Technology
Division, Los Alamos, NM

Neural injuries can have catastrophic outcomes due in part to the challenges
regenerating motor neurons (MNs). These terminally differentiated cells
have limited in vivo repair mechanisms and are laborious to culture in vitro.
One solution is to differentiate stem cells into MNs. While this
transformation can be accomplished using small molecules and growth
factors, it is often unreliable and multiple subtypes of MNs can be
generated. An alternative approach is to genetically engineer stem cells to
express key transcription factors required for spinal MN differentiation.
Transcription factors which promote MN specification include Isl1 and
Lhx3, and are therefore used as markers of mature MNs, and we used
CRISPR-Cas9 gene editing to overexpress Isl1 and Lhx3. Two strategies for
lineage-directed stem cell reprograming were applied: 1) CRISPR-Cas9
insertion of Isl1 and Lhx3, or 2) over-expression of endogenous Isl1 and
Lhx3 using deactivated Cas9 linked to a transcription activator (dCas9-
VPR). Ideal culture conditions for the CRISPR-Cas9 edited cells also
included small molecules and growth factors to reliably produce mature,
functional MNs. Ultimately, mature MNs will be co-cultured with myocytes
to establish neuromuscular junctions (NMJs). These human NMJ models
will be a valuable tool to investigate neuronal dismantling due to injury or
disease, and will provide reproducible and robust assays for high throughput
drug discovery.

LA-UR-20-23862

73
INTERROGATION OF THE 6Q23 AUTOIMMUNE DISEASE
SUSCEPTIBILITY LOCUS USING A MIRRORED CRISPR
CHROMATIN MODIFICATION SCREEN

James Ding, Kate Duffus, Paul Martin, Gisela Orozco

Centre for Genetics and Genomics Versus Arthritis, Centre for Musculoskeletal
Research, Manchester Academic Health Science Centre, University of
Manchester, Manchester, United Kingdom

A collection of variants in high linkage disequilibrium with rs6920220 at

chromosomal location 6q23 are significantly associated with increased risk of
inflammatory bowel disease, rheumatoid arthritis and ulcerative colitis, among
other autoimmune diseases. Existing studies aimed at identifying which of these
variants are most likely to mediate the increased risk associated with this locus
have reached contradictory conclusions: highlighting both single nucleotide
polymorphism rs6927172 and insertion/deletion rs35926684 as most likely to
mediate risk. These variants are separated by approximately 2.5kb and overlap
intergenic enhancers that are active in disease relevant subsets of primary
human T helper cells . In multiple relevant cell types these enhancers have been
shown to interact with TNFAIP3, IFNGR1 and IL20RA, located approximately
200, 500, and 650 kb away. In order to better understand the mechanism
mediating risk at this locus it was included in a mirrored CRISPR chromatin
modification screen.
T helper cell-like JurkatE6.1 cells constitutively expressing either dCas9-VP64
or dCas9-KRAB were transduced with a lentiviral library containing guide
RNAs (gRNAs) targeting variants within the 6q23 locus and various external
and internal controls: included were four gRNAs targeting each of the three
variants highlighted above, four gRNAs targeting the transcription start sites
(TSSs) of TNFAIP3, IFNGR1 and IL20RA (two upstream and two
downstream) and four scrambled, non-targeting guides. Following antibiotic
selection the effect of individual guides was assessed using single cell RNA-seq
(scRNA-seq) at a depth of approximately 100k reads per cell . After filtering,
including the removal of cells that could not confidently be assigned to an
individual gRNA, over 30 transcriptomes were captured for each of the 28
gRNAs described above, across both experiments.
Successful activation of chromatin in cells expressing dCas9-VP64 was evident
through increased expression of IFNGR1 in cells containing guides targeting
upstream of its TSS (1.47-fold). Similarly inactivation of chromatin in cells
expressing dCas9-KRAB was evident through reduced IFNGR1 expression in
cells containing guides targeting the TSS of IFNGR1 (0.83-fold). The effect of
perturbing the intergenic enhancer was expected to be less than that of directly
targeting TSSs and it was therefore unsurprising that there were no obvious
direct effects on these putative target genes alone. The broader impact of
individual gRNAs was further evaluated on a transcriptome wide basis aiming
to characterise perturbations downstream of these putative target genes. The
results of this study are informative regarding not only the likely mechanisms of
disease susceptibility at this locus, but also the application of scRNA-seq
chromatin modification screens to the study of enhancers generally.

74
CANCER-SPECIFIC LOSS OF TERT ACTIVATION SENSITIZES
GLIOBLASTOMA TO DNA DAMAGE

Alexandra Amen*1,2, Christof Fellmann*1,3,4, Katarzyna Soczek1, Shawn

Ren1, Rachel Lew3, Gavin Knott1, Jesslyn Park1, Andrew McKinney2,
Andrew Mancini2, Jennifer Doudna#1,3,5,6, Joseph Costello#2
1
University of California, Berkeley, Department of Molecular and Cell
Biology, Berkeley, CA, 2University of California, San Francisco,
Department of Neurological Surgery, San Francisco, CA, 3Gladstone
Institutes, GIDB, San Francisco, CA, 4University of California, San
Francisco, Department of Cellular and Molecular Pharmacology, San
Francisco, CA, 5University of California, Berkeley, Howard Hughes
Medical Institute & Innovative Genomics Institute & Department of
Chemistry, Berkeley, CA, 6Lawrence Berkeley National Laboratory,
Molecular Biophysics & Integrated Bioimaging Division, Berkeley, CA

Most glioblastomas (GBMs) achieve cellular immortality by acquiring a

mutation in the telomerase reverse transcriptase (TERT) promoter. TERT
promoter mutations create a binding site for a GA binding protein (GABP)
transcription factor complex, whose expression is associated with TERT
reactivation and telomere maintenance. Here, using biochemical and cell
biology approaches, we show direct evidence that a specific GABP complex
containing the subunit protein GABPB1L forms predominantly at the
mutant TERT promoter, leading to TERT re-expression. Furthermore, we
find that TERT promoter mutant GBM cells, unlike wild-type cells, are
immediately dependent on GABPB1L for proliferation in cell culture and
post-tumor establishment in vivo. Notably, when combined with frontline
temozolomide (TMZ) chemotherapy, GABPB1L knockdown and the
associated TERT reduction lead to an impaired DNA damage response that
results in profoundly reduced growth of intracranial GBM tumors.
Together, these findings provide new insights into the mechanism of
cancer-specific TERT regulation, uncover rapid effects of TERT suppression
in GBM maintenance, and establish GABPB1L inhibition, alone or in
combination with chemotherapy, as a therapeutic strategy for TERT
promoter mutant GBM.

* These authors contributed equally.

# Correspondence.

75
RNA EDITING FOR REPAIRING POINT MUTATIONS CAUSING
USHER SYNDROME

Lewis E Fry, Alun R Barnard, Michelle E McClements, Robert E MacLaren

University of Oxford, Nuffield Department of Clinical Neurosciences,

Oxford, United Kingdom

Background: Usher syndrome is the leading cause of deaf-blindness,

causing progressive retinal degeneration and sensorineural deafness. Usher
syndrome is an autosomal recessive disorder commonly caused by
mutations in USH2A, which encodes the Usherin protein in photoreceptors
and the inner ear. RNA editing is a potential therapeutic strategy to correct
common pathogenic variants. Adenosine deaminase acting on RNA
(ADAR) enzymes convert adenosine to inosine, which is read as guanosine
by the cell. Deactivated Cas13b enzymes bind RNA as directed by a guide
RNA, and when fused to an ADAR deaminase domain, enable the targeted
correction of G>A mutations in RNA.

Aim: To investigate the use of RNA editing with CRISPR-dCas13b-ADAR

to correct a common premature termination codon in USH2A

Methods: A dual luciferase reporter assay was developed to contain mutant

sequences from human (hUSH2A) and mouse (mUsh2a) transcripts for
guide RNA screening. Editing activity was measured via restoration of a
firefly luciferase in HEK293T cells transfected with constructs expressing
the reporter assay, dPspCas13b-ADAR variants and guide RNAs. RNA
editing rates were analysed by trace decomposition of Sanger sequencing
reads of cDNA.

Results: Guide RNAs targeting hUSH2A and mUsh2a were designed and
screened, demonstrating a range of on-target activity (2-55%) determined
by the mismatch distance between the target adenosine and the guide RNA
scaffold. RNA sequencing demonstrated the restoration of the target
premature termination codon (TAG) to the wildtype tryptophan codon
(TGG) at a mean (SD) rate of 77% (1.5) and 86% (2.1) of hUSH2A and
mUsh2a transcripts respectively. Constructs containing ADAR with the
hyperactive E488Q mutation demonstrated high on-target editing activity
but also off-target editing of adenosines across the transcript. Constructs
with the specific E488Q/T375G mutation demonstrated a 50% loss of on-
target activity relative to constructs with on the E488Q mutation, but off-
target editing was undetectable. Editing was not detectable with non-
targeting guide RNAs, or with expression of guide RNAs without dCas13-
ADAR.

Conclusion: These data support RNA editing as a potential therapeutic

approach for pathogenic G>A variants in Usher syndrome.

76
EXTENSIVE RIBOSE CHEMICAL MODIFICATION OF THE CAS12A
crRNA 5' HANDLE PSEUDOKNOT SUPPORTS GENE EDITING AND
MODULATES CIS-TRANS ACTIVITY

Eman A Ageely1, Leonora Abdullahu2, Sunit Jana2, Ramadevi

Chilamkurthy3, Daniel O'Reilly2, Philip J Jensik4, Masad J Damha2, Keith T
Gagnon1,3
1
Southern Illinois University, Chemistry and Biochemistry, Carbondale, IL,
2
McGill University, Chemistry, Montreal, Canada, 3Southern Illinois
University, Biochemistry and Molecular Biology, Carbondale, IL,
4
Southern Illinois University, Physiology, Carbondale, IL

CRISPR-Cas systems have emerged as a leading technology for therapeutic

editing of disease-linked genes. However, CRISPR-Cas enzymes are large,
non-traditional drug molecules that pose pharmacologic delivery
challenges. Therapeutic applications may benefit from less conventional
methods, including direct delivery of CRISPR RNA (crRNA) guides. To
enable direct delivery methods, which must protect against degradation, we
explored ribose chemical modification of the 5' handle of the crRNA guide
of Acidominococcus species (As) Cas12a. The 5' handle anchors crRNA
binding to Cas12a through formation of a unique, non-canonical
pseudoknot. Significant 2'-deoxy modification, as well as complete
conversion to 2'-fluoro-ribose, was well-tolerated in vitro but not in cell-
based editing. Editing in cells required that several residues be maintained
as a native RNA ribose. Replacing some of those residues with ribose-
modified nucleotides designed to partially maintain hydrogen-bonding,
including oxepane nucleic acid, arabinonucleic acid, 2'-fluoro, and 2'-
amino, identified the most sensitive positions and revealed chemical
compatibility. crRNA 5' handles with as little as six out of nineteen native
RNA ribose residues supported robust gene editing in cells. Assembly of
AsCas12a with modified 5' handle crRNAs further resulted in examples
where guide-directed sequence-specific (cis) cleavage activity and non-
sequence-specific (trans) activity were differentially affected in vitro. These
results indicate that substantial chemical modification is achievable in the
sensitive crRNA 5' handle, which should facilitate further therapeutic
development of CRISPR-Cas12a. In addition, modulating the cis-trans
activity of CRISPR-Cas12a with chemical modifications to the 5' handle,
which lies outside of the guide region, might enable new or better
diagnostic applications.

77
HARNESSING THE DIVERSITY OF CAS9 ORTHOLOGS FOR
GENOME EDITING

Giedrius Gasiunas1, Joshua K Young2, Darius Kazlauskas3, Tomas

Urbaitis1,3, Monika Jasnauskaite1, Migl Stitilyte1, Sushmitha Paulraj2,
Zhenglin Hou2, Shane K Dooley5, Clara Alarcon2, Doane Chilcoat2, Jennifer
L Curcuru4, Megumu Mabuchi4, Ryan T Fuchs4, Ezra Schildkraut4, Brett
Robb4, eslovas Venclovas3, Virginijus Siksnys1,3
1
CasZyme, Vilnius, Lithuania, 2Corteva Agriscience™, Department of
Molecular Engineering, Johnston, IA, 3Vilnius Univesity, Institue of
Biotechnology, Vilnius, Lithuania, 4New England BioLabs Inc., Ipswich,
MA, 5Iowa State University, Department of Agricultural and Biosystems
Engineering, Ames, IA

Cas9 orthologs are abundant in microbes. To explore this largely

uncharacterized diversity for genome editing applications, we established a
phylogeny-guided bioinformatic approach and developed biochemical
screens for the rapid identification and characterization of the protospacer
adjacent motif (PAM) and guide RNA (gRNA) requirements of new Cas9
proteins. This approach permitted the characterization of 79 Cas9 orthologs
with more than 50 distinct PAM sequence requirements. Computational
analyses indicated that most of this diversity came from 4 groups of
interrelated sequences providing new insight into Cas9 evolution and efforts
to engineer PAM recognition. A subset of Cas9 orthologs were purified and
their activities examined further exposing additional biochemical diversity.
This set contained nucleases that were active over broad temperature
ranges, generated staggered-end breaks and nucleases that required longer
spacers to function robustly. To establish broad applicability for use as
genome editing tools, cellular activity was examined in both plant and
human cells. Of those examined, several were capable of generating
targeted chromosomal mutations and some exhibited robust mutational
activity. Our results demonstrate that the diversity of Cas9 orthologs
provides a rich source of PAM recognition and other potentially desirable
properties that may be mined to expand the genome editing toolbox with
new programmable nucleases. Furthermore, our results provide a broad
foundation for the rational design and directed evolution of Cas9 variants
with altered PAM specificity for targeted applications such as base-editing
and prime-editing.

78
A VIRAL GUIDE RNA DELIVERY SYSTEM FOR CRISPR-BASED
TRANSCRIPTIONAL ACTIVATION AND HERITABLE TARGETED
DNA DEMETHYLATION

Basudev Ghoshal, Brandon Vong, Colette L Picard, Suhua Feng, Janet M

Tam, Steven E Jacobsen

HHMI/UCLA, Molecular Cell and Developmental Biology, Los Angeles,

CA

Plant viruses can be used as vectors to deliver genome and epigenome

editing reagents to plants. Recently, both plant RNA and DNA viruses were
used to deliver guide RNAs for CRISPR-CAS9 genome editing. However,
viral delivery of guide RNAs for CRISPR based epigenome editing
purposes such as site-specific DNA demethylation remains to be developed.
Besides, currently available viral delivery tools for genome editing show
meager rates of heritability. Here, we have developed a tobacco rattle virus
(TRV)-based guide RNA delivery system for both transcriptional activation
and targeted DNA demethylation. To facilitate heritable epigenome editing
specifically within plant meristems and the germline, we used the tRNA-
guide RNA expression system to express guide RNAs from the viral
genome. Using this system, we were able to demonstrate the targeted
removal of DNA methylation from the promoter of a gene and the
consequent activation of the gene in viral guide RNA delivered plants.
Furthermore, we observed high rates of heritability of the induced
phenotype in the progeny of virus-inoculated plants. Thus, the TRV
delivery system, combined with a specific tRNA-gRNA architecture,
provides fast and effective epigenome editing. Progress in the development
of this tool will be presented.

79
OPTIMIZED FLUORESCENT CRISPR ENZYME FUSIONS ALLOW
FOR ENRICHMENT OF EDITED CELLS BY FLUORESCENCE
ACTIVATED CELL SORTING

Steve E Glenn, Michael A Collingwood, Christopher A Vakulskas, Sarah F

Beaudoin, Nicole M Bode, Mark A Behlke

Integrated DNA Technologies, Molecular Genetics, Coralville, IA

It is often desirable to visualize CRISPR enzymes in live cells as a means to

verify efficient delivery of CRISPR reagents. The most common method to
accomplish this goal with protein cargo is to make an in-frame fusion to
fluorescent proteins like GFP or mCherry. These types of fusions have also
been successfully used in cell-sorting experiments to enrich for populations
of cells that obtain the most cargo. Producing these types of fusions is not
trivial especially when the fluorescent protein is fused to an enzyme (ie –
Cas9) that needs to retain high levels of functionality to be practically
useful. To date, most CRISPR protein fusions to GFP produce poor cellular
imaging when delivered as ribonucleoprotein (RNP) and retain relatively
low percent activity when compared to the untagged enzyme. Here we
present an optimized set of CRISPR enzyme fusions to GFP that have been
carefully fine-tuned to allow visualization in live cells and retain near
wildtype levels of on-target cleavage activity. We demonstrate that
fluorescent CRISPR enzyme fusion proteins can be used in combination
with fluorescence activated cell sorting to achieve levels of editing greater
than with the untagged CRISPR enzyme. We expect that these CRISPR
protein fusions will be a valuable resource in both academic research and
commercial product development.

80
CRISPOR UPDATES IN 2020 - MORE AND MORE GENOMES, GENE
MODEL DISPLAY AND BETTER EXPORT OPTIONS

Maximilian Haeussler1, Jean-Paul Concordet2

1
University of California at Santa Cruz, Genomics Institute, Santa Cruz,
CA, 2Museum National d'Histoire Naturelle, CNRS UMR 7196 / INSERM
U1154, Paris, France

The website https://crispor.org is a popular guide picker for CRISPR/Cas9

single-guide genome editing assay preparation. Our tool supports many
different enzymes, designs validation primers, includes many different
efficiency scoring algorithms for enzymes beyond just SpCas9 and includes
indel outcome predictors. In 2019/2020, we have added more than 100
additional genomes, the Graf et al inefficient guide criteria, Lindel scoring,
improved support for saCas9 and Cpf1 and are planning to release gene
exon display next. We are always interested in better predictions or PAM
sites and encourage authors of new algorithms and/or new enzymes to
contact us.

81
USING A CRISPR-CAS9-BASED GENE DRIVE TO TARGET STRESS
RESPONSE GENES IN CANDIDA ALBICANS

Viola Halder, Rebecca Shapiro

University of Guelph, Molecular & Cellular Biology - CBS, Guelph,

Canada

Candida albicans is an opportunistic fungal pathogen found in the oral

mucosa, the gut, the vaginal mucosa and the skin of humans. While C.
albicans can cause mild and superficial infections, severe invasive
infections can occur in immunocompromised patients. Understanding the
survival and pathogenesis of C. albicans is critical for novel antifungal drug
discovery and is the main objective of this project. Here, we exploit a
CRISPR-Cas9-based genome editing platform to create stress response
deletion libraries in C. albicans, in order to study their role in pathogen
survival. Our strategy uses a CRISPR-Cas9-based gene drive array
platform: a single plasmid with CAS9 and a pair of guide RNAs that direct
Cas9 to create double-strand breaks flanking the open reading frame (ORF)
of the gene of interest. Once the ORF is removed, the gene drive acts as a
repair template to replace the gene of interest. The gene drive can further
propagate to delete additional wild-type copies of the ORF, and thus, after
the single-gene mutants are created, a diploid double-gene mutant library
will be generated using a combinatorial mating strategy between mutant
haploid strains. This library of single and double stress response mutants
will be screened under diverse growth conditions to assess their relative
fitness. Genetic interaction analysis will be exploited to map out genetic
interactions between fungal genes involved in growth, survival and
pathogenesis. We will identify combinations of fungal stress response genes
with negative interaction or synthetic lethal interaction to uncover putative
targets for combination antifungal therapy.

82
THE APPLICATION OF CRISPR/CAS9 FOR MOLECULAR
CORRECTION THERAPY OF NEUROMUSCULAR DISORDERS

Britt Hanson1, Sofia Stenler2, Anna M Coenen-Stass3, Nina Ahlskog1,

Matthew J Wood1, Thomas C Roberts1
1
University of Oxford, Paediatrics, Oxford, United Kingdom, 2Uppsala
University, Pharmaceutical Biosciences, Uppsala, Sweden, 3AstraZeneca,
Bioscience, Oncology, IMED Biotech Unit, Cambridge, United Kingdom

Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA)

are highly debilitating, fatal and currently incurable hereditary
neuromuscular disorders. DMD is caused by out-of-frame mutations in the
dystrophin gene, encoding an essential protein for skeletal muscle
functioning. Dystrophin is comprised of multiple redundant internal repeat
domains and removal of diseased exons could result in production of a
truncated, yet adequately functional, protein. Conversely, SMA manifests
from knockout mutations within the neuronal survival motor neuron 1
(SMN1) gene. This is partially compensated by SMN2 but a C to T
polymorphism within SMN2 exon 7 induces aberrant splicing and only 10-
15% functional protein production. DMD and SMA disease genotypes are
amenable to splice correction. Current therapies using antisense
oligonucleotide-mediated skipping of disease-causing exons, whilst highly
effective, require lifelong repeat administration. To circumvent this
therapeutic hurdle, this study applies CRISPR/Cas9 gene editing for single-
intervention permanent correction of DMD and SMA. The Staphylococcus
aureus-derived CRISPR/Cas9 system, delivered using clinically safe and
effective AAVs, is being employed in a dual-cut strategy for excision of the
murine disease causing Dmd exon 23 in a severe double knock out mouse
model lacking both dystrophin as well as the developmental orthologue,
utrophin. Preliminary results show editing in the heart and diaphragm
(critical for lifespan extension), and also peripheral skeletal muscles. An
ongoing study with increased vector dosing and altered Cas9:guide RNA
ratios is being carried out to determine whether the scope of editing and
effect on pathology can be further improved. Concurrently, the application
of homology independent target integration (HITI)- and microhomology-
mediated end joining (MMEJ)-directed knock in of the wild-type SMN1
cDNA sequence is being investigated for SMA therapeutic development.
This is a novel approach to in situ genetic correction of SMA, independent
of patient genotype. Prime editing of a negative SMN2 splice regulator is
being investigated alongside as an alternative SMA therapeutic strategy
with expected higher fidelity. The overarching goal of this study is to
contribute towards developing and gaining a deeper understanding of novel
CRISPR/Cas9 therapeutic applications for these, and potentially a plethora
of other, devastating and currently incurable human genetic diseases.

83
GENOMIC CORRECTION OF SIALIC ACID STORAGE DISEASE
KNOCK-IN HUMAN FIBROBLASTS VIA CRISPR-CAS9 BASE
EDITING-DIRECTED REPAIR

Jerry F Harb, Elvin P Morales, Jeffrey Y Huang, Raymond Y Wang, Shih-

Hsin Kan

CHOC Children's Hospital, Metabolics, Orange, CA

Sialic acid storage diseases are a subset of lysosomal storage disorders that
result from mutations in Solute Carrier Family 17 Member 5 (SLC17A5)
gene. This gene is responsible for the production of sialin, a trans-
membrane protein that facilitates the transport of free sialic acid out of the
lysosome. SLC17A5 variants generate a non-functional or partially
functional sialin protein, resulting in the accumulation of free sialic acid
within the lysosome. Dependent on the specific form of Sialic acid storage
disease, affected individuals exhibit an array of symptoms from hypotonia,
severe delay in normal development, and gradual neurological issues,
among other complications. Currently, there are no available therapeutic
interventions for sialic acid storage diseases, and treatment is directed
towards managing symptoms and fostering a supportive quality of life
through care for patients. With the advent of CRISPR technology came a
novel therapeutic approach for genomic modification. Yet, classical genome
engineering utilizing CRISPR-Cas9 homology directed repair (HDR) has
demonstrated its limitations with efficacy and efficiency of correction.
Despite this, CRISPR-Cas9 continues to evolve as an essential instrument in
the molecular toolbox, particularly with the development of base editing.
CRISPR-Cas9 base editing allows for precise genomic modification of
single base pairs without double stranded breaks through base excision
repair. Base editors are comprised of two classes, cytosine base editors
(CBE) and adenine base editors (ABE), in which a catalytically dead Cas9
(dCas9) or Cas9 nickase (Cas9n) is fused to either a cytidine or adenine
deaminase, respectively. To take advantage of CRISPR-Cas9 base editing,
our group utilizes this technology to perform genomic correction of a
predominantly Finnish variant of SLC17A5, c.115CtoT (R39C), and
attempt to improve editing efficiency and decrease indel formation when
compared to CRISPR-Cas9 HDR. Future studies will aim at further
enhancing the editing specificity and efficiency using base editing
alternatives or through the potential use of prime editing systems.

84
CRISPR-MEDIATED TRANSCRIPTIONAL ACTIVATION WITH
SYNTHETIC GUIDE RNAs

Kevin Hemphill, Žaklina Strezoska, Sarah Dickerson Matheny, Elena

Maksimova, Eldon Chou, Maren Mayer Gross, Travis Hardcastle, Matthew
Perkett, Jesse Stombaugh, Emily M Anderson, Annaleen Vermeulen, Anja
van Brabant Smith

Dharmacon, a Horizon Discovery Group Company, R&D, Lafayette, CO

The CRISPR-Cas9 system has been adapted for transcriptional activation

(CRISPRa), and several second-generation CRISPRa systems (including
VPR, SunTag, and SAM) have been developed to recruit multiple
transcriptional activators to a transcriptional start site. Several studies have
demonstrated the benefit of CRISPRa in a pooled, lentiviral expression
context; however, the use of synthetic CRISPR guide RNA for activation
has not been reported. Here we show the effective use of synthetic guide
RNA for gene activation with several second-generation CRISPRa systems.
We demonstrate the use of CRISPRa crRNA with canonical tracrRNA in
the VPR system or with an extended tracrRNA containing an MS2 aptamer
sequence for the SAM system. Using chemistries and processes developed
over decades of research for the efficient, cost-effective, and high
throughput synthesis of guide RNAs over 100 nucleotides long, we
tested adding either one or two MS2 aptamers to different regions of the
tracrRNA to maximize gene expression with the SAM system. We find that
transcriptional activation with a single MS2 aptamer in the synthetic
crRNA: SAM tracrRNA complex is comparable to or higher than activation
levels achieved from sgRNA expression vectors containing two MS2
aptamers. Furthermore, in both the VPR and SAM systems, combining
several crRNA sequences targeting the same gene can enhance
transcriptional activation levels, indicating the effectiveness of synthetic
guide RNA for gain-of-function studies. Additionally, when working with
cell types or situations where viral transduction is not feasible, we show that
transcriptional activation can be achieved in the VPR system with the use of
dCas9-VPR mRNA and synthetic crRNA:tracrRNA. We also demonstrate
the use of synthetic guide RNA libraries in an arrayed CRISPR screening
format, identifying both positive and negative regulators of the IL-6
cytokine. Together, these results and tools demonstrate the suitability of
synthetic CRISPRa guide RNAs for a variety of transcriptional activation
applications.

85
HIGHLY EFFICIENT “HIT-AND-RUN” GENOME EDITING WITH
UNCONCENTRATED LENTIVECTORS CARRYING VPR.PROT.CAS9
PROTEIN PRODUCED FROM RRE-CONTAINING TRANSCRIPTS

Ivana Indikova, Stanislav Indik

University of Veterinary Medicine, Institute of Virology, Vienna, Austria

The application of gene-editing technology is currently limited by the lack

of safe and efficient methods to deliver RNA-guided endonucleases to
target cells. We engineered lentivirus-based nanoparticles to co-package the
U6-sgRNA template and the CRISPR-associated protein 9 (Cas9) fused
with a virion-targeted protein Vpr (Vpr.Prot.Cas9), for simultaneous
delivery to cells. Equal spatiotemporal control of the vpr.prot.cas9 and
gag/pol gene expression (the presence of Rev responsive element, RRE)
greatly enhanced the encapsidation of the fusion protein and resulted in the
production of highly efficient lentivector nanoparticles. Transduction of the
unconcentrated, Vpr.Prot.Cas9-containing vectors led to >98% disruption of
the EGFP gene in reporter HEK293-EGFP cells with minimal cytotoxicity.
Furthermore, we detected indels in the targeted endogenous loci at
frequencies of up to 100% in cell lines derived from lymphocytes and
monocytes and up to 15% in primary CD4+ T cells by high-throughput
sequencing. This approach may provide a platform for the efficient, dose-
controlled and tissue-specific delivery of genome editing enzymes to cells
and it may be suitable for simultaneous endogenous gene disruption and a
transgene delivery.

86
CHARACTERIZATION OF SYNTHETIC GUIDE RNAs FOR
CRISPR/CAS GENOME EDITING – AN EXTENSIVE EVALUATION
OF gRNA FORMATS, PURITY, AND DELIVERY METHODS

Ashley Jacobi, Rolf Turk, Nathan Roberts, Garrett Rettig

Integrated DNA Technologies, Molecular Genetics, Coralville, IA

The CRISPR system is a powerful tool for manipulating mammalian

genomes. Genome editing requires delivery of both the CRISPR nuclease
and the guide RNA (gRNA) to the cell. The gRNA can take multiple forms
with each offering well described advantages. Chemically modified gRNAs
provide the highest level of genome editing with the lowest toxicity and
immune activation. The gRNA can be synthesized as two separate
components, the crRNA and tracrRNA, and then annealed together through
simple hybridization. Alternatively, the gRNA can be chemically
synthesized as a single molecule, called a single guide RNA (sgRNA),
which contains a linker loop fusing the two components together. Here, we
compare editing efficiency of these two guide RNA formats delivered as
ribonucleoprotein complexes (RNP) at 255 different genomic loci in two
cell types, as well as showcase data from chemically synthesized CRISPR
gRNA screening libraries. Additionally, we compared editing efficiency of
gRNA forms when co-delivered with Cas9 mRNA. In this case, highly
modified sgRNAs consistently give the highest editing efficiency. Genome
editing with the CRISPR/Cas system is moving towards therapeutics
applications, and the sgRNA form may have added advantages for this
route. High quality sgRNAs that can be customized to many experiments
with varied scale, purification and chemical modification options are critical
for pre-clinical studies. IDT has developed a method for generating high
quality sgRNAs with rapid synthesis that can be customized for many
applications. We present data highlighting different sgRNA purification
options and provide comparison of editing efficiencies. Furthermore, a new
technology, Prime Editing, has required the need for longer gRNA
constructs. We have studied how modifications and purification of the
chemically synthesized pegRNAs effect the ability for efficient prime
editing.

87
ENHANCEMENT OF CRISPR-DRIVEN HOMOLOGY DIRECTED
REPAIR USING A NEW CLASS OF CLINICAL DNA-PK INHIBITORS

Qingzhou Ji1, Jacob T Lamberth1, Fuqiang Chen1, Christoph Vahl2, Thomas

Fuchss2, Frank T Zenke2, Gregory D Davis1
1
MilliporeSigma, Genome Engineering, St. Louis, MO, 2Merck KGaA,
Healthcare, Frankfurter, Germany

Creative problem solving in genome editing is inspired largely by our

ability to rapidly and accurately synthesize almost any DNA template
sequence we can imagine. The general hope within the scientific
community is that those synthetic templates can be efficiently copied into
the genomic DNA of any desired somatic cell type. Sadly, over the last 15
years, the complexity of DNA repair mechanisms across various cell types
has limited successful template integration for many projects. The
magnitude of this problem can be felt by the efforts deployed to develop an
array of alternative approaches such as homology-independent targeted
insertion (HITI), microhomology-mediated end-joining (MMEJ), obligate
ligation-gated recombination (ObLiGaRe), zinc finger recombinases, Base
Editing, and Prime Editing. In an astonishing success in 2019, one of the
most well characterized genetic diseases, sickle cell anemia, saw partial
phenotypic correction in a human patient not by template-based repair of
the causative single single-base mutation, but by non-homologous end
joining (NHEJ)-based perturbation of the control region of a negative
regulator (BCL11A) of an alternate native gene (fetal hemoglobin). These
are only some of the inventive measures implemented to bypass current
limitations of homology directed repair (HDR).

One additional approach which has gained strong interest involves the use
of small molecules to manipulate cellular DNA repair status. For example,
inhibitors of DNA-protein kinase (DNA-PK) such as NU7441 have been
shown to shift repair away from the disruptive random insertions / deletions
of NHEJ towards precise edits facilitated by HDR. While this approach
does not represent a universal HDR solution for all cell types, it can be
customized for specific cell types of therapeutic importance. To advance
this approach further, we have explored the ability of new classes of DNA-
PK inhibitors to enhance HDR. These new DNA-PK inhibitors are derived
from our advanced clinical oncology programs and thus have potential for
an accelerated translational path to enhance ex vivo and in vivo gene
therapies using HDR approaches. In this presentation, we illustrate data
characterizing the impact of these new DNA-PK inhibitors on NHEJ, HDR,
and cell health.

88
MASSIVELY PARALLEL KINETIC PROFILING OF NATURAL AND
ENGINEERED CRISPR NUCLEASES

Stephen K Jones Jr1,2,3, John A Hawkins1,2,3,4, Nicole V Johnson1,2,3,

Cheulhee Jung5, Kuang Hu1,2,3, James R Rybarski1,2,3, Janice S Chen6,
Jennifer A Doudna6,7,8,9, William H Press2,4, Ilya J Finkelstein1,2,3
1
University of Texas, Department of Molecular Biosciences, Austin, TX,
2
University of Texas, Institute for Cellular and Molecular Biology, Austin,
TX, 3University of Texas, Center for Systems and Synthetic Biology,
Austin, TX, 4University of Texas, Oden Institute for Comput, Austin, TX,
5
Korea University, Division of Biotechnology, Seoul, South Korea,
6
University of California, Molecular and Cell Biology, Berkeley, CA,
7
University of California, Chemistry, Berkeley, CA, 8Howard Hughes
Medical Institute, Berkeley, CA, 9Lawrence Berkeley national Laboratory,
Chemistry, Berkeley, CA

Engineered SpCas9s and AsCas12a edit fewer mispaired “off-target” DNAs

in human cells than wildtype SpCas9 (wtCas9). However, understanding of
their fidelity, mechanisms, and cleavage outcomes requires systematic
profiling across mispaired DNAs. We describe NucleaSeq—nuclease
digestion and deep sequencing—a massively parallel platform that
measures the cleavage kinetics and time-resolved cleavage outcomes for
over ten thousand mispaired DNAs with gRNA-relative mismatches,
insertions, and deletions. To capture binding specificities, we pair
NucleaSeq with our chip-hybridized association mapping platform
(CHAMP). We profile five SpCas9 variants and AsCas12a, and build a
biophysical model to reveal mechanistic insights into nuclease fidelity:
Engineered Cas9s, especially Cas9-HF1, dramatically increase cleavage
specificity, but not binding specificity, compared to wtCas9. Surprisingly,
AsCas12a and wtCas9 cleavage specificities resemble one another. The
initial sites of cleavage and further trimming depend on the nuclease, guide
RNA, and DNA mispairs. More broadly, NucleaSeq enables rapid,
quantitative, and systematic comparisons of cleavage specificity and
outcomes across nucleases – guiding scientists to pick the right instrument
from the CRISPR toolbox.

89
COMPARISON OF CAS9 ORTHOLOGUES FOR GENE SUPPRESSION
ACTIVITY

Ariel Kantor, Michelle E McClements, Robert E MacLaren

Nuffield Laboratory of Ophthalmology, University of Oxford, Nuffield

Department of Clinical Neurosciences, Oxford, United Kingdom

The development of CRISPR-Cas systems provides a powerful tool for

basic research as well as a potential avenue for therapy of genetic diseases.
A number of different Cas nucleases have been used for genome and
epigenome perturbations. Nevertheless, their delivery via adeno-associated
vector (AAV) is limited due to the small packaging capacity of the delivery
platform. Furthermore, most published studies have utilized different
genetic targets, construct designs and regulatory elements making it
difficult to draw conclusions about the comparative efficiencies and
specificities of each nuclease. Here we report the development and
optimization of an all-in-one AAV system harboring Campylobacter jejuni
(CjCas9): one of the smallest Cas9 nucleases. The system has been
validated on HEK293 cells carrying a copy of a GFP reporter. Furthermore,
we built similar counterparts harboring two different Cas9 variants: SpCas9,
and SaCas9. All constructs used exactly the same regulatory elements and
are assembled into identical vectors for delivery. We assayed comparative
efficiencies of each nuclease by plasmid transfection into HEK293T-GFP
cells. Genomic DNA was isolated 3 days post transfection and regions in
close proximity to target gRNAs were PCR-amplified for analysis of
induction of insertions and deletions (indels) by TIDE analysis.
Additionally, protein was isolated from the HEK293T-EGFP cells and GFP
fluorescence was quantified using fluorescence spectroscopy. Our
experimental strategies allowed us to compare the efficiency of different
components for genome engineering on the same target and indicate that
SpCas9 may have the highest efficiency among the tested Cas9 variants,
although the SaCas9 and CjCas9 orthologues were also able to generate a
high level of targeted indels within eGFP. We aim to further assess the
efficiency and specificity of CjCas9 paired with repressor domains DNA-
methyltransferase3A (DNMT3A) and Krüppel-associated box (KRAB) for
targeted epigenetic editing via AAV delivery.

90
GENOME EDITING OF THE PRADER-WILLI SYNDROME
IMPRINTING CENTER (PWS-IC) RESULTS IN LOSS OF
TRANSCRIPTIONAL CONTROL FOR THE SNURF-SNRPN-snoRNA-
lncRNA LOCUS IN A FELINE CELL CULTURE MODEL

Erik A Koppes1, Marie A Johnson1, Dale W Lewis2, Susanne M Golin2,

Robert D Nicholls1
1
University of Pittsburgh School of Medicine and UPMC Children's
Hospital of Pittsburgh, Division of Medical Genetics, Department of
Pediatrics, Pittsburgh, PA, 2University of Pittsburgh Graduate School of
Public Health, Department of Human Genetics, Pittsburgh, PA

The large imprinted domain at human 15q11.2 associated with Prader-Willi

syndrome and Angelman syndrome (PWS/AS) is highly conserved in cats
(Felis catus) in chromosome B3. However, there have been no studies
investigating imprinted genes in cats and whether the PWS/AS locus is
regulated in an imprinted manner with parent-of-origin-specific DNA
methylation and gene expression patterns. We have employed
CRISPR/Cas9 genome-editing to generate mutations at the PWS imprinting
center (PWS-IC) in the Crandell feline kidney (CRFK) cell line model to
investigate the imprinting status of the loci and to test the feasibility of
generating a PWS model in domestic cat. Plasmid vectors encompassing the
CRISPR-Cas9 system with guide RNAs targeting the DNA sequences on
both sides of the 2.2 kb PWS-IC were transfected into CRFK and resulted
in robust deletion of the region, as ascertained by genomic PCR. Following
transfection, single GFP(+) cells were flow-sorted in order to isolate clonal
populations. Out of 86 recovered clonal lines, 16 contained a PWS-IC
deletion and 18 had a PWS-IC inversion. From this subset we used a
combination of ddPCR, bisulfite genomic sequencing, and reverse-
transcription (RT)-PCR to examine the copy number as well as imprinted
DNA methylation and gene expression patterns of the PWS locus. From this
analysis we were able to confirm 8 lines with loss of paternal gene
expression concomitant with either deletion or inversion of the
unmethylated paternal allele. All 8 cell lines with disruption of the paternal
PWS-IC lost expression of the extended SNURF-SNRPN-lncRNA transcript
including SNURF, SNRPN, SNORD107, SNORD64, SNORD109,
SNORD116 and IPW but continued to express genes located at the proximal
PWS gene cluster, including MAGEL2, MKRN3 and NDN. In contrast,
deletion or inversion of the methylated maternal allele had no effect on
PWS gene expression. Many of the clonal lines we recovered had genome-
editing events on both parental alleles resulting in an array of different
combinations of deletion, inversion and scarred alleles. In conclusion, by
studying clonally isolated genome-edited CRFK cells, we determined that
CRFK lines show strict mono-allelic methylation, and that deletion or
inversion of the unmethylated allele results in ablation of PWS-gene
expression downstream of the PWS-IC.

91
CONTROLLING ENDOGENOUS LEVELS OF CYTIDINE
DEAMINASES BY CRISPRa FOR ELIMINATING HEPATITIS B
VIRUS

Dmitry Kostyushev1, Sergey Brezgin1,2, Anastasiya Kostyusheva1, Ekaterina

Bayurova3,4, Ilya Gordeychuk3,4,5, Lena Soppa6, Dieter Glebe6, Vladimir
Chulanov1,5
1
National Medical Research Center of Tuberculosis and Infectious Diseases,
Moscow, Russia, 2Federal Medical Biological Agency, Institute of
Immunology, Moscow, Russia, 3NF Gamaleya Research Center of
Epidemiology and Microbiology, Moscow, Russia, 4Chumakov Federal
Scientific Center for Research and Development of Immune-and-Biological
Products of RAS, Moscow, Russia, 5 Sechenov First Moscow State Medical
University, Moscow, Russia, 6Justus Liebig University Giessen, Institute of
Medical Virology, National Reference Center for Hepatitis B Viruses and
Hepatitis D Viruses, Giessen, Germany

APOBEC/AID deaminases are natural host restriction factors with broad antiviral
activity. They convert cytosines to uracil in ssDNA causing mutational inactivation
and DNA breaks in viral genomes. The use of APOBEC/AID as antiviral tools is
limited, as stimulation of their expression is hard to achieve and uncontrolled
expression is associated with carcinogenesis. Developing a cure for chronic hepatitis
B virus infection, a disease that annually leads to over one million deaths
worldwide, is a critical goal of global health care. APOBEC/AID are able to
specifically deaminate and destroy HBV DNA, not affecting the host genome. We
leveraged CRISPRa systems to induce endogenous levels of APOBEC/AID. Using a
CRISPRa with selected sgRNAs we achieved 4-100,000-fold activation of
APOBEC3A (A3A), APOBEC3B (A3B), APOBEC3G (A3G) and AID in vitro.
Elevated APOBEC/AID expression resulted in >99% decline in HBV replication.
Next, we assessed deamination activity of CRISPRa-induced APOBEC/AID and
detected abundant deamination of HBV genomes with typical G->A and C->T
mutations. We also observed frequent deletions in HBV DNA, indicating that
reduction in viral replication is associated with degradation of HBV genomes. But,
AID induced expression of a pro-apoptotic factor, whereas A3G inflicted DNA
damage. Although A3A and A3B did not exhibit detectable toxicity, it is unclear
whether prolonged upregulation of these factors is less damaging to the infected
cells. We hypothesized that a certain reduction in APOBEC/AID activation might
not affect cell viability but retains its antiviral activity. Using machine learning, it
was established that introducing single-nucleotide mismatches in sgRNA enables
titration of CRISPRa activity with a 10% decrement. We designed libraries of
attenuated sgRNAs with mismatched nucleotides targeting APOBEC/AID and tested
their effects on mRNA levels and antiviral properties. In general, titration of mRNA
levels mostly followed the predicted model with a decrement 10-20% to 50% using
attenuated sgRNAs. Antiviral activity of A3B, A3G and AID was mostly unaltered
even upon dramatic (>2-20-fold) decline in target gene activation, whereas anti-
HBV activity of A3A was consistently reduced.

In conclusion, activation of APOBEC/AID deaminases by CRISPRa tools is an

effective approach for inactivating HBV in experiment. Acknowledgements:
CRISPRa work was supported by RFBR-DFG grant 20-515-12010 and GL
595/9-1 design of attenuated sgRNAs was supported by RFBR grant 20-015-
00442.
92
COMPARING THE OVERALL HR/NHEJ RATIO OF DIFFERENT
CRISPR ENDONUCLEASES

Sarah L Krausz1,2, Éva Varga1,3,4, Hannah N Stabb1, Ervin Welker1,4

1
Institute of Enzymology, Research Centre for Natural Sciences, Budapest,
Hungary, 2School of Ph.D. Studies, Semmelweis University, Budapest,
Hungary, 3Doctoral School of Multidisciplinary Medical Science,
University of Szeged, Szeged, Hungary, 4Institute of Biochemistry,
Biological Research Centre, Szeged, Hungary

Homologous recombination (HR) and Non-homologous end joining (NHEJ)

are the two main repair systems of the mammalian cell, that correct the
DNA double strand break and frequently restore the original DNA
sequence. By inducing cleavage using a designer endonuclease, these
systems could potentially be exploited for site-specific gene modification,
which in mammalian cells, is preferentially obtained as the result of the
NHEJ repair. For numerous medical and scientific applications, achieving a
highly efficient HR over NHEJ is critical, that has triggered the
development of several approaches to boost HR over NHEJ. Endonucleases
differ in many aspects, the overhang they make and the position of the
cleavage from the PAM sequence. The most frequently used designer
endonucleases are the CRISPR nucleases, we are interested in which
CRISPR nuclease’s cleavage results in the most preferred HR/NHEJ ratio.

We aim to monitor the HR and NHEJ inducing capacity of various CRISPR

endonucleases on different targets. The NHEJ events are observed through
the extent of GFP disruption of an integrated copy of a GFP in N2a cells.
The efficiency of HR depends on the features of the particular homology
arms employed, its length and its distance from the cleavage site. To keep
these parameters constant, it is necessary to reconstruct the homologous arm
for every target site, which is a laborious process. To rationalize the
execution of these experiments we designed an easy to use plasmid-based
assay to measure HR events. The system is based on two plasmids
containing homology arms; each plasmid contains one half of the coding
region of mScarlet with an overlap between the mScarlet halves. All target
sites used in these experiments are positioned downstream of the first
mScarlet half, at the same distance from the homology arms. The cleavage
of the target induces HR-repair between the identical regions leading to the
recovery of a functioning mScarlet. The assays used to observe the NHEJ
and HR events are applied to the same target site, in the same cells. By
measuring the number of mScarlet positive as well as GFP negative cells by
flow cytometry we can discern the NHEJ and HR events of a specific
endonuclease on a particular target sequence allowing us to conclude an
overall HR/NHEJ ratio of a specific endonuclease.

93
SYSTEMATIC MAPPING OF GENETIC INTERACTIONS IN HUMAN
CANCER CELLS REVEALS CONTEXT DEPENDENT CANCER
SIGNALING PATHWAYS

Samson H Fong*1,2, Brent M Kuenzi*1, John Lee1, Kyle Sanchez1, Ana

Bojorquez-Gomez1, Kyle Ford2, Brenton P Munson1,2, Katherine Licon1,
Jeff Hager4, John Paul Shen1, Jason F Kreisberg1, Prashant Mali2, Trey
Ideker1,2,3
1
UC San Diego, Medicine, La Jolla, CA, 2UC San Diego, Bioengineering,
La Jolla, CA, 3UC San Diego, Computer Science and Engineering, La Jolla,
CA, 4IDEAYA Biosciences, San Francisco, CA

Genetic interaction screens have long been used to map gene functions and
cellular pathways in model organisms and more recently in human cells.
However, due to the technical challenges of these maps, most studies in
human cells have focused on a single cell line, limiting the ability to
determine how different genetic and cellular contexts influence genetic
interactions. Here, we engineered a 110,728-element combinatorial
CRISPR-Cas9 library to systematically map 12,282 genetic interactions
among tumor suppressor genes and druggable targets. We mapped the
landscape of genetic interactions in cell lines from breast cancer, head and
neck squamous carcinoma, and non-small cell lung cancer as well as in non-
cancerous, epithelial cells. The resulting genetic interaction maps emphasize
the context dependency of these interactions. Of the interactions that are
conserved across multiple cell lines, many include frequently essential
genes, suggesting that these interactions may be critical for fundamental
cellular functions. We found that genetic interaction maps from cancer cell
lines are more similar to one another than non-cancer interaction maps, even
between cancer and normal cells from the same tissue. Many of the
identified genetic interactions suggest therapeutic interventions, such as the
combination of CDK4 and MAPK inhibitors. These genetic interaction
maps provide a broad resource to investigate context dependency in cancer
signaling pathways and in cancer drug response.

* authors contributed equally

94
TO STUDY THE ROLE OF DNA TOPOLOGY IN REGULATING
LncRNAs IMPLICATED IN NEURAL DEVELOPMENT USING
CRISPR/CAS9 EDITED CORTICAL ORGANOIDS.

Manoj Kumar1,2, Debojyoti Chakraborty1

1
CSIR-Institute of Genomics and Integrative Biology, Genomics and
Molecular Medicine, New Delhi, India, 2Academy of Scientific and
Innovative Research (AcSIR), Ghaziabad, India

Long non-coding RNAs (lncRNAs) are transcripts longer than 200

nucleotides having low protein-coding aptitude. If we try to enumerate all
the annotated lncRNAs to date, we can predict a dearth of exclusivity in
their way of functioning, highlighting plausible but yet unexplored
functional redundancy. This can be explained through spatiotemporal
regulation of lncRNAs functions, as supported by many recent studies.
Likewise, there is also adequate literature supporting the lncRNA-
dependent neural cell-type-specific expression, helping to maintain cellular
identity. Additionally, when human stem cells (hESCs) undergo neural
differentiation either by artificial or natural means, there are changes in
chromatin architecture and if taken together with spatiotemporal regulation
of lncRNAs during different cell stages of early neurogenesis, it makes
sense that changes in chromatin architecture can act as a cue for this spatial-
temporal existence of these lncRNAs. Experiments done in mice show that
indeed chromatin changes can regulate expression and existence of
lncRNAs during neural development. However, the confined set of
lncRNAs maintaining a major cell pool of neural stem cells, intermediate
progenitors and neurons are not yet well characterised.
The mechanism of lncRNA expression as a consequence of divergent DNA
topological changes will allow us to identify the cues that explain the
confounding expression of functionally similar lncRNAs.
To commence this study, a proper model recapitulating stage of early
neurogenesis and cytoarchitecture of the human brain was needed. In that,
we were successful in developing cortical organoids from hESCs. Three
major cell pools (NSCs, IPCs and Neurons) of neural development have
been isolated based on endogenous lineage-specific fluorescently tagged
protein markers using CRISPR/Cas9 induced HDR based targeted knock-in.
We further aim to study the presence of different known and novel
lncRNAs and their role in neural development using the technique of total
RNA sequencing.

95
HIGHLY ORGANIZED, VARIOUSLY PATTERNED
ACCUMULATION PLATFORMS OF TRANSCRIPTION ACTIVATORS
USING CLASS 1 AND CLASS 2 CRISPR SYSTEMS

Atsushi Kunii*1, Shiho Nakamura*2, Kazuto Yoshimi3, Tomoji Mashimo3,

Takashi Yamamoto2, Tetsushi Sakuma2
1
Hiroshima University, Graduate School of Science, Hiroshima, Japan,
2
Hiroshima University, Graduate School of Integrated Sciences for Life,
Hiroshima, Japan, 3The University of Tokyo, Institute of Medical Science,
Tokyo, Japan

*Authors contributed equally

In the last half decade, several types of artificial transcription activators,

based on CRISPR-Cas9, have been developed and refined. Of these, in
SAM and SunTag systems, the effector proteins, expressed in trans, can be
recruited to the target sites via the MS2 RNA-binding system and GCN4-
scFv antibody system, respectively. In our previous study, we have created
a strong transcriptional activation system, achieved by fusing GCN4 repeat
to MS2 coat protein to accumulate numbers of activators, fused to scFv
antibodies. We showed that our novel system, named “TREE,” resulted in
greater effect of activating exogenous reporters and endogenous genes
(Kunii et al., CRISPR J, 2018). Here, we report two main updates of the
TREE system: 1) expansion of the lineup of RNA aptamers and protein tags
to increase the variety of orthogonal TREE systems, and 2) diversion of the
TREE system into Escherichia coli Type I-E CRISPR. We have so far
implemented PP7, boxB, and com aptamers in addition to MS2, as well as
split fluorescent protein (GFP11) tag and gp41 (MoonTag) in addition to
GCN4 (SunTag). We demonstrated the functionality of 12 patterns as
combinations of the RNA aptamers and the protein tags in human cells,
forming the basis for the future expansion of TREE system, enabling
simultaneous, orthogonal, and flexible control of multiple genes.
Furthermore, we created engineered Type I-E crRNA scaffolds containing
various aptamers such as MS2, PP7, boxB, and com, and recruited
transcriptional activators with or without adaptor tag arrays. Consistent with
the original TREE system based on Type II CRISPR-Cas9, stronger
transcriptional activation was observed using our highly accumulable
platform of transcriptional activators via adaptor tag arrays compared to the
activity of the system without tag arrays. Our expanded TREEs, named
FOREST, will open up a new avenue of scalable and highly efficient
transcriptional control and beyond.

96
DISSECTING THE ROLE OF RECURRENT HYPERPLOIDY IN
TUMORIGENESIS

Asad A Lakhani, Vishruth Girish, Christine Scaduto, Jason Sheltzer

Cold Spring Harbor Laboratory, Cancer Center, Cold Spring Harbor, NY

Approximately 90% of human tumors exhibit aneuploidy, an alteration in

the copy number status of whole chromosomes or chromosomal arms. The
experimental induction of aneuploidy, however, has thus far failed to reveal
the exact mechanisms by which chromosomal variability contributes to
tumorigenesis. The systematic characterization of the pan-cancer
consequences of aneuploidy remains challenging for multiple reasons. The
simultaneous alteration in the dosage of hundreds of genes on the aneuploid
chromosome means that differentiating between driver and passenger genes
is difficult. This is further complicated by the technical challenge of
inducing targeted chromosomal arm gain or loss, and of distinguishing the
phenotypic effects of the aneuploid chromosome from effects of genomic
instability. To navigate these challenges, we have developed a novel
chromosomal arm loss strategy to generate genetically-matched cell lines
that differ in the copy number of a single chromosomal arm in a near-
diploid, genomically stable background. We present proof-of-principle for
this approach using the ovarian adenocarcinoma cell line A2780, which has
three copies of chromosome 1q. Trisomy of chromosome 1q is observed
across breast, lung, and germ cell tumors, and in nearly 25% of ovarian
cancers. To generate A2780 cells disomic for chromosome 1q, we utilized
CRISPR-Cas9 for HDR-mediated integration of a positive-negative
selection cassette, induced arm loss using a centromere-proximal guide, and
negatively selected for cells that had lost the selection cassette. We
subsequently isolated clonal populations of cells that had lost a copy of
chromosome 1q, and found that these 1q disomic cells proliferated slower
in vitro, and formed both fewer and smaller colonies under anchorage-
independent conditions compared to their 1q trisomic counterparts.
Furthermore, these cells failed to form tumors following subcutaneous
xenografts in nude mice. We identify MDM4 and MCL1 as putative,
dosage-sensitive driver genes for 1q hyperploidy. MDM4 enhances p53
ubiquitination by its negative regulator MDM2, and directly inhibits p53 by
binding to its transactivation domain. MCL1 is an anti-apoptotic protein of
the BCL2 family, and heterodimerizes with other BCL2 family proteins.
Single copy deletions of MDM4 in a 1q trisomic background compromised
clonogenicity in anchorage-independent conditions, whereas MDM4
overexpression in 1q disomic cells partially rescued this phenotype.
Similarly, CRISRPi-mediated knockdown of MCL1 compromised
clonogenicity in 1q trisomic cells, whereas MCL1 overexpression in 1q
disomic cells partially rescued this phenotype. These results suggest MDM4
and MCL1 may be two of several drivers for the gain of chromosome 1q.
We thereby demonstrate a clinically relevant approach for the investigation
of hyperploid chromosomal contributions towards tumorigenesis.
97
CHANGE-SEQ REVEALS GENETIC AND EPIGENETIC EFFECTS ON
CRISPR-CAS9 GENOME-WIDE ACTIVITY

Cicera Lazzarotto1, Nikolay Malinin1, Yichao Li1, Ruochi Zhang2, Yang Yang2,
GaHyun Lee1, Eleanor Cowley3, Yanghua He1, Kasey Jividen1, Varun Katta1,
Natalia Kolmakova4, Christopher Petersen1, Qian Qi1, Samantha Maragh4,
Giedre Krenciute1, Jian Ma2, Yong Cheng1, Shengdar Tsai1
1
St Jude Children's Research Hospital, Memphis, TN, 2Carnegie Mellon
University, Pittsburgh, PA, 3Roche Diagnostics, Indianapolis, IN, 4National
Institute of Standards and Technology, Gaithersburg, MD

Current methods for defining the genome-wide activity of CRISPR-Cas9

nucleases are not easily scalable to the throughput needed to fully understand
fundamental principles that determine Cas9 cellular specificity. Here we
describe CHANGE-seq (Circularization for High-throughput Analysis of
Nuclease Genome-wide Effects by Sequencing), a fast, streamlined, Tn5
tagmentation-based assay for measuring the genome-wide activity of Cas9 in
vitro that is easily scalable to many targets and samples.
To systematically evaluate Cas9 genome-wide activity, we performed
CHANGE-seq on 110 targets in human primary T-cells. Analysis of the
201,934 off-target sites identified by CHANGE-seq revealed that G-base
frequency and nucleotide diversity in the target site are strongly associated with
specificity. The large-scale CHANGE-seq dataset we generated also enabled the
training of a highly accurate machine learning model to predict off-target
activity.
Next, we performed GUIDE-seq on two sets of sites previously evaluated by
CHANGE-seq, a set chosen without a priori knowledge of specificity, and a set
based on specificity as defined by CHANGE-seq. The number of sites detected
by CHANGE-seq and GUIDE-seq were strongly correlated (R2=0.85),
indicating that CHANGE-seq can identify highly-specific targets. To validate
the sensitivity of CHANGE-seq for identifying sites of bona fide cellular off-
target mutations, we selected 648 on- and off-target sites from six sgRNAs
target sites, for analysis by targeted tag sequencing. Of the 648 CHANGE-seq
detected sites we examined, we confirmed 278 (42.5%) by targeted tag
sequencing, including an average of 98.3% of the sites detected by both
CHANGE-seq and GUIDE-seq. Notably, we confirmed 84 of 427 (18.3%) off-
target sites detected exclusively by CHANGE-seq and not GUIDE-seq that we
analyzed by targeted tag sequencing. Our results demonstrate that CHANGE-
seq genome-wide activity profiles are strong predictors of cellular activity and
that CHANGE-seq is more sensitive than GUIDE-seq for identifying sites of
low-frequency cellular off-target activity. Additionally, we evaluated the impact
of chromatin accessibility on cellular off-target activity by comparing
CHANGE-seq with matched GUIDE-seq cellular off-target, chromatin, and
transcriptional profiles generated from the same human primary T-cells. We
found that cellular off-target activity was two to four times more likely to occur
near active promoters, enhancers, and transcribed regions.
Finally, CHANGE-seq profiling of 6 targets across 8 individual genomes
revealed that ~15.2% of human single-nucleotide variants analyzed had
substantial effects on nuclease activity. CHANGE-seq is a simplified, sensitive,
and scalable approach to understanding the specificity of genome editors.
98
INVESTIGATING PROLIFERATION SUPPRESSOR BEHAVIOR IN
WHOLE GENOME GENETIC FITNESS SCREENS

Walter F Lenoir1,2, Micaela Morgado1, Megan E McLaughlin1, Eiru Kim1,

Traver Hart1,2,3
1
The University of Texas MD Anderson Cancer Center, Bioinformatics and
Computational Biology, Houston, TX, 2Quantitative Sciences, The
University of Texas MD Anderson Cancer Center UTHealth Graduate
School of Biomedical Sciences, The University of Texas MD Anderson
Cancer Center, Houston, TX, 3The University of Texas MD Anderson
Cancer Center, Cancer Biology, Houston, TX

Innovation of CRISPR gene-editing technology has rapidly advanced our

ability to systematically study mammalian functional genomics. Genome-
wide CRISPR knockout screens in hundreds of cell lines have identified
context specific genetic vulnerabilities in cancer and have provided new
insights into gene function. Much of the research effort thus far has been
spent on optimizing phenotype distinctions between essential genes,
required for cell fitness, and non-essential genes, exhibiting no effect on cell
fitness, gene sets. Essential genes represent less than 20% of available
observations from a single screen, suggesting that there is data that has
largely been excluded from previous analyses.

To date, there has been sparse investigation of proliferation suppressor

genes, whose knockout results in increased cellular growth rates, in the vast
collection of genome-wide genetic screens coming from the Sanger and
Broad Institute’s DepMap. We conducted a systematic survey of
proliferation suppressor genes based on COSMIC defined tumor suppressor
genes’ (TSG) measurements of genetic fitness. Our working hypothesis is
that we can leverage the distinct fitness score signature of proliferation
suppressor genes in a screen to identify pathway level proliferation
suppressor biology and novel proliferation suppressor functions for specific
genes. We have identified distinct patterns of genetic growth fitness
distributions that depend on a TSG’s functional status. Additionally, we are
able to leverage the signature of fitness suppressors to detect other
consistent genetic proliferation suppressors of cancer cells.

We have developed a method that can detect proliferation suppressor

behavior within whole genome genetic fitness screens that we validated
through mutation and expression profiles. From these specific hit calls, we
designed a proliferation suppressor network based on co-occurrence of
proliferation suppressor gene status that demonstrates related cellular
functions between gene pairs. Finally, we have identified a short list of
genes that have not been noted previously to have proliferation suppressor
behavior, representing candidate TSGs. Included in these novel hits are
fatty acid synthesis genes demonstrating proliferation suppressor scores
within a select group of acute myeloid leukemia cell lines.
99
COPYCATCHERS: NOVEL ACTIVE GENETIC ELEMENTS FOR
DETECTING AND QUANTIFYING HOMOLOG-DIRECTED SOMATIC
GENE CONVERSION

Zhiqian Li1, Valentino M Gantz1, Ethan Bier1,2

1
University of California San Diego, Cell and Developmental Biology, San
Diego, CA, 2University of California San Diego, Tata Institute for Genetics
and Society-UCSD, San Diego, CA

CRISPR-based active genetic elements, or gene-drives, copied via

homology-directed repair (HDR) in the germline are transmitted to progeny
at super-Mendelian frequencies. Active genetic elements also can generate
widespread somatic mutations, but the genetic basis for such phenotypes
remains largely unknown. Because CRISPR induced double-stranded
breaks (DSBs) are predominantly repaired in somatic cells via non-
homologous end-joining (NHEJ) rather than HDR, it generally has been
assumed that somatic effects of gene-drives also primarily reflect NHEJ
outcomes. Here we quantify somatic gene conversion (SGC) events using
active genetic reporter elements called CopyCatchers. CopyCatchers
inserted into three independent genetic loci reveal unexpectedly high rates
of somatic cell copying. SGC efficiency depends on both temporal and
spatial aspects of Cas9 protein expression, and the dosage of several novel
functionally conserved candidate genes. These results suggest potential
gene therapy strategies wherein corrective genetic information might be
provided by homologous chromosomes.

100
A ROBUST METHOD FOR TAGGING ENDOGENOUS GENES
THROUGH PROMOTER TRAPPING AND SHORT HOMOLOGY
ARMS

Xiquan Liang, Narges Anbardar, Sridhar Ranganathan, Mohan C Vemuri,

Potter Jason, Jonathan D Chesnut

Thermo Fisher Scientific, Cell Biology, Carlsbad, CA

Proteins are spatially and temporally regulated within a cell. To study the
function of a protein, it is important to monitor the dynamic changes in
expression and localization in living cells. Here, we developed a rapid and
highly efficient method to add fluorescent tags to endogenous genes by
combined use of promoter trapping and short homology arms that
dramatically increase the efficiency and specificity of integration. The
donor design was greatly assisted by the use of TrueDesign Genome Editor,
a newly developed software platform that aids in gRNA ranking and
subsequent homology arm identification for knock-in experiments. A donor
DNA is prepared by a simple one-step PCR to add approximately 35nt
homology arms to a TrueTag™ donor template with no need for
construction of a donor plasmid. The efficiencies of tagging endogenous
genes with a 1.4 kb promoterless GFP, RFP or OFP reporter cassette range
from 50% to 100% upon antibiotic selection with higher level of specificity
occurring at the C-terminus than at the N-terminus. The method has been
validated using multiple targets in a variety of cell lines, including human
induced pluripotent stem cells, primary T cells and hematopoietic stem cells
(HSC). The HSCs expressing an EmGFP reporter could be enriched by cell
sorting and subsequently differentiated into different lineages of mature
blood cells.
Taken together, the method described here is extremely useful in
developing reporter cell lines for monitoring the subcellular location of
proteins as well as the dynamic progression during differentiation. It has
even broader applications in transgene expression and immune cell therapy.

101
ENHANCING MAIZE GRAIN YIELD BY CRISPR/CAS9 GENOMIC
EDITING OF CLE (CLAVATA3/EMBRYO-SURROUNDING REGION)
GENES FOR MAIZE BREEDING

Lei Liu, Edgar Demesa Arevalo, Richelle Chen, Tara Skopelitis, Qingyu
Wu, David Jackson

Cold Spring Harbor Laboratory, Jackson Lab, Cold Spring Harbor, NY

One of the most remarkable achievements during maize domestication and

improvement was producing maize with enlarged ears to dramatically
enhance grain productivity. Ear size is largely determined during early
development when the inflorescence meristem (IM) proliferates and
produces spikelets. Therefore, increase in IM activity is likely to generate
larger ears with higher yield. Meristems are maintained by the well known
CLAVATA–WUSCHEL feedback signaling pathways, where secreted CLE
peptides function as signals in cell-cell communication, proliferation and
differentiation. Null mutants of Zmfcp1 and Zmcle7, CLV3 homologs in
maize, result in over-proliferated IM and fasciated ears with more kernel
rows, but shorter ear with less grain yield. However, weak alleles of
fasciated-ear genes, FEA2 and FEA3, can increase grain yield.
We therefore created weak alleles of ZmCLE7 and ZmFCP1 by promoter
bashing using CRISPR/Cas9. We found multiple alleles with promoter
deletions and inversions, and some of them showed significantly enlarged
but normal ears. The most striking allele, which had a 300bp deletion in the
ZmCLE7 promoter, led to a slight decrease of ZmCLE7 expression, and
increase of IM activity to generate a ~25% increase in grain yield in B73
background, by producing deeper kernels on a bigger ear, an effect that is
higher than any other reported natural allele.
We also identified another maize CLE, ZmCLE1E5, with a similar
expression pattern to ZmCLE7 that is upregulated in Zmcle7 mutants,
suggesting a potentially redundant role in meristem homeostasis.
ZmCLE1E5 CRISPR null alleles had significantly longer ears with more
kernel rows. Our studies suggest that genomic editing can modulate
fundamental stem cell pathways by targeting cis-regulatory or coding
regions to significantly enhance complex yield traits, not in only maize
breeding but also de novo domestication of teosinte to re-shape modern
maize.

102
GENOME EDITING FOR FVIII AND AAT GENE THERAPY CURES

Reka Lorincz, David Curiel

Washington University in St Louis, Department of Radiation Oncology, St

Louis, MO

The CRISPR/Cas9 nuclease system is a powerful tool for manipulating

genomes by DNA editing. This allows for functional knock-in for gene
therapy correction. However, current limits of in vivo gene delivery
methods have practically restricted clinical application. In this regard, the
recombinant adeno-associated viral (AAV) vectors have been mainly
applied for in vivo CRISPR/Cas9-mediated gene therapy studies. However,
native AAV tropism limits the possibility of organ selective gene editing. In
addition, AAV’s innate liver tropism is associated with hepatotoxicity with
potential risk of integration to the host genome. As an anodyne, we
explored adenoviral (Ad) vector to achieve effective CRISPR/Cas9-based
knock-in. The unique capacity to alter Ad tropism can address both of these
limits. For alpha1-antitrypsin (AAT) deficiency lung disease, the
augmentation of AAT within the lower respiratory tract is the key biologic
mandate, thus we targeted Ad vector to the pulmonary endothelium. This
pulmonary targeted Ad approach has been designed to accomplish gene
therapy for AAT lung disease in a murine model of this disorder
(SERPINA1a-e KO mice). For hemophilia A gene therapy, we have
pursued an approach to limit potential hepatotoxicity. Specifically, we have
sought a non-hepatic source of the corrective factor VIII (FVIII). Thus we
explored a pan-endothelium delivery so that vascular endothelium can serve
this goal. We designed an optimal co-delivery of CRISPR/Cas9 and FVIII
therapeutic gene by a myeloid cell-binding Ad that allows effective and
selective in vivo editing for endothelial target cells to achieve phenotypic
correction in hemophilia A murine model. Here, we propose a highly
original vectorology to traverse key barriers for efficient in vivo genome
editing-based gene therapy in order to achieve long term genetic correction
of hemophilia A and AAT deficiency.

103
DEVELOPMENT OF AN EFFICIENT GENOME EDITING METHOD IN
A TILAPIA CELL LINE.

Angela L Man*1, Tarang K Mehta*1, Wilfried Haerty1, Federica Di

Palma1,2,3
1
The Earlham Institute, Organisms and Ecosystems, Norwich, United
Kingdom, 2University of East Anglia, Norwich Medical School, Norwich,
United Kingdom, 3University of East Anglia, School of Biological Sciences,
Norwich, United Kingdom

The Oreochromis tilapias are an economically important group of fish for

aquaculture, whose production has expanded dramatically in the last two
decades. To further improve tilapia aquaculture by breeding-in desirable
traits, it is necessary to better understand the genes and regulatory networks
driving valuable mechanisms of disease resistance, growth, and tolerance to
varying water conditions, e.g., salinity. Comparative functional genomics,
transcriptomics and epigenomics are powerful tools to study tissue-specific
regulatory networks. We recently reported striking cases of rapid network
rewiring events of adaptive trait genes, e.g., visual systems in East African
cichlids, including a tilapia species commonly used in aquaculture,
Oreochromis niloticus (Nile tilapia). Moreover, we identified many discrete
regulatory variants associated with network rewiring events, and in vitro
assays confirm that transcription factor binding site mutations disrupt
regulatory edges.

To functionally and phenotypically test several regulatory edges that could

be associated with adaptive traits in a related species, we look to develop an
efficient and reproducible genome editing method in an immortalised cell
line derived from Oreochromis mossambicus (Mozambique tilapia) brain
cells (OmB) (Gardell et al., 2014). Our aim is to establish and optimise
genome editing methodologies in the OmB cell line focussing on protocols
for (1) electroporation of Cas9-sgRNA RNP complexes; (2) lentiviral
transduction of Cas9 and gRNAs; and (3) use of Cas9 nickases and dual-
guide RNAs for editing of regulatory regions. Development of protocols for
genome-wide CRISPR knock out (GeCKO) (Doench, 2018) and Perturb-
Seq (Dixit et al., 2016) approaches would also be a focus.

By developing an efficient genome editing method in the OmB cell line, we

aim to functionally validate (1) several predicted regulatory edges that could
be associated with aquaculture-relevant adaptive traits; and (2) regulatory
regions that could be associated with domestication and adaptation of
several tilapia species/strains. The identification and validation of trait-
associated genomic regions could be ultimately useful for breeding-in
desirable traits as part of genomic selection programmes in Tilapia
aquaculture.

104
RELEASING A gRNA FROM AN mRNA TRANSCRIPT FOR
ASSOCIATION WITH DCAS9

Michelle E McClements1,2, Caroline F Peddle1,2, Robert E MacLaren1,2

1
University of Oxford, Nuffield Laboratory of Ophthalmology, Department
of Clinical Neurosciences, Oxford, United Kingdom, 2Oxford University
Hospitals NHS Trust and NIHR Biomedical Research Centre, Oxford Eye
Hospital, Oxford, United Kingdom

For autosomal dominant diseases unsuitable for gene supplementation

strategies, suppression of the disease allele with dCas9 in combination with
a suitable gRNA is an inviting therapeutic option. This strategy would
require long-term provision of the dCas9 and the gRNA and would
therefore benefit from AAV delivery to the desired target cell type. With
long-term expression of the dCas9 and gRNA, cell-specific expression
would be important to limit unwanted non-specific effects over time. Whilst
the dCas9 can be expressed from a cell-specific RNA polymerase II
promoter, the gRNA is typically expressed from a ubiquitous RNA
polymerase III promoter. This raises possible safety concerns as the
consequences of constant unregulated expression of gRNA in non-targeted
cell types are currently unknown. To expand transgene design options, we
have attempted to release a gRNA from within an mRNA transcript for
association with dCas9.

A gRNA sequence was inserted in a GFP reporter construct in two forms,

with additional nucleotides at the 3’ end of the scaffold (G1.GFP) and with
no additional nucleotides (G2.GFP). The resulting constructs were used to
transfect HEK293T cells and harvested after 48 hours with GFP expression
confirming production of functional transcripts. Cytoplasmic and nuclear
enriched fractions were prepared and RT-PCR of RNA samples confirmed
successful removal of G1 gRNA but not G2 gRNA from the GFP
transcripts. QPCR assessments using three primer sets targeting GFP,
unspliced gRNA-GFP and gRNA sequences were performed. Total RNA
ratios of these three targets were 1:0.9:1.3 for samples transfected with the
G2.GFP construct and were 1:0.2:0.3 for the G1.GFP transfected samples.
The significant difference in levels of gRNA to GFP in G1.GFP transfected
cells (p=0.0077) suggested degradation of the gRNA. Co-transfection of the
G1.GFP construct with pCMV.dCas9 and a luciferase reporter containing
the target sequence for the gRNA in the Renilla expression cassette revealed
no knockdown of Renilla activity. Co-transfection of the pCMV.dCas9 and
luciferase reporter construct with the G1 gRNA expressed from a U6
promoter also induced no significant repression of Renilla activity.

We present encouraging data showing that a gRNA can be released from an

mRNA transcript but the gRNA used here did not induce knockdown of
target gene expression with dCas9. These preliminary studies encourage
further investigations to build on these data.
105
HIGH-THROUGHPUT ANALYSIS OF REPAIRED CRISPR DOUBLE-
STRANDED BREAKS REVEALS NUCLEASE-SPECIFIC
COMPLEXITY

Matthew S McNeill1, Gavin Kurgan1, Heng Li2, Yu Wang1, Mark Behlke3

1
Integrated DNA Technologies, Bioinformatics, Coralville, IA, 2Dana-
Farber Cancer Institute, Biomedical Informatics, Boston, MA, 3Integrated
DNA Technologies, Molecular Genetics, Coralville, IA

CRISPR systems enable targeted editing in a wide variety of organisms by

introducing single- or double-strand DNA breaks, which are repaired using
native molecular pathways. We know that molecular pathway usage and
expression varies with cell type and genomic target location. We add to this
knowledge by characterizing the unique allelic patterns that result from
non-homologous end joining (NHEJ) after double stranded breaks (DSBs)
introduced by Cas9 or Cas12a. We analyzed editing outcomes using
rhAmpSeq™ assays, amplifying 273 Cas9 and 243 Cas12a gRNAs
delivered into Jurkat and Hap1 cells. Our results demonstrate that deletion
repair outcomes are larger after Cas12a, as compared to Cas9, DSBs.
Additionally, insertions typically occur within 1 nt of the Cas9 DSB, while
insertions typically occur in bimodal peaks near the two nicks introduced by
Cas12a. Finally, we find that deletions can occur > 4 nt away from the Cas9
DSB and > 3 nt away from each nick introduced by Cas12a. Using these
insights, we developed synthetic data sets representing 11 Cas9 and Cas12a
on- and off-targets (603 total targets), and we developed a biologically
informed analysis pipeline (CRISPAltRations). Using synthetic and real
data, we demonstrate that CRISPAltRations outperforms other publicly
available tools while having as good or better runtime. In addition, we
extend the pipeline’s usefulness by adding: quantification of frame-shifting
indels, a comparison of observed versus in-silico predicted indel profiles
(e.g. FORECasT), detailed characterization/visualization of perfect vs
imperfect homology directed repair (HDR) events, support for multi-guide-
introduced deletions, and more.

106
IN SITU GENERATION OF CAR T-CELLS

Samir A Mendonca, David T Curiel

Washington University in St. Louis School of Medicine, Raditation

Oncology, Saint Louis, MO

The remarkable performance of the chimeric Antigen Receptor (CAR) T-

cell technology in the clinical oncology sphere has provoked an expanding
need for agent availability. Presently methods of ex vivo genetic
engineering of T-cells are employed, owing to the absence of off-the-shelf
universal cellular reagents. In either case, resource consuming
manufacturing and upscaling processes represent practical barriers to
translational and commercial development of this promising approach. On
this basis, the concept of in situ transduction of T-cells, for CAR-T
induction, has been proposed as a novel strategy to feasibilise this approach.
In this regard, only a limited repertoire of presently available vectors
possess the in vivo efficiency and selectivity, in combination with long term
gene expression capacity, mandated by the in situ transduction strategy. On
this basis, we are seeking to develop an adenovirus system to address this
requirement, capitalizing on the unparalleled targetability of adenoviral
vectors (Ad). As a proof-of-principle strategy we are evaluating the delivery
efficiency of the GFP gene reporter to lymphocytes (CD3+ cells) by the
liver hexon modified liver untargeted Ad though systemic delivery in either
wild type and transgenic hCAR +/- C57BL/J mice.

107
ASSESSING EFFICIENCY AND FIDELITY OF CRISPR BASED
THERAPIES USING MOLECULAR COMBING

Imen Mestiri, Prakhar Bisht, Engin Altunlu, Samira Mzalouat, Aurélien

Petit, Laurence Maggiorella, Florence Mahé, Aaron Bensimon

Genomic Vision, Bagneux, France

Recent advances in genome editing technologies, in particular the

CRISPR/Cas toolkit, are driving innovations in precision medicine. New
methods continue to be developed with the aim of providing consistent and
safer gene and cell therapies. However, these methods can still result in a
wide range of unintended modifications that hamper their full translation
into the clinic.
While early results from clinical trials are encouraging, a comprehensive
analysis of genome editing outcomes in in vitro and preclinical models is
still needed to mitigate the risk.
Most of current assays measuring gene editing outcomes focus on off-target
edits or unintended indels in the immediate vicinity of the targeted site but
more complex changes can occur further away.

Molecular combing, a proprietary technology to directly visualize single

DNA molecules, offers the possibility to quantify and visualize these
changes without any amplification bias, providing an orthogonal analytical
method to sequencing based approaches.

We will present and discuss different unpublished studies that illustrate how
our molecular combing based quality control assay allows to quantify (i) the
editing efficiency, (ii) rare and complex on-target rearrangements including
large events like balanced and unbalanced translocations, iii) exogenous
DNA integration (e.g. AAV vectors) at the targeted site or elsewhere in the
genome.

108
EFFICIENT PRODUCTION AND APPLICATION OF ssDNA for
CRISPR/CAS KNOCKINS IN HUMAN PRIMARY T CELLS

Montse Morell, Tatiana Garachtchenko, Lily Lee, Thomas P Quinn,

Michael Haugwtiz, Andrew Farmer

TakaraBio USA, Cell Biology, Mountain View, CA

The application of CRISPR/Cas9-based technology to engineer site-specific

insertions of genes or sequences longer than 200 bp via homology-directed
repair (HDR) is often very difficult. A primary challenge involves achieving
efficient delivery of the DNA template used for HDR following Cas9-
sgRNA-mediated cleavage at the genomic target site. Electroporation of
ribonucleoprotein complexes (RNPs) is the preferred method for delivery of
Cas9 and sgRNA in vitro due to the easy preparation of the reagents and
their transient nature in the target cells, which limits the possibility of off-
target effects. Two popular approaches that leverage this method to
engineer knockins involve co-electroporation of the DNA donor template
along with the Cas9-sgRNA RNPs, or adeno-associated virus (AAV)-
mediated transduction of the donor as ssDNA following RNP
electroporation. While the use of AAV often results in high editing
efficiencies, it requires cloning into the appropriate vectors and producing
viral particles prior to genome editing.
For the nonviral approach, co-electroporation of the Cas9-sgRNA RNP and
donor sequence can be performed with either double-stranded DNA
(dsDNA) or single-stranded DNA (ssDNA) as the HDR donor template.
However, the application of ssDNA is associated with two important
advantages: lower rates of random or off-target integration (Chen et al.
2011; Roth et al. 2018) and lower cytotoxicity (Roth et al. 2018), especially
when working with hiPSCs or primary cells.
Here we present the Guide-it™ Long ssDNA Production System v2 which
provides a simple and fast method for producing long ssDNA (up to 5,000
nt) for use as donor templates in gene editing applications. This system uses
PCR followed by enzymatic degradation of one of the strands of the
amplified product. The resulting ssDNA can be used in knockin
experiments targeting endogenous genes. As a proof of concept, we
specifically tagged in primary T cells two endogenous genes (TUB1 and
SEC61B) with a fluorescent protein and performed a knockin of murine -
TCR into the human endogenous TRAC locus (Schober et al. 2019).
References
Eyquem, J. et al. Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances
tumour rejection. Nature. 543(7643), 113–117 (2017).

Roth, TL. et al. Reprogramming human T cell function and specificity with non-
viral genome targeting. Nature. 559(7714), 405-409 (2018).

Schober, K. et al. Orthotopic replacement of T-cell receptor a- and b- chains with

preservation of near-physiological T-cell function. Nat. Biomed. Eng.543, 974–984
(2019).
109
METAL-ORGANIC FRAMEWORKS; A NOVEL CRISPR/CAS9
DELIVERY APPROACHES IN THERAPEUTIC GENOME EDITING

Muhammad Naeem, Irshad Ahmad

King Fahd University of Petroleum and Minerals (KFUPM), Life Sciences,

Dhahran, Saudi Arabia

Gene-editing technology is of significant interest to target a specific

location in the genome by deletion or addition that has revolutionized the
era of basic biomedical research and therapeutic applications. The clustered
regularly interspaced short palindromic repeats/CRISPR associated protein
9, CRISPR/Cas9 emerged as a substantial tool due to its simplicity in use,
less cost, and extraordinary efficiency as compared to the conventional
gene-editing tools, including Zinc Finger Nuclease (ZFNs) and
Transcription Activator-Like Effector Nuclease (TALEN). However,
Optimization for effective and specific cell type CRISPR/Cas9 delivery
vectors remains an open, challenging problem for efficient and effective
genome editing. Usually, viral vectors used to deliver CRISPR/Cas9
reagents to a specific cell with high efficiency; they cause several
drawbacks such as the risk of carcinogenesis, high immune response, and
off-target effects. Alternatively, non-viral vectors are less mutagenic, less
immunogenic, safe, and simple in CRISPR/Cas9 delivery. Recently
synthetic metal-organic frameworks (MOFs) have been constructed through
linkers, and metals have attracted the scientific communities due to their
unique properties including, high porosity, great internal surface area,
biodegradability, biocompatibility, and high thermal and chemical stability.
In this paper, we will highlight the progresses of MOFs constructed to
transfer the CRISPR/Cas9 components in vitro and in vivo for therapeutic
genome editing. We will also highlight the future prospects and challenges
of designing of MOFs CRISPR/Cas9 delivery vehicles for clinical
applications.

*Authors contributed equally

110
EXTENDED ANALYSIS OF AMPLICON SEQUENCING DATA WITH
MACHIATO FOR PRIME EDITING

Kazuki Nakamae1, Mitsumasa Takenaga2, Shota Nakade3,4, Ichiro Nazuka5,

Akinori Awazu2, Naoaki Sakamoto2, Tetsushi Sakuma2, Takashi Yamamoto1,2
1
Hiroshima University, Genome Editing Innovation Center, Hiroshima, Japan,
2
Hiroshima University, Graduate School of Integrated Sciences for Life,
Hiroshima, Japan, 3Hiroshima University, Graduate School of Science,
Hiroshima, Japan, 4Massachusetts Institute of Technology(MIT), Department of
Biological Engineering, Cambridge, MA, 5TOPPAN PRINTING CO., LTD.,
Information & Communication Division, Tokyo, Japan

CRISPR gene editing technology has been widely spreading in various fields.
Along with that, many bioinformatics resources have been developed to analyze
the genomic sequences edited with various forms of CRISPR methods. Of
these, CRISPResso2, recently developed tool to analyze the amplicon
sequencing data (Clement et al., 2019), can easily estimate the editing efficiency
and validate the editing quality. Such bioinformatics methodologies have been
applied in various strategies of CRISPR gene editing, including Cas9-mediated
gene knockout, HDR-mediated incorporation of exogenous DNA templates, and
even Prime Editing. Prime Editing is a unique method reported very recently, in
which Cas9 nickase fused to reverse transcriptase enables “search-and-replace”
gene editing. However, conventionally used data analysis tools were not enough
to obtain the comprehensive understanding of the various tendency on the
editing efficiency such as its relationship with genomic contexts including
thermodynamics and secondary structure, because their analysis pipeline does
not include a process compiling and profiling the variable results obtained from
multiple samples.
Here, we present a comprehensive pipeline, named MaChIAto, to extend the
functionality of the data analysis with CRISPResso2. The joint pipeline,
CRISPResso2-MaChIAto, mainly has two functions: (1) more precise
quantification of the edited reads based on the multi-step re-classification with
Bayesian optimization, and (2) relationship analysis with >70 kinds of genomic
contexts and unexpected mutation patterns. To demonstrate the capability of
MaChIAto, we applied MaChIAto to profile the publicly available Prime
Editing data (Anzalone et al., 2019). Our re-classification pipeline successfully
enabled to reduce the percentages of the misidentified reads erroneously
classified as precisely edited alleles from 8.6% to 0% in the best case (VEGFA
+1T to A rep1) compared to the original CRISPResso2. There were no
unexpected InDel sequences found in the misclassification of all samples
obtained with MaChIAto. Furthermore, MaChIAto showed the difference
between the groups with high, moderate, and low efficiency in the context of
InDel distributions and predicted values for structural properties. The results
implied that the efficiency of Prime Editing depends on some structural features
and the InDel patterns at the target locus. Our extended analysis software of
amplicon sequencing data would be one of the essential tools to assess the
upcoming editing methods like Prime Editing and enables to quickly find
critical characteristics related to the efficiency of CRISPR gene editing.

111
ENGINEERING A MINIATURIZED SACAS9 ADENINE BASE
EDITOR WITH REDUCED RNA OFF-TARGETS AND INCREASED
ON-TARGET DNA EDITING EFFICIENCIES

Minh Thuan Nguyen Tran1, Mohd Khairul Nizam Mohd Khalid1, Qi

Wang1, Jacqueline K Walker1, Predrag Kalajdzic2, Grace E Lidgerwood3,4,
Kimberley Dilworth2, Leszek Lisowski2,5, Alice Pebay3,4, Alex W Hewitt1
1
Menzies Institute for Medical Research, The University of Tasmania,
School of Medicine, Hobart, Australia, 2Translational Vectorology Research
Unit, The University of Sydney, Children's Medical Research Institute,
Sydney, Australia, 3The University of Melbourne, Department of Surgery,
Melbourne, Australia, 4The University of Melbourne, Department of
Anatomy and Neuroscience, Melbourne, Australia, 5Military Institute of
Hygiene and Epidemiology, The Biological Threats Identification and
Countermeasure Centre, Pulawy, Poland

Precision genome engineering has dramatically advanced with the

development of CRISPR/Cas base editing systems that include cytosine
base editors and adenine base editors (ABEs). We compared the editing
profile of circularly permuted and domain inlaid Cas9 base editors, and
found that on-target editing can be largely maintained following their
intradomain insertion, but that structural permutation of the ABE can affect
differing RNA off-target events. With this insight, structure-guided design
was used to engineer an SaCas9 ABE variant (microABE I744) that has
dramatically improved on-target editing efficiency and a reduced RNA-off
target footprint compared to current N-terminal linked SaCas9 ABEmax
(7.10) variants. This represents one of the smallest AAV-deliverable Cas9-
ABEs available, which has been optimized for robust on-target activity and
RNA-fidelity using a novel method that does not involve amino acid
substitutions.

112
HIGH-THROUGHPUT MAPPING OF NODE OF RANVIER AND
AXON INITIAL SEGMENT ASSOCIATED PROTEINS BY HiUGE
GENOME EDITING

Yuki Ogawa, Matthew Rasband

Baylor College of Medicine, Neuroscience, Houston, TX

The nodes of Ranvier and axon initial segment (AIS) are important for the
generation and propagation of action potentials, and regulation of neuronal
polarity. It is well established that AnkyrinG (AnkG) and 4 spectrin play
key roles in the formation and maintenance of the nodes and AIS.
Interestingly, nodes and AIS share a common molecular organization.
However, some new node-specific and AIS-specific or -enriched proteins
have been discovered (e.g. Trim46, Ranbp2, NuMA1, TREK1) and the
molecular mechanisms that stabilize the AIS and control neuronal polarity
remain obscure. Recently, we identified candidate AIS-associated proteins
using AnkG-knock out mice, proximity biotinylation, and proteomics. In
the current study, to further investigate these candidates, we have used the
AAV-based CRISPR-mediated homology independent universal genome
engineering (HiUGE) method for high-throughput modification of
endogenous proteins. Although most candidates we tested were not AIS-
specific or -enriched proteins, we observed that Itsn1 was enriched at the
AIS. Separately, while Arhgap21 was found throughout neurons, detergent
extraction revealed a detergent-resistant pool of Arhgap21 in the soma
adjacent to the AIS. This approach may provide new insights into the
mechanisms underlying AIS and node of Ranvier formation and
stabilization.

113
VALIDATION OF CRISPR GENE KNOCKOUTS IN U2OS CELL
LINES WITH DIA MS-BASED PROTEOMICS

Jennifer Oki1, Vito Dozio2, David Conant1, Travis Maures1, Michelle Gut2,
Yuehan Feng2, Kristina Beeler2, Scott Pattison1, Nicholas Dupuis2
1
Synthego Corporation, Research, Menlo Park, CA, 2Biognosys AG,
Research, Schlieren (Zurich), Switzerland

CRISPR-Cas9 has developed into an essential tool for gene modification in

cellular systems with applications in screening and target identification,
target validation, and generation of disease models. Key areas of research
and development in CRISPR technology include the improvement of
knockout efficiency and gene specificity. The results of a CRISPR gene
modification are commonly validated using readily available off-the-shelf
antibodies which may not always be available, or custom designed assays
that are time-consuming and expensive. Here, we have developed an
optimized RNP (ribonucleoprotein) delivery protocol that yields gene
knockouts (KOs) which can be reliably assessed using inexpensive
genotypic analysis and are functional at the pool level. Further, we
demonstrate the utility of quantitative global proteomic characterization
using data-independent acquisition mass spectrometry (DIA-MS) to validate
knockout of protein expression levels in U2OS cells. We compared this
proteomics analysis with RNA-Sequencing and concluded that DIA-MS is
suitable for elucidating specific changes after CRISPR editing with a small
number of samples and demonstrating functional gene KO. The global
proteomics data set not only enables rapid validation of target knockouts but
also enables the characterization of secondary consequences of gene
modification.

114
A GENE SIGNAL AMPLIFIER PLATFORM FOR MONITORING THE
UNFOLDED PROTEIN RESPONSE

Carlos A Origel Marmolejo1,2, Bhagyashree Bachhav3, Sahiti D Patibandla2,

Alexander L Yang4, Laura Segatori5
1
Stanford University School of Medicine, Department of Radiation
Oncology, Stanford, CA, 2Rice University, Department of BioSciences,
Houston, TX, 3Rice University, Department of Chemical and Biomolecular
Engineering, Houston, TX, 4Rice University, Department of Computational
and Applied Mathematics, Houston, TX, 5Rice University, Department of
Bioengineering, Houston, TX

Gene expression in mammalian cells results from coordinated protein-

driven processes guided by diverse mechanisms of regulation, including
protein-protein interactions, protein localization, DNA modifications, and
chromatin rearrangement. Increasingly the study of gene expression
signatures and profiles is becoming the focal point in biological research,
particularly in the characterization of stress-response signaling pathways.
Current technologies to monitor gene expression rely on transcriptomic
analysis (e.g. DNA microarrays, RNA-seq, and quantitative RT-PCR) that
do not provide temporal resolution of gene expression dynamics. To address
the need to monitor chromosomal gene expression generating a readily
detectable signal output that recapitulates gene expression dynamics, I
developed a gene signal amplifier platform that links transcriptional and
post-translational regulation of a fluorescent output to the expression of a
chromosomal target gene. The platform is composed of a tunable
orthogonal gene network to amplify the output signal, while relying on the
chromosomal integration of the main regulator using CRISPR-Cas9 to
readily link the genetic circuit to a target gene, thus adapting the system to
monitor any cellular target. I generated a multiplex reporter system for
monitoring markers of the unfolded protein response (UPR), a complex
signal transduction pathway that remodels gene expression in response to
proteotoxic stress in the endoplasmic reticulum. By recapitulating the
transcriptional and translational control mechanisms underlying the
expression of a target gene with high sensitivity, this platform provides a
novel technology for monitoring gene expression with superior sensitivity
and dynamic resolution.

115
SCREENING SNP-BINDING CRISPR/Cas9 gRNAs TARGETING
RHODOPSIN AS PART OF AN ALLELE-SPECIFIC STRATEGY TO
TREAT AUTOSOMAL DOMINANT RETINITIS PIGMENTOSA.

Caroline F Peddle1, Michelle E McClements1, Robert E MacLaren1,2

1
University of Oxford, Nuffield Department of Clinical Neurosciences,
Oxford, United Kingdom, 2Oxford Eye Hospital, Oxford, United Kingdom

Dominant mutations in rhodopsin (RHO), a light-sensitive protein found in

rod photoreceptors in the retina, cause retinitis pigmentosa. This disease
affects roughly 1 in 16 000 individuals and causes progressive loss of
photoreceptors over several decades, resulting in blindness. Successful gene
therapy treatment of this condition will require allele-specific knock down
of the mutant rhodopsin strand. As there are over 150 identified RHO
mutations, the ideal treatment strategy should be mutation-independent.
Common, non-pathogenic single nucleotide polymorphisms (SNPs) present
on the mutant RHO strand can be targeted with CRISPR/Cas9. The absence
of this SNP on the wild-type strand will create a 1 bp mismatch between the
gRNA and DNA which may be sufficient to prevent Cas9-driven gene
disruption.

The human RHO gene was screened for common SNPs, which occurred
either in the gRNA binding region, or generated or removed a PAM site.
Seven SNPs and one 10 bp deletion were identified that enabled the design
of 10 gRNAs (RHO_1-10). Encouragingly, the SNPs occurred in the gRNA
“seed region”, the 8-10 bp at the 3’ end of the sequence where gRNA:DNA
mismatches are least tolerated, in all gRNAs except RHO_7. The 10 bp
deletion spanned the gRNA binding region of gRNA RHO_3 and removed
the PAM site from gRNA RHO_4, making these the most promising
candidates for allele-specific disruption of RHO.

HEK293-EGFP cells were genotyped and found to be homozygous for all

SNPs. gRNAs targeting the SNPs present in HEK293-EGFP cells were
cloned into an all-in-one plasmid expressing both SaCas9 and gRNA, and
transfected into HEK293-EGFP cells. TIDE analysis was conducted on 7 of
the 10 gRNAs, which had rhodopsin disruption levels of 16.4 % with gRNA
RHO_2 to 44.2 % with gRNA RHO_7. The remaining 3 gRNAs could not
be assessed by TIDE analysis as highly repetitive regions adjacent to the cut
site prevented successful Sanger sequencing (a requirement of TIDE
analysis). Instead these were subcloned into the pGEM-T Easy vector and
sequenced. This method detected higher editing efficiencies of 66.6 % to
73.3 %.

All gRNAs were able to disrupt rhodopsin in HEK293-EGFP cells in vitro

with varying efficiencies from 16.4 % to 73.3 %. The subcloning technique
appears to detect a higher rate of CRISPR-mediated gene disruption than
TIDE analysis.
116
RAPID, FIELD-DEPLOYABLE CRISPR DIAGNOSTICS FOR ANY
NUCLEOBASE VARIANT IN DNA OR RNA USING A HIGHLY
SPECIFIC CAS9 FROM FRANCISELLANOVICIDA(FNCAS9)

Rhythm Phutela*1,2, Mohd Azhar*1,2, Dipanjali Sinha1,2, Namrata Sharma1,2,

Manoj Kumar1,2, Debojyoti Chakraborty1
1
CSIR-Institute of Genomics & Integrative Biology, Genomics and
Molecular Medicine, New Delhi, India, 2Academy of Scientific and
Innovative Research, Ghaziabad, India

In recent years, CRISPR diagnostics has become an integral part of nucleic

acid detection, reducing the time and complexities associated with
sequencing-based approached Apart from the canonical genome editing
abilities of CRISPR, its components have been effectively used for
detection of variety of nucleic acid targets such as pathogenic bacteria or
viruses and disease-causing single nucleotide polymorphic mutations.
Current strategies that rely on using CRISPR components for nucleic acid
detection principally use the trans-cleavage readout of reporter molecules in
the presence of an active substrate: ribonucleoprotein complex. CRISPR
Cas proteins that have been employed for DNA/RNA detection in this
manner include Cas12 (for DNA)and Cas13 (for both DNA and RNA).
Each of these diagnostic platforms has its own strengths and shortcomings
that are majorly associated with sensitivity, specificity, and read-out modes.
The primary focus of these platforms is the detection of low copy numbers
of nucleic acids from body fluids where the collateral activity of fluorescent
reporters amplifies the signal of the substrate. For point-of-care (POC)
diagnostics, where high sensitivity, low cost and time of the detection
procedure with less complex experimentation is very crucial for valuable
prognosis, alternate detection platforms that are robust, doesn’t require
extensive design and has flexible readout modes are preferable.
Here we report an economical, rapid, and POC diagnostic platform for
detection of all possible pathogenic nucleic acids including nucleobase
variants with 1 base pair resolution using the highly specific binding affinity
of FnCas9. We show its success in the detection of monogenic variants in
DNA/RNA obtained from multiple sources including patient samples and
pathogenic microorganisms. We call this approach FnCas9 Editor Linked
Uniform Detection Assay (FELUDA). As an immediate application of
FELUDA, we show its successful deployment in the rapid and accurate
diagnosis of COVID-19 infected individuals through a simple paper strip
based readout.

117
CRISPRme: POPULATION AND PATIENT-SPECIFIC OFF-TARGET
SITE ANALYSIS FOR CRISPR GENOME EDITING

Samuele Cancellieri2, Elia Dirupo2, Anne Shen3, Daniel Bauer3, Nicola

Bombieri2, Rosalba Giugno2, Luca Pinello1
1
MGH/Harvard/Broad, Molecular Pathology, Boston, MA, 2University of
Verona, Computer Science, Verona, Italy, 3Boston Children's Hospital,
Hematology/Oncology, Boston, MA

CRISPR genome editing has become a widely-used tool to easily and

efficiently modify a genome of interest in a programmable way and could
be a promising therapeutic modality to produce beneficial genetic changes
in patient cells. However, off-targets (i.e., unwanted cleavages at sites with
high homology with the desired target sequence) may occur. It is important
to consider the efficiency of the guide RNA at these off-target sites,
especially when multiple guides are considered for a given application in
order to maximize the editing outcomes and assess safety.

Although several websites have been developed to aid the design of single-
guide RNAs (sgRNAs) and to predict outcomes of specific sgRNAs, these
tools do not explicitly account for personal genetic variants and are
therefore limited in assessing efficiency, specificity, and safety of sgRNAs.
In fact, their on and off-target scores are calculated based on reference
genomes only.

To overcome these limitations, we present CRISPRme, a web application

that extends CRISPRitz (Cancellieri et al, Bioinformatics 2020) with a user-
friendly interface to enumerate on- and off-target sites accounting for
mismatches, DNA/RNA bulges, and common genetic variants.

Importantly, CRISPRme performs genomic scanning while accounting for

existing haplotypes, instead of enumerating combinations of genetic
variants that may not exist in a given population.

CRISPRme analyzes super populations (based on variants from the 1000

Genome Project) as well as personal genomes, and produces reports to
quickly assess the potential risk of off-target editing.

CRISPRme can be easily deployed on any machine and customized with

personal genomes (user-defined reference genome and/or variants from
personal VCFs) to maintain personal genome privacy and security.

The CRISPRme website is available online at http://crisprme.di.univr.it/

118
DIRECT CARDIOGENIC REPROGRAMMING OF FIBROBLASTS AS
A PROMISING THERAPEUTIC STRATEGY FOR INJURED HEART

Volodymyr V Balatskyi1,2, Anna Myronova1, Oksana O Piven1

1
Department of Human Genetics, Institute of Molecular Biology and
Genetics, National Academy of Sciences of Ukraine, Kyiv, Ukraine,
2
Nencki Institute of Experimental Biology, Polish Academy of Sciences,
Laboratory of Molecular Medical Biochemistry, Warsaw, Poland

The adult heart is a dynamic organ that is capable of significant remodeling

during life. Nevertheless, cardiomyocyte deficiency underlies the most
causes of heart failure. Human left ventricle has approximately 2 to 4 billion
cardiomyocytes, myocardial infarction can kill up to 25% of them within a
few hours. All dead cardiomyocytes are replaced by fibroblasts and adipose
tissue resulting in heart fibrosis. Multiple lines of evidence suggest that the
heart of higher vertebrates has a regenerative capacity . However, this
regeneration is extremely limited and is not sufficient to heal the injured
heart.
In the present study we focused on CRISPRa system for the direct
reprogramming of fibroblasts into cardiomyocytes. We suppose that
CRISPRa system can serve as a promising platform for an alternative and
non-integrating method to reprogram fibroblasts into cardiac-like cells for
the regeneration of affected heart tissue. To this end, we used the SP-Cas9-
VPR (Addgene Plasmid No. 68463) variant that is able to effectively
activate the expression of target genes. Using CRISPR-ERA and Ensembl
Genome Browser tools, CCTop-CRISPR/Cas9 target-based online predictor
and NCBI blast tools, we identified the possible candidates for sqRNAs that
are complementary to the promoter regions of cardiac transcription factors
(GATA4, MyoD, Tbx5, Mef2c and HAND2) in humans and rats.Thus, we
have selected the most promising sqRNAs candidates for the following
synthesis and cloning into phU6-gRNA (Addgene plasmid #53188) and
Cas9 sgRNA (Addgene plasmid #68463) vectors to create sgRNA library.
Following isolation of rat embryonic fibroblasts and their liposomal
transfection, we validated sqRNAs library efficiency. Thus, we selected the
sgRNA that led to the highest expression levels of target genes in primary
cells. Next, we combined all selected sgRNA for the following transfection
into two experimental sets. We observed an increase in the expression of
cardiogenic transcription factors in rat fibroblasts on the third, sixth and
ninth day after liposomal transfection by set 1 and set 2. Moreover, we
found an elevation in the expression of cardiac marker genes (Serca, ANP
and b-MHC). However, we did not observe spontaneously contracting cells.
We suppouse, for the successful reprogramming of fibroblast into
cardiomyocytes additional conditions should be optimized (i.e.,
downregulation of the fibroblasts-specific signaling pathways, media
supplementation)

119
EXPANDING THE SCOPE OF PLANT GENOME ENGINEERING
WITH NOVEL CAS12A ORTHOLOGS AND HIGHLY
MULTIPLEXABLE EDITING SYSTEMS

Yingxiao Zhang1, Qiurong Ren2, Xu Tang2, Shishi Liu2, Aimee A

Malzahn1, Jiaheng Wang2, Desuo Yin1, Changtian Pan1, Yanhao Cheng1,
Qiudeng Que3, Yong Zhang2, Yiping Qi1
1
University of Maryland, Plant Science and Landscape Architecture,
College Park, MD, 2University of Electronic Science and Technology of
China, Biotechnology, Chengdu, China, 3Syngenta, Research Triangle Park,
NC

CRISPR-Cas12a is a promising genome editing system for targeting AT-

rich genomic regions. Comprehensive genome engineering requires
simultaneous targeting of multiple genes at defined locations. To expand the
targeting scope of Cas12a, we screened nine new Cas12a orthologs and
identified six, Lb5Cas12a, BsCas12a, ErCas12a, Mb2Cas12a, MbCas12a
and TsCas12a, that possess high editing activity in rice. Among them,
Mb2Cas12a stands out with high editing efficiency, relaxed PAM
requirements and tolerance to low temperature. To enable large-scale
genome engineering, we compared 12 multiplexed Cas12a systems and
identified a potent system that exhibited nearly 100% biallelic editing
efficiency with the ability to target as many as 14 sites in rice. This is the
highest level of multiplex edits in plants to date using Cas12a. Two compact
single transcript unit CRISPR-Cas12a interference systems were also
developed for multi-gene repression in plants. This study has greatly
expanded the targeting scope of Cas12a for crop genome engineering.

120
LARGE INSERTIONS MEDIATED BY CRISPR/CAS EDITING VIA
IMPROVED HDR TEMPLATES, AND COMPREHENSIVE
CHARACTERIZATION OF EDITING EVENTS WITH LONG READ
SEQUENCING.

Garrett R Rettig, Mollie Schubert, Gavin Kurgan, Matthew McNeill, Jessica

Woodley, Mark Behlke

Integrated DNA Technologies, Molecular Genetics, Coralville, IA

Specific genome editing outcomes can be achieved via homology-directed

repair (HDR). This repair mechanism is exploited when a repair template,
homologous to the genomic sequence adjacent to the double-stranded break
(DSB), is delivered at the same time that the DSB is generated. HDR repair
outcomes with CRISPR/Cas systems are most efficiently driven by single-
stranded DNA (ssDNA) templates when small insertions (up to ~120 bp),
deletions, or SNP changes are desired edits. Larger insertions can be
incorporated via HDR using enzymatically-generated ssDNA or double-
stranded DNA (dsDNA) donor templates. Here, we present work
demonstrating that improved efficiency in repair by large insertions is
obtained when dsDNA donor templates include novel end-modifications.
These modifications improve the frequency of HDR and abrogate blunt
insertion repair outcomes. Using Alt-R modified dsDNA templates, we
observe more than a 4-fold increase in the ratio of HDR:blunt repair
outcomes as compared to an unmodified dsDNA template. In addition, off-
target editing analysis shows that the repair has improved specificity with
these modifications. Characterizing the integrity of these insertions while
maintaining phasing of the DNA sequence can be achieved with long-read
sequencing. Long-read sequencing technology allows for a more
comprehensive analysis of the outcome of large insertions or deletions
created by CRISPR/Cas9 genome editing. Here, we describe a target
enrichment approach to selectively sequence a region of interest (ROI)
around the CRISPR edited site to measure the rates of precise insertion by
HDR.

121
MODULATING THE ACTIVITY OF CRISPR-CAS WITH CHEMICAL
MODIFICATIONS IN SINGLE-GUIDE RNAs

Daniel Ryan1, David Taussig1, Suhani Thakker1, Israel Steinfeld1, Ben

Lunstad2, Rob Kaiser1, Ryan McCaffrey1, Justin Townsend2, Patrick
Chaffey1, Bo Curry1, Doug Dellinger2, Laurakay Bruhn1
1
Agilent Technologies, Agilent Labs & Genomics, Santa Clara, CA,
2
Agilent Technologies, Agilent Labs, Boulder, CO

As gene editing and control systems move rapidly from research to

therapeutic applications, adjusting the duration of their activity in vitro and
in vivo becomes increasingly important. Leveraging our capability to
robustly chemically synthesize single-guide RNAs and to incorporate
modified nucleotides at any position, we have developed novel approaches
for improving the activity and specificity of CRISPR systems using
chemical modifications in guide RNAs. Previously, we developed a novel
approach for enhancing efficiency of gene editing, especially in primary
cells, by employing site-specific chemical modifications, e.g. 2 -O-methyl-
3 phosphorothioate (MS) modification of the first and last three
internucleotide linkages at both ends of guide RNAs (Hendel et al. Nature
Biotechnology, 2015). We have performed further investigations of
different types of chemical modifications at various positions in single-
guide RNAs to identify configurations that further extend their lifetime in
cells while maintaining strong activity of the CRISPR-Cas system for
binding and cleavage of target DNA sequences. For example, single-guide
RNAs with three MS modifications at both ends (3xMS-3xMS) or three MS
modifications at the 5’ end and two, three or four MP (2 -O-methyl-
3 PACE) modifications at the 3 end (3xMS-2xMP, 3xMS-3xMP, and
3xMS-4xMP, respectively) were transfected into mammalian cells without
Cas9 protein, and these exhibited increasingly longer half-lives across this
series as measured directly by qRT-PCR of the guide RNAs, especially
when larger numbers of MP modifications were added at the 3 end.
Measurements of indel frequencies by deep sequencing of HepG2 cells co-
transfected with these modified single-guide RNAs and Cas9 mRNA
showed order of magnitude increases in editing activity by using guide
RNAs with 3xMS-4xMP modifications compared to the 3xMS-3xMS
modifications. We have also investigated the performance of guide RNAs
including modifications at both ends such as 3xMS-3xMP with additional
modifications placed throughout the guide RNA to further enhance stability
of guide RNAs while maintaining good activity and specificity. These
studies further demonstrate novel configurations of chemical modifications
in guide RNAs that increase and/or modulate the activity of CRISPR-Cas
systems, a capability that may be particularly useful when delivering guide
RNAs into difficult to edit cells or when delivering guide RNAs in vivo.

122
THE CONVERSION OF HUMAN IPSCS INTO MYOCYTES VIA
CRISPR/CAS9 TECHNOLOGY

Joseph C Sanchez1, Emilia A Solomon1, Katie L Davis-Anderson1, Sofiya

N Micheva-Viteva1, Rashi S Iyer2, Scott N Twary1
1
Los Alamos National Laboratory, Bioscience Division, Los Alamos, NM,
2
Los Alamos National Laboratory, Information Systems & Modeling, Los
Alamos, NM

Human induced pluripotent stem cells (iPSCs) have the potential to form
nearly any cell type. Commonly used strategies for the differentiation of
iPSCs into different cell types include the viral-based expression of
transcription factors and/or the addition of growth factors during the
differentiation process; however, the safety concerns of viral transfection
and expense of signaling molecules may hinder downstream applications.
Accordingly, a need remains to develop a cost-effective, reproducible and
versatile strategy to direct the fate of human stem cells. Here, we use
CRISPR/Cas9 technology to aid in the direct reprogramming of iPSCs into
muscle cells. A key step in the direct reprogramming of iPSCs into muscle
cells requires the simultaneous activation of the myoblast determining
protein MYOD1 and inhibition of the pluripotency factor OCT4. We employ
an integration-free approach to alter the gene expression of iPSCs by
combining siRNA-mediated knockdown of OCT4 and transient expression
of mRNA encoding dCas9-VPR. In addition, we test various guide RNAs
(gRNAs) that target different regions of the MYOD1 promoter and identify
the gRNA that converts iPSCs into myocytes with the highest efficiency
based on the differentiated cells morphological appearance and expression
of muscle cell-specific proteins. Furthermore, we perform RNA-seq to
examine the global gene expression profiles of the dCas9-VPR derived
muscle cells and muscle cells created using traditional approaches. We
anticipate that this comparison will reveal any “off-target” effects that
dCas9-VPR might have on the expression of other genes. The ability to
predictably control stem cell differentiation using CRISPR/Cas9 technology
has the potential to aid in the development of novel therapies for tissue and
organ repair, the discovery of novel gene and signaling networks, and
provide useful insight to the molecular mechanisms underlying genetic
diseases.

123
DEVELOPMENT OF A CRISPR/CAS9-BASED THERAPY FOR
HUTCHINSON-GILFORD PROGERIA SYNDROME

Olaya Santiago-Fernández, Fernando G. Osorio, Víctor Quesada, Francisco

Rodríguez, Sammy Basso, Daniel Maeso, Alicia R. Folgueras, José M P.
Freije, Carlos López-Otín

Universidad de Oviedo, Departamento de Bioquímica y Biología

Molecular, Facultad de Medicina, IUOPA, Oviedo, Spain

Hutchinson-Gilford progeria syndrome (HGPS) is a rare disease

characterized by several aging-like manifestations emerging in childhood.
For this reason, it has also been studied as a model of physiological aging.
Its molecular cause lies in a point mutation in the LMNA gene that leads to
the accumulation of a toxic protein called progerin, which is also expressed
during normal aging. Over the last years, different therapeutic approaches
against this syndrome have been tested, although none of them was aimed at
targeting the mutation at the DNA level. In the CRISPR era, new therapies
based on the Cas9 editing system hold an important promise for the
treatment of different genetic diseases. In this regard, HGPS is as a relevant
candidate, as downregulation of this pathogenic protein in progeroid mouse
models improves their health and lifespan.

Here, we explore the efficacy of a CRISPR/Cas9-based approach -both in

vitro and in vivo- for the treatment of HGPS. Thus, the use of an sgRNA
allowed the introduction of frameshift mutations in the LMNA gene that led
to a reduction of progerin accumulation in cells from HGPS patients and
from the LmnaG609G/G609G mouse model of this disease. Moreover, in vivo
delivery of a CRISPR/Cas9 editing system in these mice by adeno-
associated virus (AAVs), caused a reversion of several of their progeroid
alterations as well as an extension of their lifespan. These preclinical
findings suggest that CRISPR/Cas9-based strategies could constitute a
potential treatment for human progeria.

124
MANUFACTURE OF CRISPR RNPs FOR CLINICAL USE

Nicole L Kavanaugh, Samantha Foti, Shawn Shafer

Aldevron, Protein Services, Madison, WI

Aldevron specializes in manufacturing Cas9 proteins, as well as custom

protein development and manufacturing services. Although many vendors
offer research-grade CRISPR/Cas nucleases, these are not applicable to
clinical programs due to construct design and quality grade. While
Aldevron is the only manufacturer to offer GMP-grade Cas9 proteins and
select vendors offer GMP-grade synthetic single guide RNAs (sgRNAs),
there are currently no options for complexing the two as a ribonucleoprotein
(RNP) in a GMP-certified process. In an effort to address this gap in
manufacturing for therapeutic genome editing, Aldevron has created a
method to complex, QC, and characterize RNP complexes under GMP
conditions.

125
CLEAVAGE-RESISTANT TEMPLATES FOR CRISPR-CAS9 EDITING
OF SNVs IN HUMAN STEM CELLS

William C Skarnes1, Nathaniel Roberts2, Nathan Delvaux2, Enrica A

Pellegrino3, Justin A McDonough1, Ashley Jacobi2, Garrett Rettig2
1
Jackson Laboratory for Genomic Medicine, Cellular Engineering,
Farmington, CT, 2Integrated DNA Technologies, Coralville, IA, 3The
Francis Crick Institute, London, United Kingdom

Human diseases are mostly caused by single nucleotide variants (SNVs)

and modeling disease in cultured cells requires efficient modification of one
or both alleles depending on dominant or recessive inheritance. CRISPR-
Cas9 editing of SNVs is particularly challenging given the fact that the
modified allele contains only one mismatch relative to the guide RNA and
is subject to re-cleavage and damage by Cas9 nuclease1. On-target damage
is especially problematic for SNVs positioned outside of the guide RNA
seed region.

The introduction of ‘silent’ or ‘blocking’ mutations in the repair template

(e.g., protospacer adjacent motif (PAM) mutations) can be used to suppress
re-cleavage; however, these additional nucleotide changes do not exist in
nature and may have unintended consequences on gene expression, for
example, by perturbing normal mRNA splicing or altering chromatin
structure in non-coding regulatory sequences. Moreover, PAM mutations
also complicate the editing of revertant alleles which are useful as a control
for any genome instability that may arise during the editing and culture of
cells.

To minimize re-cleavage and subsequent damage of SNV alleles following

HDR, we identified standard DNA modifications of the sugar-phosphate
backbone that render the target site resistant to Cas9 endonuclease
following HDR. We demonstrate that these ‘cleavage-resistant’ repair
templates permit high-efficiency editing of SNV and revertant alleles in
human stem cells and, furthermore, can be used to control the zygosity of
clones by co-delivery of cleavage-resistant wild-type and SNV repair
templates.

1. Skarnes WC, Pellegrino E, McDonough JA. Improving homology-

directed repair efficiency in human stem cells. Methods 2019.

126
A CRISPR/CAS13-BASED APPROACH FOR FUNCTIONAL STUDY
OF RNA MODIFICATIONS

Huong Ta, Jun Li, Iain Searle

Department of Molecular and Biomedical Sciences, The University of

Adelaide, Adelaide 5005, Australia

Recently there has been renewed interest in RNA modifications in animals

and plants. We have been using second generation sequencing to undertake
transcriptome-wide identification of m5C in mice and plants (David et al.,
2017, Li et al., unpublished). To date, the functional importance of RNA
modifications has been often demonstrated by using mutants in “writer”,
“reader” and “eraser” proteins that have global effects. The effect of a
specific modification on an RNA molecule in vivo is far less well
understood. Recently, Cas13 was successfully fused with a deaminase
domain to enable A to I and C to U RNA editing (Cox et al., 2017,
Abudayyeh et al., 2019). We have fused deactivated Cas13 proteins with
various RNA modifying domains to site specifically modify RNA
modifications on mRNAs and long non-coding RNAs in Arabidopsis
thaliana. Our aim is to establish a robust method to understand the function
of RNA modifications on specific RNA molecules in a tissue specific and
temporal manner.

127
CRISPR-CAS9 ANTIMICROBIAL: RESISTANCE REVERSAL
POTENTIAL IN CHALLENGING CONDITIONS

Thaysa L Tagliaferri1,2,4, Natália R Guimarães2, Marcella M Pereira2, Liza F

Vilela3, Hans-Peter Horz*4, Simone G Santos*2, Tiago O Mendes*1
1
Federal University of Viçosa, Department of Biochemistry and Molecular
Biology, Viçosa, Brazil, 2Federal University of Minas Gerais, Department of
Microbiology, Belo Horizonte, Brazil, 3Federal University of Minas Gerais,
Department of Biochemistry and Immunology, Belo Horizonte, Brazil, 4RWTH
Aachen University Hospital, Institute of Medical Microbiology, Aachen,
Germany

*Shared last authorship

Considering that the emergence and spreading of antimicrobial resistance genes

is much faster than the discovery of new antimicrobials, alternative strategies,
such as reversing resistance using CRISPR-Cas9 may have a significant impact
for future clinical applications. Here, we exploited the system further under
challenging conditions: by targeting the blaTEM–1 AMR gene located on the
high-copy plasmid pSB1A2 (100–300 copies/cell) and by directly tackling
blaTEM–1positive clinical isolates.

The gRNA was designed to target a highly conserved region of the blaTEM and
was inserted into a CRISPR-Cas9 vector. After transformation into Escherichia
coli, the functionality of the system was verified by qPCR. The drug-resistance
phenotype was assessed by disk diffusion and bacterial growth behavior in the
presence or absence of beta-lactam. pSB1A2 presence was evaluated via FACS
and qPCR. The effect of CRISPR-Cas9 in clinical isolates was evaluated via
disk-diffusion tests, qPCR, WGS and re-sensitization extension was assessed in
Galleria mellonella.

Upon CRISPR-Cas9 insertion into E. coli harboring pSB1A2, its copy number
and, accordingly, the blaTEM–1 gene expression decreased but did not become
extinct in a subpopulation of CRISPR-Cas9 treated bacteria. Sequence
alterations in blaTEM–1 were observed, likely resulting in a dysfunction of the
gene product. Consequently, a full reversal to an antibiotic sensitive phenotype
was achieved, despite plasmid maintenance.

In a clinical isolate of E. coli, plasmid clearance and simultaneous re-

sensitization to five beta-lactams was possible. Reusability of antibiotics could
be confirmed by rescuing larvae of G. mellonella infected with CRISPR-Cas9-
treated E. coli, as opposed to infection with the unmodified clinical isolate. The
drug sensitivity levels could also be increased in a clinical isolate of
Enterobacter hormaechei and to a lesser extent in Klebsiella variicola, both of
which harbored additional resistance genes affecting beta-lactams. The data
show that targeting drug resistance genes is encouraging even when facing
high-copy plasmids. In clinical isolates, the simultaneous interference with
multiple genes mediating overlapping drug resistance might be the clue for
successful phenotype reversal.

128
DEVELOPMENT AND CHARACTERIZATION OF PIN-POINT BASE
EDITOR: A MODULAR CRISPR–RNA APTAMER-MEDIATED BASE
EDITING SYSTEM

Juan C Collantes1, Victor M Tan1, Huiting Xu1, Amer Alasadi1, Jingjing

Guo1, Hanlin Tao1, Melany Ruiz-Urigüen1, Katarzyna M Tyc2, Tommaso
Selmi3, John J Lambourne3, Jennifer A Harbottle3, Jesse Stombaugh3,
Jinchuan Xing2, Ceri M Wiggins3, Shengkan Jin1
1
Robert Wood Johnson Medical School, Pharmacology, Piscataway, NJ,
2
Rutgers - the State University of New Jersey, Genetics, Piscataway, NJ,
3
Horizon Discovery, R&D, Cambridge, United Kingdom

The recent development of base editing (BE) systems circumvents the

requirements of generating DNA double strand breaks and homologous
directed repair for gene editing, promising broad applications in therapeutic
developments. In the first published BE system, a cytidine deaminase
effector is directly fused to a nuclease deficient Cas9 protein and recruited
to the target DNA sequence. Here, we describe an alternative and modularly
designed base editing system, which we named Pin-pointTM.
Pin-point BE recruits the effector deaminase through the RNA component
of the CRISPR complex. This system contains a chimeric gRNA encoding
an RNA-aptamer at the 3’ end, which in turn recruits its cognate aptamer
ligand fused to a deaminase effector. Using this system, targeted nucleotide
modification was achieved with high precision in mammalian cells. We
applied our system to correct a loss of function mutation, to induce
therapeutically relevant gene knockouts, and to modulate transcription of a
target gene by editing of regulatory elements at promoter sequences. The
optimized Pin-point BEs exhibit high on-target base editing efficiency, low
indel formation rates, and low off-target activity in endogenous loci of
mammalian cells. In addition, with different effector cytidine deaminases,
Pin-point BEs can have different base editing position preferences, thus
broadening the targetable space in the genome. The configuration of Pin-
point BE is modular, each individual component of the system can be
optimized separately, and different orthologous components are inter-
exchangeable by simply re-programing the chimeric gRNA molecule . Our
studies support that Pin-point BE system, with its modular design, compact
transcription units, and variations in base editing position preferences,
offers another platform for developing base editing agents as research tools
and therapeutics.

129
RECODING ESSENTIAL GENES TO REDUCE RESISTANCE TO
CRISPR-BASED GENE DRIVES IN DROSOPHILA MELANOGASTER

Gerard Terradas1,2, Anna B Buchman1,3, Omar S Akbari1,2, Ethan Bier1,2

1
University of California San Diego, Section of Cell and Developmental
Biology, La Jolla, CA, 2University of California San Diego, TATA Institute
for Genetics and Society, La Jolla, CA, 3Verily Life Sciences, South San
Francisco, CA

There is excitement regarding the potential use of CRISPR-based gene

drive systems to control insect pest populations or disease-transmitting
vectors. However, alleles resistant to Cas9 cleavage can interrupt the spread
of the desired genetic element in wild populations. Different strategies have
already been proposed to tackle the issue and suppress the effects of
resistance. We hypothesized that inserted drives into genes essential for
viability or reproduction that carry recoded sequences, restoring function of
the endogenous gene, should be able to overcome the production of
disadvantageous NHEJ events. An important feature of such drives is that
NHEJ induced loss-of-function alleles should be eliminated from the
population in a dominant fashion by lethal/sterile mosaicism. Individuals
inheriting the recoded drive should not suffer from the such effects,
however, unless the targeted locus was haplo-insufficient. Thus, when
perfect allelic copying occurs, the drive-carrying individuals would still
produce a functional protein and hence lethality would only be associated
with non-functional NHEJ alleles.
In this study we use a hackable split gene drive system in Drosophila
melanogaster, where the gRNA is able to home but Cas9 remains static in a
separate locus, to demonstrate how resistance can be overcome if the drive
allele is dependent on a perfect copying event from donor to receiver
chromosome. This split system which enable flexible optimization of drive
performance, can also be hacked to create a full-drive system where Cas9
and gRNA are encoded in the same locus. We evaluated the driving
efficiency of the cargo in a range of autosomal loci by using different
germline-expressed Cas9. This allowed us to detect whether genomic
context (Cas9 promoter, chromosomal location) and sex of the individual
driving the allelic conversion play a role in both gene-drive efficiency and
NHEJ production rate. Due to the fact that our genetic constructs are
inserted in essential genes, we also show that the introduction of a recoded
version of the gene restores its protein function upon successful allelic
conversion. We also performed cage trials with in depth generation-based
phenotypic and genotypic screening that allowed us to pinpoint exact NHEJ
occurrence and fixation. In addition, cage trials demonstrated how the split
gene-drive system could be applied in the field in a safer, yet not as
powerful, manner compared to classic full gene drives. These studies also
shed light on the advantages associated with drives inserted into weakly
haplo-insufficient loci.

130
USING CRISPR-CAS9 GENOME EDITING TO INTRODUCE
NATURALLY OCCURRING DELETIONAL MUTATIONS
ASSOCIATED WITH ELEVATED FOETAL HAEMOGLOBIN AS AN
APPROACH FOR TREATING SICKLE CELL DISEASE AND -
THALASSEMIA

Sarah Topfer, Gabriella Martyn, Kate Quinlan, Merlin Crossley

University of New South Wales, School of Biotechnology and

Biomolecular Sciences, Sydney, Australia

-haemoglobinopathies such as sickle cell disease and -thalassemia are

among the most common genetic disorders in the world and are caused by
mutations in the -globin locus.
It was identified that increased levels of foetal haemoglobin are beneficial
for patients with these disorders, reducing the severity of their symptoms.
Foetal haemoglobin is expressed from about 12 weeks gestation. In most
individuals it is developmentally silenced after the first year following birth
and is replaced by adult haemoglobin which remains the dominant
haemoglobin throughout life. This is known as globin switching. A rare,
benign condition known as hereditary persistence of foetal haemoglobin
(HPFH) prevents the silencing of foetal haemoglobin resulting in expression
into adulthood. The increase in foetal haemoglobin in this condition results
in ameliorated symptoms of -haemoglobinopathies.
While the mechanism underlying many of the point mutations resulting in
HPFH have been explored and characterised, HPFH caused by deletional
mutations has yet to be well understood. We have introduced a number of
naturally occurring deletional HPFH mutations into a cell model via a
CRISPR-Cas9 mediated genome editing approach. Through these cell
models we have investigated whether the deletions substantially increase
HbF levels and explored the mechanisms involved in reversing globin
switching. By developing a better mechanistic understanding of globin
switching using HPFH deletional mutation cell models we aim to explore
reactivation of foetal haemoglobin as a potential therapeutic strategy for
patients with -haemoglobinopathies.

131
SIMPLE ELECTROPORATION FOR EFFICIENT CRISPR/CAS9
GENOME EDITING IN MURINE ZYGOTES

Simon E Tröder1,2, Lena K Ebert1,3, Linus Butt1,3, Laurens Wachsmuth1,5,

Bernhard Schermer1,3,4, Branko Zevnik1,2
1
University of Cologne, Cologne Excellence Cluster for Cellular Stress
Responses in Aging-Associated Diseases (CECAD), Cologne, Germany,
2
University of Cologne, in vivo Research Facility, Medical Faculty,
Cologne, Germany, 3University of Cologne, Department II of Internal
Medicine and Center for Molecular Medicine Cologne (CMMC), Cologne,
Germany, 4University of Cologne, Systems Biology of Aging Cologne
(Sybacol), Cologne, Germany, 5University of Cologne, Institute for
Genetics, Cologne, Germany

Electroporation of zygotes represents a rapid alternative to the elaborate

pronuclear injection procedure for CRISPR/Cas9-mediated genome editing
in mice. However, current protocols for electroporation either require the
investment in specialized electroporators or corrosive pre-treatment of
zygotes which compromises embryo viability. Here, we describe an easily
adaptable approach for the introduction of specific mutations in C57BL/6
mice by electroporation of intact zygotes using a common electroporator
with synthetic CRISPR/Cas9 components and minimal technical
requirement. Direct comparison to conventional pronuclear injection
demonstrates significantly reduced physical damage and thus improved
embryo development with successful genome editing in up to 100% of
living offspring. Hence, our novel approach for Easy Electroporation of
Zygotes (EEZy) allows highly efficient generation of CRISPR/Cas9
transgenic mice while reducing the numbers of animals required.

132
HIGH-THROUGHPUT OFF-TARGET DETECTION SUPPORTS
CLINICAL RESEARCH

Rolf Turk1, Garrett Rettig1, Gavin Kurgan1, Rafet Basar2, May Daher2,
Jenny Shapiro3, Ortal Iancu3, Adi Tovin-Recht3, Matt McNeill1, Mollie
Schubert1, Katy Rezvani2, Ayal Hendel3, Mark Behlke1
1
Integrated DNA Technologies, Functional Genomics, Coralville, IA, 2The
University of Texas MD Anderson Cancer Center, Department of Stem Cell
Transplantation and Cellular Therapy, Houston, TX, 3The Mina and Everard
Goodman Faculty of Life Sciences, Bar-Ilan University, Institute of
Nanotechnology and Advanced Materials, Ramat-Gan, Israel

The CRISPR/Cas9 system demonstrates unparalleled editing efficiency but

suffers from concerns related to target site specificity. Even though high-
fidelity Cas9 mutants have been developed with increased specificity,
interrogation of off-target effects remains necessary for many applications.
Empirical determination of off-target effects (OTEs) continues to be a
compulsory exercise as in silico prediction tools generally suffer from low
specificity or low sensitivity. Here we present a high-throughput variation
on a previously reported, unbiased OTE detection methodology to
empirically interrogate OTEs in a cellular environment. This assay requires
relative low amounts of genomic DNA and is based on enzymatic rather
than mechanical shearing of genomic DNA without loss of sensitivity.
Furthermore, we can more accurately detect OTEs through optimized
alignment strategies. Upon identification of OTEs, multiplexed target
enrichment by amplification followed by next-generation sequencing
(rhAmpSeq™) allows for rapid quantification of OTEs. We applied this
workflow for two systems: 1) silencing the glucocorticoid receptor to render
viral-specific T-cells resistant to the lymphocytotoxic effects of
glucocorticoids, which are commonly used to treat graft-versus-host
disease, and 2) optimizing editing of RAG1/2 loci in HSPCs as mutations in
these genes lead to severe combined immunodeficiency.

133
TARGETED PERTURB-SEQ ENABLES GENOME-SCALE GENETIC
SCREENS IN SINGLE CELLS

Daniel Schraivogel1, Andreas R Gschwind2, Lars Velten3, Lars M

Steinmetz1,2
1
EMBL, Genome Biology, Heidelberg, Germany, 2Stanford University
School of Medicine, Genetics, Stanford, CA, 3CRG, Bioinformatics and
Genomics, Barcelona, Spain

The transcriptome contains rich information on molecular, cellular, and

organismal phenotypes. However, experimental and statistical
considerations limit sensitivity and throughput of genetic screening with
single-cell transcriptomics readout. To overcome these limitations, we
introduce targeted Perturb-seq (TAP-seq), a sensitive, inexpensive, and
platform-independent method focusing single-cell RNA-seq coverage on
genes of interest, thereby increasing the precision and scale of genetic
screens by orders of magnitude. TAP-seq permits routine analysis of 1,000s
of CRISPR-mediated perturbations within a single experiment, detects weak
effects and lowly expressed genes, and decreases sequencing requirements
up to 50-fold. We apply TAP-seq to generate perturbation-based enhancer-
target gene maps for 1,778 enhancers within 2.5% of the human genome.
Thereby, we show that enhancer-target association is jointly determined by
3D contact frequency and epigenetic states, allowing accurate prediction of
enhancer targets throughout the genome. In addition, we demonstrate that
TAP-seq can identify cell subtypes with only 100 sequencing reads per cell.

134
ELIMINATING GENOME-WIDE AND TRANSCRIPTOME-WIDE OFF-
TARGET MUTATIONS BY BASE EDITOR

Lijie Wang*1,2,3, Wei Xue*4, Hongxia Zhang*5,6, Runze Gao*1,2,3, Lina

Zhou1,2,3, Yun-Ni Lei1,4, Jia Wei4, Jing Wu1, Hanhui Ma1, Xingxu Huang1,2,
Bei Yang*7, Hao Yin*5,6, Li Yang*4, Jia Chen*1,2
1
ShanghaiTech University, School of Life Science and Technology,
Shanghai, China, 2Chinese Academy of Sciences, Shanghai Institute of
Biochemistry and Cell Biology, CAS Center for Excellence in Molecular
Cell Science, Shanghai, China, 3University of Chinese Academy of
Sciences, Beijing, China, 4Chinese Academy of Sciences, University of
Chinese Academy of Sciences, CAS Key Laboratory of Computational
Biology, CAS-MPG Partner Institute for Computational Biology, Shanghai
Institute of, Shanghai, China, 5Wuhan University, Zhongnan Hospital of
Wuhan University, Department of Urology, Frontier Science Center for
Immunology and Metabolism Medical Research Institute, Wuhan, China,
6
Wuhan University, Zhongnan Hospital of Wuhan University, Department
of Pathology, Frontier Science Center for Immunology and Metabolism
Medical Research Institute, Wuhan, China, 7ShanghaiTech University,
Shanghai Institute for Advanced Immunochemical Studies, Shanghai, China

Conjugation of CRISPR-Cas9 with cytidine deaminases leads to base

editors (BEs) for programmable C-to-T editing, which holds great potentials
in clinical applications, but suffers from off-target mutations. By taking
advantage of a deoxycytidine deaminase inhibitor (dCDI) domain, a
transformer BE (tBE) system is developed to induce efficient editing with
only background levels of genome-wide and transcriptome-wide off-target
mutations. After being produced, tBE remains inactive at off-target sites
with a cleavable fusion of dCDI, thus eliminating unintended mutations.
Only when binding at on-target sites, tBE is transformed to cleave off the
dCDI domain and catalyzes targeted deamination for precise editing. After
delivery into mice via a dual-AAV system, tBE created a premature stop
codon in Pcsk9 and significantly reduced serum PCSK9 level, which
resulted in ~30% decrease of total cholesterol. Together, the development of
tBE establishes a highly-precise base editing system and its in vivo
efficiency displays potentials for clinical applications.

135
DISSECTING EPIGENETIC AND CHROMATIN INFLUENCES ON
CRISPR/CAS9 GENOME EDITING AND DNA REPAIR.

Trevor Weiss1,2,3, Peter Crisp4, Nathan Springer1,2,3, Feng Zhang1,2,3

1
University of Minnesota, Department of Plant and Microbial Biology,
Minneapolis, MN, 2University of Minnesota, Center for Precision Plant
Genomics, Minneapolis, MN, 3University of Minnesota, Microbial and
Plant Genomics Institute, Minneapolis, MN, 4University of Minnesota,
Microbial and Plant Genomics Institute, Minneapolis, MN

In recent years, newly developed genome editing technologies, such as

CRISPR/Cas9, have allowed for advanced functional genomics and
accelerated translational research. While much work has been performed to
understand the rules governing the efficacy of different CRISPR/Cas
systems in various organisms, little is known about the impact of
chromosomal contexts, such as epigenetic modifications and chromatin
structure, on genome editing and repair outcomes. Previous work suggests
chromatin accessibility may play a more important role than DNA
methylation in determining the CRISPR/Cas9-mediated editing efficiency.
However, these studies investigated only a few genomic sites with different
CRISPR/Cas9 targeted sequences, making it difficult to separate the
epigenetic effects from sequence variation. In this research, we sought to
address this question by examining identical CRISPR/Cas9 target sequences
with distinct epigenetic features located throughout the Arabidopsis thaliana
genome. Our results indicate a combinatory effect of DNA methylation and
chromatin accessibility could lead to significant variations, i.e. 10-100 fold
changes, in editing efficacy and mutation profiles. By tapping into the high
resolution epigenome information and the wealth of epigenetic resources in
Arabidopsis, our study provides insight towards better understanding the
influence of epigenetic and chromatin factors on genome editing, enabling
new strategies for improved editing outcomes at refractory sites.

136
CHARACTERIZING AND IMPROVING THE GENOME-WIDE
SPECIFICITIES OF NEAR-PAMLESS CRISPR-CAS9 VARIANTS

Madelynn N Whittaker1,2, Russell T Walton1,2, Kathleen A Christie1,2,3,

Benjamin P Kleinstiver1,2,3
1
Massachusetts General Hospital, Center for Genomic Medicine, Boston,
MA, 2Massachusetts General Hospital, Department of Pathology, Boston,
MA, 3Harvard Medical School, Department of Pathology, Boston, MA

The safety of CRISPR enzymes is an important consideration for research

and therapeutic applications of genome editing technologies. It has been
demonstrated that wild-type CRISPR-Cas enzymes can interact with off-
target sites that resemble the intended on-target site, leading to undesirable
off-target editing. The extent to which this unwanted activity is observed
and potentially exacerbated when using SpCas9 variants with less stringent
PAM requirements merits investigation. We recently engineered SpCas9 to
relax its canonical requirement for an NGG PAM, leading to variants that
can recognize NGN and NRN>NYN PAMs (named SpG and SpRY,
respectively) (1). Reducing the stringency of the PAM should in principle
increase off target editing, since these new variants can now access nearly
4-8x more target sites compared to wild-type SpCas9. To understand if the
minimal PAM requirements of these engineered variants influenced their
genome-wide specificities, we characterized their propensity for off-target
editing using GUIDE-seq. With SpG and SpRY, we observed an increased
number of off-target sites that were generally attributable to the expanded
targeting ranges of these variants. To mitigate editing at these new off-
target sites, we constructed high-fidelity versions of SpG and SpRY based
on our previous work engineering SpCas9-HF1 (2). Importantly, the HF1
versions of SpG and SpRY retained on-target activity while eliminating
nearly all GUIDE-seq detected off-target sites (with total off-target editing
in any of the HF1 samples not reaching more than 6% of total GUIDE-seq
reads for a given sample). Together, while we demonstrate that the relaxed
PAM requirements of new SpCas9 variants can lead to editing of novel off-
target sites, this phenotype can be suppressed for applications that require
highly specific editing when using SpG-HF1 and SpRY-HF1 derivatives
with improved fidelity. We anticipate that the continued development of
improved enzymes will provide safe and efficacious genome editing
reagents for research and therapeutic use, and we will provide an update on
our recent progress towards this goal.

(1) Walton et al., Science (2020) 368(6488):290-6

(2) Kleinstiver & Pattanayak et al., Nature (2016) 529(7587):490-5

137
CYTOSINE BASE EDITORS WITH MINIMIZED UNGUIDED DNA
AND RNA OFF-TARGET EVENTS AND HIGH ON-TARGET
ACTIVITY

Yi Yu, Thomas C Leete, David A Born, Lauren Young, Luis A Barrera,

Seung-Joo Lee, Holly A Rees, Giuseppe Ciaramella, Nicole M Gaudelli

Beam Therapeutics, Cambridge, MA

Cytosine base editors are molecular machines which enable efficient and
programmable reversion of T•A to C•G point mutations in the human
genome without induction of DNA double strand breaks. Recently, the
foundational base editors ‘BE3’ and ‘BE4max’, containing rAPOBEC1,
were reported to induce low but detectable genome-wide DNA and
transcriptome-wide RNA cytosine deamination. To mitigate CBE-induced
unbiased off-target events, we developed a highly-sensitive, high-
throughput cellular assay to select candidate next-generation cytosine base
editors with reduced spurious off-target editing profiles relative to BE3/4,
whilst maintaining equivalent or superior on-target editing. Here we report a
systematic screen of 153 putative cytidine deaminases with diverse
sequences and identify, highlight, and characterize four core new CBEs.
These next-generation CBEs (BE4-RrA3F, BE4-AmAPOBEC1, BE4-
SsAPOBEC2, BE4-ppAPOBEC1) were further optimized through
structure-guided mutagenesis of the deaminase domain. These next-
generation CBEs display comparable DNA on-target editing frequencies,
whilst eliciting a 12- to 69-fold reduction in C-to-U edits in the
transcriptome, and up to a 45-fold overall reduction in unguided off-target
DNA deamination relative to BE4 containing rAPOBEC1. Further, no
enrichment of genome-wide C•G to T•A edits are observed in mammalian
cells following transfection of mRNA encoding five of these next-
generation editors. Taken together, these next-generation CBEs represent a
collection of base editing tools for applications in which minimized off-
target and high on-target activity are required.

138
MEASURING GERMLINE VARIATION OF HUMAN GENE
ESSENTIALITY BY GENOME-WIDE CRISPR/CAS9 SCREENING IN
INDUCED PLURIPOTENT STEM CELLS

Yan Zhou*, Jing Eugene Kwa*, Felicity Allen, Luca Crepaldi, Elin Madli
Peets, Gemma Turner, Verity Goodwin, Rebecca McRae, Katie Urgo,
Jackie Bryant, Kay Clarke, Adam Hunter, Oliver Dovey, Leopold Parts

Wellcome Genome Campus, Wellcome Sanger Institute, Cambridge,

United Kingdom

* These authors contributed equally to this work

The effects of a gene knockout on phenotype are highly variable across

different yeast strains, as well as cancer cell lines. The extent to which
cellular mutation outcome changes between random individuals in a
population, and the genetic determinants that suppress or enhance the
deleterious effects of an allele are rarely understood. We generated 37
SpCas9-expressing monoclonal induced pluripotent stem cell lines with
high nuclease activity, derived from 23 unrelated healthy individuals from
the HIPSCI project. We then measured the variation of gene essentiality
across all lines by performing pooled CRISPR/Cas9 knockout screens using
a minimized genome-wide library. We established a linear mixed effects
model to estimate the magnitude of variance contributed by biological and
technical sources, and identified screen quality, viral titer, cell line
pluripotency, gender, and time point as main technical confounders. After
accounting for these, we could establish the distribution of broad sense
heritability estimates of gene essentiality, and identified over 1,000 genes
with more than 20% of non-technical variation due to the cell line donor.
Ongoing analyses are evaluating the link of variable essentiality to cell line
properties, such as gene expression and copy number, as well as other
genomic features. Our results illuminate which genes’ requirement for
survival and growth is malleable in principle, and how this relates to
genomic properties of the cells, as well as variation across tumor cell lines.
This is important both for understanding the variable penetrance of
developmental disorder mutations, as well as for separating potential
synthetic lethal interactions in cancer from natural variation in gene
essentiality.

139
STRUCTURE AND MECHANISM OF TWO TYPE III CRISPR
DEFENCE NUCLEASES

Wenlong Zhu*1, Stephen A McMahon*1, Stuart McQuarrie*1, Sabine

Gruschow1, Shirley Graham1, Robert Rambo2, Januka S Athukoralage1,
Tracey M Gloster1, Malcolm F White1
1
University of St Andrews, School of Biology, St Andrews, United
Kingdom, 2Diamond Light Source Ltd, Diamond House, Oxford, United
Kingdom

Type III CRISPR-Cas system is one of the most common CRISPR systems,
providing adaptive immunity against mobile genetic elements in
prokaryotes. On binding target RNA species, multi-protein interference
complex generates cyclic oligoadenylate signalling molecules. Cyclic
oligoadenylate, in turn, binds to and activates downstream effector proteins
via a CRISPR-associated Rossman-fold (CARF) domain leading to non-
specific RNA and DNA cleavage to provide immunity. Here we describe
the structure and mechanism of two novel cOA-activated CRISPR defence
nucleases, Can1 (“CRISPR ancillary nuclease 1”) and Can2 (“CRISPR
ancillary nuclease 2”). Can1 has a unique monomeric structure with two
CARF domains, one nuclease domain and one nuclease-like domain. Can2
forms a symmetric parallel homodimer. Each monomer has one CARF
domain and one nuclease domain. Both crystal structures of the enzymes
have been captured in the activated state, with a cyclic tetra-adenylate (cA4)
molecule bound at the core of Can1 and at the dimer interface of the CARF
domains of Can2 dimer. cA4 binding to the dimeric CARF domains
allosterically stimulates a metal-dependent non-specific nuclease activity
for both proteins. Can1 licenses a metal-dependent DNA nuclease activity
specific for nicking of supercoiled DNA which is predicted to slow down
viral replication kinetics by leading to the collapse of DNA replication
forks. Can2 degrades both supercoiled DNA and ssRNA rapidly which may
result in cell dormancy or death of the infected cells. In addition, we
demonstrate that Can2 confers immunity in a recombinant CRISPR system
in E. coli by interfering with phage infection.

140
TEG-SEQ: A WORKFLOW FOR IN CELLULO MAPPING OF CRISPR
SPECIFICITY

Pei-zhong Tang, Bo Ding, Lansha Peng, Vadim Mozhayskiy, Jason Potter,

Jonathan D Chesnut

ThermoFisher, R&D, Cell Biology, Carlsbad, CA

Engineered nucleases, including the CRISPR/Cas9 system, have been

widely used for genome editing, and is now being developed to create gene
and cell therapies to treat human disease. However, lack of specificity
leading to off-target cleavage is still a concern. To measure this, an in
cellulo method, genome-wide unbiased identification of double stranded
breaks enabled by sequencing (GUIDE-seq) was developed and has been
widely used (Tasi Q et.al). However, this method as originally reported was
associated with a significant level of non-specific target amplification which
reduced sensitivity and increased the cost to detect low-frequency off-target
events. In an attempt to improve robustness and sensitivity, we developed a
modified method termed Target-Enriched GUIDE-seq (TEG-seq). The
modification improves the sensitivity approximately 10 fold compared to
GUIDE-seq. In addition to the increased specificity, we developed high-
throughput workflow and data analysis tool that led TEG-seq to become
more cost-effective. Using TEG-seq, we evaluated a panel of Cas9 mutants
to identify potential high-fidelity Cas9 protein that will be a critical for
genome editing, especially for gene and cell therapy. We also used TEG-seq
to map on- and off-target cleavage events on 22 gRNAs targeting a set of
therapeutically relevant SNPs. Finally, TEG-seq was used to evaluate
CRISPR off-target profiling for therapeutic applications in different cells
including iPSC and CAR-T cells and an animal model. TEG-seq off-target
detection with the use of high-fidelity Cas9 proteins will be one of the
crucial steps in genome-editing and gene therapy.

141
WHOLE-GENOME CRISPR KNOCK-IN ARRAYED LIBRARY
CREATION BY HiUGE

Akiyoshi Uezu1,2, Alan Ma1, Richard Kim1, Maia Clarkin1, Laukik

Nagawekar1, Ed Field1, Scott Soderling1,2,3
1
CasTag Biosciences, Durham, NC, 2Duke University Medical School,
Department of Cell Biology, Durham, NC, 3Duke University Medical
School, Department of Neurobiology, Durham, NC

CasTag Bioscience has developed a CRISPR/Cas9-based endogenous

protein labeling method called Homology-independent Universal Genome
Engineering (HiUGE). This method employs gene-specific guide RNAs
(GS-gRNAs) to create insertion sites in the coding regions of genes of
interest (GOI). These insertion sites serve as knock-in targets for CasTag’s
catalog of application-specific “payloads.” Employing rapidly assembled
GS-gRNAs and a growing number of the catalog of payloads with various
functional moieties, HiUGE is straightforward, scalable, and highly flexible
in terms of protein targeting and applications. CasTag is developing a
whole-genome HiUGE platform comprising GS-gRNAs for all 20,000
human protein-coding genes for various unbiased screening applications.
These GS-gRNAs will be paired with a HiUGE GFP donor to create cell
lines that will serve as a powerful screening repository for phenotypic
analysis. Through prioritizing genes by the Pharos database, MANE
selected transcripts, and gRNA designing tools, CasTag will rapidly
generate GS-gRNA targeting vectors by an automated workflow. These
vectors will be paired with our GFP HiUGE payloads to co-transfect
designated cell lines and screen for proper payload insertion, validate
expression using an automated cell-imaging system.

142
IDENTIFICATION OF MAMMALIAN HOST FACTORS REQUIRED
FOR LEGIONELLA PNEUMOPHILA INFECTION THROUGH A
FUNCTIONAL GENOMIC SCREEN

Devanand D Bondage1, Caroline Esnault2, Ryan Dale2, Matthias Machner1

1
Division of Molecular and Cellular Biology, Eunice Kennedy Shriver
National Institute of Child Health and Human Development, National
Institutes of Health, Bethesda, MD, 2Bioinformatics and Scientific
Programming Core, Eunice Kennedy Shriver National Institute of Child
Health and Human Development, National Institutes of Health, Bethesda,
MD

Legionella pneumophila is a gram-negative bacterial pathogen that can

colonize the human lung after accidental inhalation. While there is
extensive research describing bacterial virulence factors and their
contribution to pathogenesis, little is known about what host factors that are
involved in the infection process. Using the GeCKO CRISPR guide
(g)RNA library, we performed a genome-wide loss-of-function screen for
genes in Raw264.7 mouse macrophages that render cells resistant against L.
pneumophila infection. Upon challenge with GFP-producing flagellin-
deficient L. pneumophila, infected macrophages were incubated for 24 hrs
to enrich for survivors, their gRNA were amplified, sequenced, and
matched to their target genes. We identified 758 candidate genes (identified
in 4 out of 4 experiments at FDR < 0.2) including those previously linked to
L. pneumophila pathogenesis, such as TRAPPC2L (a component of
trafficking protein complex), NCKAP1L (a component of SCAR/WAVE
complex), and SUPT7L (a component of chromatin-modifying multiprotein
complex) as well as several unknown hits. Moving forward, once these
candidates have been validated in a secondary screen, gene deletions will be
constructed for a select set of candidates in the original RAW264.7
monoclone background using CRISPR/Cas9 and/or RNAi methods to
confirm that suppression of L. pneumophila growth was not caused by off-
target effects. The most promising gene candidates or pathway will be
studied in detail (protein biochemistry, fluorescence microscopy, etc.) for
their role during infection and why their disruption attenuates L.
pneumophila intracellular propagation.

143
TECHNICAL ABSTRACTS
FOR WORKSHOPS
T-1
SINGLE CELL CLONING FOR AUTOMATED AND EFFICIENT
CRISPR WORKFLOWS

Cell Microsystems, Inc. Research Triangle Park, North Carolina

Performing efficient single cell cloning workflows is often hindered by poor

cell viability, slow clonal outgrowth, stress-mediated phenotypic selection,
an inability to verify clonality, and overall manual labor input required.
Traditional methods like limiting dilution and more high throughput
technologies such as flow sorting and droplet dispensing technologies are
common approaches used in single cell cloning but none completely solve
these challenges. The CellRaft AIR™ System is a bench-top instrument
capable of sorting single cells into individual microwells, imaging those
cells in the microwells, and then isolating clonal colonies for downstream
applications. The system is based on Cell Microsystems’ core CellRaft
technology which employs a microwell array for culturing and phenotyping
cells using brightfield and/or fluorescent microscopy prior to isolation.
The CellRaft approach for CRISPR workflows provides detailed phenotypic
analysis, higher viability, faster time to clone, and verifiable clonality – all
in a hands-free platform. For especially fragile cells or low efficiency
edited populations, the CellRaft AIR System allows screening of 1000s of
single cells as they recover post-edit and propagate into clones. An
introduction to the CellRaft technology, applications, and comparisons to
traditional methods will be presented.

T-2
Participant List
Dr. Norzehan Abdul-Manan Dr. Matthew Anderson
Vertex Pharmaceuticals Inc. National Cancer Institute
[email protected] [email protected]

Dr. Patricia Abete-Luzi Mr. Joshua Ang Xin De

National Cancer Institute The Pirbright Institute
[email protected] [email protected]

Dr. Brittany Adamson Mr. Jacob Antony

Princeton University Washington State University
[email protected] [email protected]

Mr. Opeoluwa Adewale Fasoro Dr. Mandana Arbab

Johns Hopkins University Broad Institute
[email protected] [email protected]

Ms. Belen Adiego Dr. Michael Aregger

Wageningen University Research University of Toronto
[email protected] [email protected]

Dr. Delasa Aghamirzaie Pol Arranz-Gibert

illumina Yale University
[email protected] [email protected]

Dr. Ali Ahmed Mr. Januka Athukoralage

University of California Davis University of St Andrews
[email protected] [email protected]

Dr. Tomomi Aida Dr. Francesco Aulicino

Massachusetts Institute of Technology University of Bristol
[email protected] [email protected]

Mr. Davide Aiello Dr. Hua Bai

IBF-CNR Iowa State University
[email protected] [email protected]

Mr. Ahmad Al Kawam Dr. Anindya Bandyopadhyay

Blueprint Medicines Reliance Industries Ltd.
[email protected] [email protected]
Ms. Karina Barbosa Guerra Dr. Alex Bortvin
Sanford Burnham Prebys Medical Carnegie Institution for Science
Discovery Institute [email protected]
[email protected]
Dr. Justin Bosch
Dr. Kamal Barley Harvard Medical School
Stony Brook University—SUNY [email protected]
[email protected]
Dr. Chris Boshoff
Dr. Scott Behie NIH
Cell Press [email protected]
[email protected]
Prof. Jimmy Botella
Alisa Bell University of Queensland
Cystic Fibrosis Foundation [email protected]
[email protected]
Mr. Amine Bouchareb
Dr. Erica Bello University of Oxford
Wellcome Sanger Institute [email protected]
[email protected]
Ms. Lexi Bounds
Mr. Rohan Bhattacharya Duke University
Duke University [email protected]
[email protected]
Mr. Tyson Bowen
Dr. Maximilian Billmann LifeEDIT, Inc.
University of Minnesota [email protected]
[email protected]
Dr. Michael Boylan
Mr. Kevin Bishop National Cancer Institute
NIH [email protected]
[email protected]
Dr. Nicole Brackett
Dr. Jonas Blomme Indoor Biotechnologies Inc.
University of Ghent - VIB [email protected]
[email protected]
Dr. Chris Brown
Dr. Annekatrien Boel Metagenomi
Ghent University [email protected]
[email protected]
Dr. Laurakay Bruhn
Dr. Devanand Bondage Agilent Technologies
National Institutes of Health [email protected]
[email protected]
Ms. Michaela Bruntraeger Mr. Julien Capin
Wellcome Sanger Institute University of Bristol
[email protected] [email protected]

David Bryson Dr. Daviel Cardenas

Beam Therapeutics New England Biolabs
[email protected] [email protected]

Ms. Krista Budinich Dr. Antonio Casini

University of Pennsylvania Alia Therapeutics
[email protected] [email protected]

Mr. Mattijs Bulcaen Dr. Kathy Ceballos

KU Leuven Cold Spring Harbor Laboratory
[email protected] [email protected]

Brian Burger Dr. Tomas Cermak

Genus plc Inari Agriculture Inc.
[email protected] [email protected]

Dr. Meg Byrne Mr. Zhuangzhuang Chai

Wellcome Sanger Institute Institute of Genetics and Developmental
[email protected] Biology
[email protected]
Mr. Alan Cabrera
Rice University Ms. Nicola Chan
[email protected] Wellcome Sanger Institute
[email protected]
Dr. Adam Camblin
Beam Therapeutics Mr. Chen Chang
[email protected] Southern University of Science and
Technology
Ms. Diana Campos-Iglesias [email protected]
Universidad de Oviedo
Dr. Rui Chen
[email protected]
Baylor College of Medicine
Mr. Gracian Camps [email protected]
FIMA-Universidad de Navarra
[email protected] Mr. Peter Chen
Harvard University
Ms. Hera Canaj [email protected]
Chan Zuckerberg Biohub
[email protected]
Prof. Jia Chen Prof. Byung-Kwan Cho
ShanghaiTech University KAIST
[email protected] [email protected]

Dr. Janice Chen Dr. Peter Choi

Mammoth Biosciences University of Pennsylvania
[email protected] [email protected]

Ms. Pan Chen Dr. Shaorong Chong

University of Wyoming Arbor Biotechnologies
[email protected] [email protected]

Lo-I Cheng Dr. Amit Choudhary

Beam Therapeutics The Broad Institute
[email protected] [email protected]

Dr. Albert Cheng Dr. Kathleen Christie

The Jackson Laboratory Massachusetts General Hospital
[email protected] [email protected]

Mr. Teri Cheng Dr. Ci Chu

Cold Spring Harbor Laboratory Insitro Inc.
[email protected] [email protected]

Dr. Adam Chernick Dr. Haihua Chu

Integrated DNA Technologies Beam Therapeutics
[email protected] [email protected]

Dr. Jessica Chery Dr. Deanna Church

Food and Drug Administration (FDA) Inscripta, Inc
[email protected] [email protected]

Dr. Hongbo Chi Dr. Alberto Ciccia

St. Jude Children's Research Hospital Columbia University Irving Medical Center
[email protected] [email protected]

Dr. Mengna Chi Dr. Hannah Clark

Cincinnati Children's Hospital Nature Protocols, Springer Nature
[email protected] [email protected]

Dr. Doane Chilcoat Dr. Claire Clelland

Corteva Agriscience UCSF/Gladstone
[email protected] [email protected]
Dr. Kendell Clement Dr. TJ Cradick
Massachusetts General Hospital Excision BioTherapeutics
[email protected] [email protected]

Dr. Ross Cloney Dr. Xiaoxia Cui

Nature Research Washington University in St. Louis
[email protected] [email protected]

Dr. Matthew Coelho Ms. Kaitlin Dailey

Wellcome Sanger Institute North Dakota State University
[email protected] [email protected]

Dr. Bruce Conklin Mr. Eric Danner

Gladstone Institutes Max Delbrück Center
[email protected] [email protected]

Eve Coomber Ms. Crystal Davey

Wellcome Sanger Institute University of Utah
[email protected] [email protected]

Dr. Robert Cooper Dr. Jessica Davis

UC San Diego Sangamo Therapeutics
[email protected] [email protected]

Dr. Sybilla Corbett Dr. Katie Davis-Anderson

The European Bioinformatics Institute Los Alamos National Laboratory
(EMBL-EBI) [email protected]
[email protected]
Mr. David De Vleesschauwer
Prof. Jacob Corn BASF
ETH Zurich [email protected]
[email protected]
Ms. Merve Dede
Dr. Cecilia Cotta-Ramusino The University of Texas MD Anderson
Tessera Therapeutics Cancer Center
[email protected] [email protected]

Dr. Sergio Covarrubias Ms. Fatemeh Dehbandi

BioMarin Phamaceuticals George Mason University
[email protected] [email protected]

Ms. Abigail Cozart Dr. Jennifer DeLeon

St. Jude Children's Research Hospital Cold Spring Harbor Laboratory Press
[email protected] [email protected]
Dr. Ahmet Denli Osama El Demerdash
Cold Spring Harbor Laboratory Press Cold Spring Harbor Laboratory
[email protected] [email protected]

Dr. Aniruddha Deshpande Ms. Elizabeth Epstein

Sanford Burnham Prebys Medical ABS Global, Inc.
Discovery Institute [email protected]
[email protected]
Mr. Niklaus Evitt
Dr. Katelijn D'Halluin Perelman School of Medicine at UPenn
BASF [email protected]
[email protected]
Dr. Alejandra Falla
Dr. Erica DiCesare Vor Biopharma
Stony Brook University [email protected]
[email protected]
Ms. Iana Fedorova
Mr. James Ding Skoltech
University of Manchester [email protected]
[email protected]
Ms. Lynn Fellman
Dr. Augusto Diniz Fellman Studio Inc.
Cold Spring Harbor Laboratory [email protected]
[email protected]
Dr. Christof Fellmann
Mr. David Dooley Gladstone Institutes / UCSF
University of Tennessee Knoxville [email protected]
[email protected]
Mr. Thomas Fernandez
Dr. Jennifer Doudna Beam Therapeutics
University of California, Berkeley / HHMI [email protected]
[email protected]
Dr. Antonio Fernandez Dumont
Dr. Oliver Dovey European Food Safety Authority
Wellcome Sanger Institute [email protected]
[email protected]
Ms. Joana Ferreira da Silva
Mr. Gediminas Drabavicius CeMM, Research Center for Molecular
Vilnius University, Life Sciences Center Medicine
[email protected] [email protected]

Dr. Daniel Durocher Ms. Kathryn Fichter

Lunenfeld-Tananbaum Research Institute Cincinnati Children's Hospital
[email protected] [email protected]
Prof. Ilya Finkelstein Dr. Sara Garcia
University of Texas at Austin Verve Therapeutics
[email protected] [email protected]

Ms. Amber Flores Dr. Jason Gardiner

University of California Davis University of California Los Angeles
[email protected] [email protected]

Mr. David Fraser Ken Gareau

Wellcome Sanger Institute Editas Medicine
[email protected] [email protected]

Ms. Elizabeth Frias Dr. Giedrius Gasiunas

Novartis Institutes for Biomedical Research CasZyme
[email protected] [email protected]

Mr. Lewis Fry Dr. Nicole Gaudelli

University of Oxford Beam Therapeutics
[email protected] [email protected]

Dr. Shinichiro Fuse Dr. Basudev Ghoshal

MPM Capital University of California, Los Angeles
[email protected] [email protected]

Dr. Keith Gagnon Dr. Larry Gilbertson

Southern Illinois University Bayer Crop Science
[email protected] [email protected]

Ms. Danielle Gallagher Mr. Vishruth Girish

Brandeis University Cold Spring Harbor Laboratory
[email protected] [email protected]

Ms. Feng Gao Daniel Gitterman

Bayer Wellcome Sanger Institute
[email protected] [email protected]

Dr. Yudong Gao Dr. Steve Glenn

Duke University Integrated DNA Technologies
[email protected] [email protected]

Dr. Huirong Gao Dr. Tasos Gogakos

Corteva Agriscience MGH
[email protected] [email protected]
Dr. Thomas Gonatopoulos-Pournatzis Ms. Britt Hanson
University of Toronto University of Oxford
[email protected] [email protected]

Dr. Guochun Gong Mr. Jerry Harb

Regeneron Pharmaceuticals Inc CHOC Children's Hospital
[email protected] [email protected]

Dr. Brian Goodman Fred Harbinski

MPM Capital Maze Therapeutics
[email protected] [email protected]

Ms. Rosa Selenia Guerra Resendez Lisa Hardy

Rice University Beam Therapeutics
[email protected] [email protected]

Dr. Qiuyu Guo Dr. Asma Hatoum-Aslan

University of California Los Angeles University of Illinois at Urbana-Champaign
[email protected] [email protected]

Dr. David Gutstein Mr. Julian Havens

Regeneron Pharmaceuticles Cincinnati Children's Hospital
[email protected] [email protected]

Dr. Mary Haak-Frendscho Dr. Matthias Heidenreich

Spotlight Therapeutics, Inc. Vertex Pharmaceuticals
[email protected] [email protected]

Dr. Emma Haapaniemi Mr. Kevin Hemphill

University of Oslo Horizon Discovery
[email protected] [email protected]

Dr. Maximilian Haeussler Dr. Andrew Hempstead

University of California at Santa Cruz Addgene
[email protected] [email protected]

Ms. Viola Halder Jacob Herman

University of Guelph Fred Hutch Cancer Research Center
[email protected] [email protected]

Dr. Chun Han Dr. Marc Hild

Cornell University Novartis Institutes for BioMedical
[email protected] Research, Inc.
[email protected]
Ms. Uyen Linh Ho Dr. Jeffrey Hussmann
Universite de Lausanne UCSF
[email protected] [email protected]

Kara Hoar Mr. Soonkyu Hwang

Beam Therapeutics KAIST
[email protected] [email protected]

Dr. Megan Hochstrasser Dr. Stanislav Indik

University of California, Berkeley University of Veterinary Medicine
[email protected] [email protected]

Dr. Klaus Hoeflich Prof. Walter Isaacson

Blueprint Medicines Tulane University
[email protected] [email protected]

Mr. Jonathan Hsu Prof. Stephen Jackson

Massachusetts Institute of Technology University of Cambridge
[email protected] [email protected]

Dr. Patrick Hsu Dr. Leni Jacob

University of California, Berkeley Blueprint Medicines`
[email protected] [email protected]

Dr. Wenhui Hu Ms. Ashley Jacobi

Temple University School of Medicine Integrated DNA Technologies, Inc.
[email protected] [email protected]

Dr. Yueh-Chiang Hu Dr. Steven Jacobsen

Cincinnati Children's Hospital Medical HHMI/UCLA
Center [email protected]
[email protected]
Dr. Maria Jasin
Dr. Chunling Hu Memorial Sloan Kettering Cancer Center
Mayo Clinic College of Medicine [email protected]
[email protected]
Dr. Kathy Jastrzebski
Dr. Yuhan Huang NKI-AVL
Cold Spring Harbor Laboratory [email protected]
[email protected]
Dr. Marie Javelle
Dr. Avery Hunker LIMAGRAIN
University of Washington [email protected]
[email protected]
Dr. Hari Jayaram Ms. Natalie Kaiser
Spotlight Therapeutics Inc. Michigan State University
[email protected] [email protected]

Dr. Qingzhou Ji Dr. Roarke Kamber

MilliporeSigma Stanford University
[email protected] [email protected]

Prof. Martin Jinek Ms. Shalini Kamu Reddy

University of Zurich Wellcome Sanger Institute
[email protected] [email protected]

Dr. Kasey Jividen Mr. Ariel Kantor

St Jude Children's Research Hospital University of Oxford
[email protected] [email protected]

Mr. Ross Johnson Dr. Bahri Karacay

Benson Hill Integrated DNA Technologies
[email protected] [email protected]

Dr. Stephen Jones Mr. Artur Karasyov

University of Texas at Austin Maze Therapeutics
[email protected] [email protected]

Dr. J. Keith Joung Dr. Alborz Karimzadeh

Massachusetts General Hospital & Harvard Joslin Diabetes Center
Medical School [email protected]
[email protected]
Subhasis Karmakar
Dr. Casey Jowdy National Rice Research Institute
MilliporeSigma [email protected]
[email protected]
Mr. Varun Katta
Dr. Florian Jupe St. Jude Children's Research Hospital
Bayer CropScience [email protected]
[email protected]
Dr. Katharina Kawall
Dr. Bhagyashree Kaduskar Fachstelle Gentechnik und Umwelt
Tata Institute of Genetics UCSD [email protected]
[email protected]
Antwon Kennedy
Dr. Christine Kaiser [email protected]
Merck
[email protected]
Mr. Kangsan Kim Dr. Dmitry Kostyushev
KAIST NMRC for Tb and Infectious Diseases
[email protected] [email protected]

Ms. Woori Kim Ms. Kateryna Kratzer

KAIST Dalhousie University
[email protected] [email protected]

Dr. Jinoh Kim Ms. Sarah Krausz

Iowa State University Research Centre for Natural Sciences
[email protected] [email protected]

Dr. Bill Kim Mr. Jason Kreisberg

Pairwise Plants University of California, San Diego
[email protected] [email protected]

Dr. Ben Kleinstiver Dr. Ralf Kuehn

Massachusetts General Hospital and Max Delbrueck Center for Molecular
Harvard Medical Medicine
[email protected] [email protected]

Ms. Zuzana Kocsisova Ms. Anne-Marie Kuijpers

Benson Hill BASF
[email protected] [email protected]

Ms. Natalia Kolmakova Mr. Manoj Kumar

NIST CSIR-Institute of Genomics & Integrative
[email protected] Biology
[email protected]
Dr. Alexis Komor
University of California, San Diego Dr. Maneesh Kumar
[email protected] Bristol Myers Squibb
[email protected]
Dr. Kazunari Kondo
National Institute of Health Sciences Dr. Sanjay Kumar
[email protected] Thermo Fisher Scientific
[email protected]
Mr. Colin Konishi
New York University School of Medicine Mr. Atsushi Kunii
[email protected] Graduate School of Science, Hiroshima
University
Dr. Erik Koppes [email protected]
University of Pittsburgh
[email protected]
Mr. Stephen Laderman GaHyun Lee
Agilent Technologies St. Jude Children's Research Hospital
[email protected] [email protected]

Ms. Natalie Lai Ms. So Jung Lee

Maze Therapeutics Columbia University Irving Medical Center
[email protected] [email protected]

Mr. Asad Lakhani Matt Lee

Cold Spring Harbor Laboratory Beam Therapeutics
[email protected] [email protected]

Mr. Dieter Lam Mr. Thomas Leete

Beam Therapeutics Beam Therapeutics
[email protected] [email protected]

Dr. Audrone Lapinaite Mr. Mitchell Leibowitz

Arizona State University Dana-Farber Cancer Institute
[email protected] [email protected]

Dr. David Larson Mr. Rodrigo Leite de Oliveira

Benson Hill NKI
[email protected] [email protected]

Dr. Chrysa Latrick Dr. Mathieu Lemaire

Editas Medicine University of Toronto
[email protected] [email protected]

Dr. Cicera Lazzarotto Mr. Walter Lenoir

St Jude Children`s Research Hospital MD Anderson Cancer Center
[email protected] [email protected]

Sophie Leacock Dr. Manuel Leonetti

Wellcome Sanger Institute Chan Zuckerberg Biohub
[email protected] [email protected]

Amanda Lee Ms. Tiffany Leong

University of Pennsylvania University of Texas at Dallas
[email protected] [email protected]

Ms. Andrea Lee Dr. Rachel Levine

St Jude Children's Research Hospital St Jude Children's Research Hospital
[email protected] [email protected]
Dr. Mark Lewandoski Dr. Aaron Lin
National Cancer Institute/NIH Princeton University
[email protected] [email protected]

Dr. Wei Li Dr. Ran Lin

Children's National Medical Center/GWU Rockefeller University
[email protected] [email protected]

Ms. Zhuokun LI Mr. Dexing Lin

Centre for Molecular Medicine Norway Institute of Genetics and Developmental
(NCMM) - UiO Biology
[email protected] [email protected]

Dr. Zhongsen Li Dr. John Lindbo

Kenfeng Seed HM Clause
[email protected] [email protected]

Dr. Wei Li Dr. Samantha Linder

Nature Genetics Newpath Partners
[email protected] [email protected]

Ms. Yan Li Dr. Zachary Lippman

Cold Spring Harbor Laboratory Cold Spring Harbor Laboratory
[email protected] [email protected]

Dr. Hanqin Li Dr. Mathew Littlejohn

University of California, Berkeley Livestock Improvement Corporation
[email protected] [email protected]

Dr. Shengdi Li Dr. Dan Liu

European Molecular Biology Laboratory Baylor College of Medicine
[email protected] [email protected]

Dr. Zheng Li Dr. Lei Liu

UCLA Cold Spring Harbor Laboratory
[email protected] [email protected]

Dr. Zhiqian Li Dr. David Liu

University of California Broad Institute of Harvard and MIT
[email protected] [email protected]

Dr. Xiquan Liang Dr. Junjie Liu

Thermo Fisher Scientific UC Berkeley
[email protected] [email protected]
Ms. Ze Liu Dr. Jens Magnusson
Complete Genomics Stanford University
[email protected] [email protected]

Ms. Reka Lorincz Dr. Barun Mahata

Washington University in St Louis Rice University
[email protected] [email protected]

Ms. Marialucrezia Losito Dr. Barbara Mair

AstraZeneca Boehringer Ingelheim
[email protected] [email protected]

Mr. Benjamin Low Dr. So Makabe

The Jackson Laboratory BEX CO., LTD.
[email protected] [email protected]

Jiamiao Lu Dr. Kira Makarova

AMGEN Inc. National Center for Biotechnology
[email protected] Information/NIH
[email protected]
Dr. Eric Lubeck
insitro, inc. Dr. Yukimasa Makita
[email protected] Takeda Pharmaceutical Company
[email protected]
Ms. Romane Lyautey
Friedrich Miescher Institute for Biomed. Dr. Punam Malik
Research Cincinnati Children's Hospital
[email protected] [email protected]

Dr. Qing Lyu Dr. Nikolay Malinin

Augusta University St Jude Children's Research Hospital
[email protected] [email protected]

Ms. Megumu Mabuchi Dr. Angela Man

New England Biolabs Earlham Institute
[email protected] [email protected]

Prof. Robert MacLaren Dr. Siva Manjunath

University of Oxford Bayer Corporation
[email protected] [email protected]

Dr. Morgan Maeder Dr. Yingwei Mao

Newpath Newco Penn State University
[email protected] [email protected]
Dr. Samantha Maragh Dr. Justin McDonough
NIST -National Institute of Standards & The Jackson Laboratory for Genomic
Technology Medicine
[email protected] [email protected]

Dr. Marcello Maresca Dr. Matthew McNeill

AstraZeneca Integrated DNA Technologies
[email protected] [email protected]

Dr. Maria Martinez Dr. Jasmine McQuerry

Centre de Recherche en Cancerologie. Children's Mercy
[email protected] [email protected]

Catherine Mason Dr. John Means

USDA Children's Mercy Research institute
[email protected] [email protected]

Dr. Caline Matar Dr. Tarang Mehta

Vertex Pharmaceuticals Earlham Institute
[email protected] [email protected]

Dr. Karen Maxwell Dr. Yu Mei

University of Toronto KWS
[email protected] [email protected]

Mr. Sam Mayo Dr. Drew Mendelsohn

Retired CSHL
[email protected] [email protected]

Ms. Emma Mayoux-Andrews Dr. Samir Mendonca

Wellcome Sanger Institute Washington University School of Medicine
[email protected] [email protected]

Dr. Lorenz Mayr Ms. Rina Mepani

ETH Zurich Spotlight Therapeutics
[email protected] [email protected]

Dr. Michelle McClements Dr. Imen Mestiri

University of Oxford Genomic Vision
[email protected] [email protected]

Dr. William McCombie Mr. Frank Meulewaeter

Cold Spring Harbor Laboratory BASF
[email protected] [email protected]
Dr. Joseph Miano Mr. Joshua Modell
Medical College of Georgia at Augusta Johns Hopkins School of Medicine
University [email protected]
[email protected]
Dr. Sina Mohammadi
Dr. Dan Michael Merck & Co.
The Weizmann Institute of Science [email protected]
[email protected]
Dr. Prarthana Mohanraju
Dr. Richard Michelmore Leiden University Medical Centre (LUMC)
University of California, Davis [email protected]
[email protected]
Dr. Sylvain Moineau
Ms. Jessica Miciak Université Laval
Johns Hopkins School of Medicine [email protected]
[email protected]
Dr. Claudio Monetti
Dr. Sierra Miller Bluerock Therapeutics
NIST [email protected]
[email protected]
Dr. Maria Montiel
Dr. Michael Mina Beam Therapeutics
Harvard University, T.H. Chan School of [email protected]
Public Health
[email protected] Dr. Guillermo Montoya
Novo Nordisk Center for Protein Research
Dr. Elena Minones-Moyano [email protected]
Synthego
[email protected] Dr. Elvin Morales
Children's Hospital of Orange County
Gabe Mintier [email protected]
Bristol-Myers Squibb Co
[email protected] Dr. Montserrat Morell
TakaraBio USA
Ms. Maria Miquel
[email protected]
UK Erlangen
[email protected] Dr. Alexander Morgan
Khosla Ventures
Dr. Yuichiro Miyaoka [email protected]
Tokyo Metropolitan Institute of Medical
Science
Mr. Yasuhiro Moriizumi
[email protected]
BEX Co., Ltd.
[email protected]
Dr. Sonia Muliyil Dr. Roberto Nitsch
Cell Press AstraZeneca
[email protected] [email protected]

Dr. Cameron Myhrvold Dr. Seitaro Nomura

Broad Institute of MIT and Harvard The University of Tokyo
University [email protected]
[email protected]
Mr. Mateusz Nowaczyk
Dr. Satya Swathi Nadakuduti The Institute of Bioorganic Chemistry,
University of Florida Polish Acad
[email protected] [email protected]

Mr. Muhammad Naeem Dr. James Nunez

KFUPM Saudia Arabia University of California, San Francisco
[email protected] [email protected]

Dr. Shota Nakade Dr. Osamu Nureki

Massachusetts Institute of Technology University of Tokyo
[email protected] [email protected]

Dr. Kazuki Nakamae Dr. Andre Nussenzweig

Hiroshima University National Institutes of Health (NIH)
[email protected] [email protected]

Ms. Varsha Neelakantan Dr. Lauryl Nutter

Maze Therapeutics The Hospital for Sick Children
[email protected] [email protected]

Mr. Benjamin Newman Dr. Yuki Ogawa

Wellcome Trust Sanger Institute Baylor College of Medicine
[email protected] [email protected]

Mr. Minh Thuan Nguyen Tran Kiera O'Keefe

Menzies Institute for Medical Research St. Jude Childrens Research Hospital
[email protected] [email protected]

Ms. Tabea Nimz Jennifer Oki

Wellcome Sanger Institute Synthego
[email protected] [email protected]

Masataka Nishiga Gabriella Oliveira

Stanford Univeristy Beam Therapeutics
[email protected] [email protected]
 Adrian Oliver Dr. Stamatis Papathanasiou
Duke University Dana Farber Cancer Institute, HMS
[email protected] [email protected]

Carlos Origel Mr. Joon Park

Stanford School of Medicine Forage Genetics International
[email protected] [email protected]

Prof. Keishi Osakabe Dr. Jan Parker-Thornburg

Tokushima University MD Anderson Cancer Center
[email protected] [email protected]

Prof. Yuriko Osakabe Dr. Cindy Park-Windhol

Tokushima University Beam Tx
[email protected] [email protected]

Dr. Melanie Ott Dr. Laura Parrilla-Monge

University of California, San Francisco Stony Brook University
[email protected] [email protected]

Jon Oyer Ms. Victoria Parsons

Pfizer Inc University of North Carolina at Chapel Hill
[email protected] [email protected]

Prof. Mehmet Ozsoz Ms. Simona Patange

Near East University University of Maryland / NIH
[email protected] [email protected]

Dr. Michael Packer Dr. Patrick Pausch

Beam Therapeutics University of California, Berkeley
[email protected] [email protected]

Dr. Patrick Paddison Ms. Caroline Peddle

Fred Hutchinson Cancer Research Center Oxford University
[email protected] [email protected]

Ms. Juan Pan Ms. Oana Pelea

New England Biolabs The University of Oxford
[email protected] [email protected]

Ms. Smriti Pandey Ms. Enrica Pellegrino

Harvard University The Crick Institute
[email protected] [email protected]
Prof. Jerry Pelletier Dr. Gabriele Proetzel
McGill University Takeda
[email protected] [email protected]

Prof. Jason Peters Mr. Niraj Punjya

University of Wisconsin-Madison UC Davis
[email protected] [email protected]

Dr. Karl Petri Dr. Stanley Lei Qi

MGH Stanford University
[email protected] [email protected]

Erin Petruccione Prof. Yiping Qi

University of Oregon University of Maryland, College Park
[email protected] [email protected]

Ms. Rhythm Phutela Dr. Yongjun Qian

CSIR-Institute of Genomics & Integrative Cold Spring Harbor Laboratory
Biology [email protected]
[email protected]
Mr. Jason Qian
Dr. Federica Piccioni Harvard Medical School
Merck [email protected]
[email protected]
Prof. Zhenpeng Qin
Dr. Luca Pinello University of Texas at Dallas
MGH/Harvard/Broad [email protected]
[email protected]
Dr. Kate Quinlan
Dr. Oksana Piven UNSW Sydney
Institute of Molecular Biology and [email protected]
Genetics, NASU
[email protected] Ms. Krishnaveni Ramanan
Beam Therapeutics
Mr. Robert Potter
[email protected]
ThermoFisher Scientific
[email protected] Dr. Alexandros Rampotas
University of Oxford
Mr. Chris Poulin [email protected]
Patterns and Predictions
[email protected] Dr. Apoorva Ravi Shankar
National Institutes of Health
[email protected]
Dr. Tom Ream Ms. Tanja Rothgangl
Bayer UZH Zurich
[email protected] [email protected]

Dr. Holly Rees Dr. Kevin Rozwadowski

Beam Tx Agriculture and Agri-Food Canada
[email protected] [email protected]

Mr. Santiago Restrepo-Castillo Dr. Jacob Rubens

Mayo Clinic Tessera Therapeutics
[email protected] [email protected]

Dr. Garrett Rettig Dr. Daniel Ryan

Integrated DNA Technologies Agilent Technologies
[email protected] [email protected]

Dr. Christopher Richie Dr. Qila Sa

National Institute on Drug Abuse Glaxosmithkline
[email protected] [email protected]

Dr. Christie Rizk Dr. Nathan Sallee

GenomeWeb Maze Therapeutics
[email protected] [email protected]

Dr. Brock Roberts Dr. Joseph Sanchez

Allen Institute for Cell Science Los Alamos National Laboratory
[email protected] [email protected]

Ms. Michelle Roenspies Henry Sanchez

Karlsruhe Institute of Technology Children's Hospital of Philadelphia
[email protected] [email protected]

Ms. Elena Romero Fernandez Ms. Saranya Santhosh Kumar

RCI Regensburg Center for Interventional Children's Hospital of Philadelphia
Immunolog [email protected]
[email protected]
Ms. Olaya Santiago Fernández
Dr. Pamela Ronald University of Oviedo
University of California, Davis [email protected]
[email protected]
Dr. Andrew Santiago-Frangos
Mr. Wesley Rosales Montana State University
Novartis Institutes for Biomedial Research [email protected]
[email protected]
Mr. Shayan Sarkar Dr. Kathy Shair
Stony Brook University University of Pittsburgh
[email protected] [email protected]

Mr. Tomoaki Sasaki Dr. Peng Shang

Vertex Pharmaceuticals Leiden University Medical Centre
[email protected] [email protected]

Dr. Ezra Schildkraut Mamta Sharma

New England Biolabs Wellcome Sanger Institute
[email protected] [email protected]

Mr. Moritz Schlapansky Dr. Tripti Sharma

ETH Zurich UT Southwestern Medical Center
[email protected] [email protected]

Ms. Mary Schmidt Dr. Jason Sheltzer

Abbvie Cold Spring Harbor Laboratory
[email protected] [email protected]

Dr. Bernhard Schmierer Dr. Richard Shen

Karolinska Institutet RS Technology Ventures, LLC.
[email protected] [email protected]

Dr. David Scott Dr. Chenjie Shen

Arbor MIT
[email protected] [email protected]

Dr. Max Sellman Dr. Amir Sherman

Aldevron ARO
[email protected] [email protected]

Dr. Monica Sentmanat Dr. Richard Sherwood

Washington University Brigham and Women's Hospital, Harvard
[email protected] Med School
[email protected]
Dr. Yevgeniy Serebrenik
CHOP Dr. Sumangala Shetty
[email protected] Rutgers-RWJMS
[email protected]
Dr. Shawn Shafer
Aldevron Dr. Peyton Shieh
[email protected] Massachusetts Institute of Technology
[email protected]
Dr. Sung Bong Shin Mr. Guoxu Song
USDA ARS Institute of Biophysics, Chinese Academy
[email protected] of Scienc
[email protected]
Dr. Jay Shockey
USDA Dr. Bicna Song
[email protected] Children's National Medical Center
[email protected]
Mr. Andrew Siaz
NIH Youngzee Song
[email protected] ThermoFisher Scientific
[email protected]
Dr. Frederic Sigoillot
Novartis Institutes for Biomedical Research Dr. Erik Sontheimer
[email protected] UMass Medical School
[email protected]
Mr. Alec Silver
Beam Therapeutics Mr. Alexander Sousa
[email protected] Harvard University
[email protected]
Mr. Brandon Simone
Mayo Clinic Dr. Hans Spiegel
[email protected] KGS-NIH
[email protected]
Dr. William Skarnes
The Jackson Laboratory for Genomic Mr. Simon Sretenovic
Medicine University of Maryland - College Park
[email protected] [email protected]

Mr. Jason Skelton Ms. Martyna Sroka

Wellcome Sanger Institute Cold Spring Harbor Laboratory
[email protected] [email protected]

Laura Smith Mr. Zach Stevenson

AbbVie University of Oregon
[email protected] [email protected]

Mr. Aaron Solomon Dr. J Seth Strattan

Agilent Technologies Two Bear Capital
[email protected] [email protected]

Dr. Sabine Studer

Dana-Farber Cancer Institute
[email protected]
 Jack Sullivan Dr. Lei Tang
Beam Therapeutics Springer Nature
[email protected] [email protected]

Ms. Hong Sun Dr. Yaoliang Tang

Merck & Co. Augusta University
[email protected] [email protected]

Dr. Ming Sun Ms. Sara Tavella

Beam Therapeutics IFOM
[email protected] [email protected]

Dr. Hillary Sussman Ms. Selina Shiqing Teh

Cold Spring Harbor Laboratory Press Johns Hopkins University School of
[email protected] Medicine
[email protected]
Dr. Susanne Swalley
Biogen Dr. Gerard Terradas
[email protected] University of California, San Diego
[email protected]
Ms. Huong Ta
The University of Adelaide Dr. Mark Thomas
[email protected] Wellcome Sanger Institute
[email protected]
Dr. Thaysa Tagliaferri
Federal University of Vicosa Dr. Yee Mon Thu
[email protected] Allegheny College
[email protected]
Dr. Akshay Tambe
Spotlight Therapeutics Ms. Shelby Tonelli
[email protected] Beam Therapeutics
[email protected]
Mr. Kaiwen Tan
China Agricultural University Ms. Sarah Topfer
[email protected] UNSW Sydney
[email protected]
Mr. Victor Tan
Rutgers University Ms. Laura Torella
[email protected] FIMA-Universidad de Navarra
[email protected]
Dr. Pei-zhong Tang
ThermoFisher Prof. Jacques Tremblay
[email protected] Universite Laval
[email protected]
Dr. Simon Troder Dr. Alessandro Umbach
University of Cologne University of Trento
[email protected] [email protected]

Dr. Bruce Tromberg Mr. Dustin Updike

NIH The MDI Biological Laboratory
[email protected] [email protected]

Dr. Yuri Trusov Mr. Tomas Urbaitis

University of Queensland CasZyme
[email protected] [email protected]

Dr. Shengdar Tsai Ms. Jana Urbanova

St. Jude Children's Research Hospital Wellcome Sanger Institute
[email protected] [email protected]

Dr. Stephen Tsang Ms. Salustra Urbin

Columbia University Medical Center Lawrence Livermore Lab
[email protected] [email protected]

Caroline Tsou Dr. Fyodor Urnov

Agilent Technologies Innovative Genomics Institute
[email protected] [email protected]

Dr. Rolf Turk Dr. Michelle Valentine

Integrated DNA Technologies Bayer Crop Science
[email protected] [email protected]

Mr. Josh Tycko Ms. Lilly Van de Venn

Stanford University ETH Zurich
[email protected] [email protected]

Dr. Otoya Ueda Dr. Gaurav Varshney

Chugai Pharmaceutical Co., Ltd. Oklahoma Medical Research Foundation
[email protected] [email protected]

Dr. Akiyoshi Uezu Dr. Lars Velten

CasTag Biosciences Centre for Genomic Regulati9on (CRG)
[email protected] [email protected]

Ms. Esther Uijttewaal Dr. Daniel Verduzco

IMBA/Vienna Biocenter Abbvie
[email protected] [email protected]
Mr. Leo Vo Mr. David Weiss
Columbia University Agilent Technologies
[email protected] [email protected]

Dr. Daniel Voytas Mr. Trevor Weiss

University of Minnesota University of Minnesota
[email protected] [email protected]

Dr. Chris Vulpe Dr. Jonathan Weissman

University of Florida, Gainesville Whitehead Institute and MIT/HHMI
[email protected] [email protected]

Mr. Yiming Wan Ms. Lauren Wensing

Stony Brook University University of Guelph
[email protected] [email protected]

Ms. Lijie Wang Dr. Malcolm White

ShanghaiTech University University of St. Andrews
[email protected] [email protected]

Prof. Yanli Wang Ms. Melissa White

Institute of Biophysics, CAS The University of Adelaide
[email protected] [email protected]

Dr. (James) Jianbin Wang Ms. Laura Whitman

BMS Agilent Technologies
[email protected] [email protected]

Dr. Doreen Ware Madelynn Whittaker

Cold Spring Harbor Laboratory/USDA/ARS Massachusetts General Hospital
[email protected] [email protected]

Mr. Kei Watanabe Dr. Blake Wiedenheft

Kyoto University Montana State University
[email protected] [email protected]

Mr. Tomoyuki Watanabe Dr. Beeke Wienert

Chugai Pharmaceutical CO., Ltd. Integral Medicines
[email protected] [email protected]

Ms. Jingyi Wei Martin Wienisch

Stanford University Massachusetts Institute of Technology
[email protected] [email protected]
Dr. Jonathan Wilde Mr. Jun Yan
Massachusetts Institute of Technology Princeton University
[email protected] [email protected]

Dr. Andy Willaert Dr. Wen-Hsuan Yang

Ghent University Cold Spring Harbor Lab
[email protected] [email protected]

Mr. Daniel Williams Mr. Raymond Yang

Shelter Island High School Beam Therapeutics
[email protected] [email protected]

Dr. Max Wilson Junyun Yang

University of California, Santa Barbara Bayer Crop Science
[email protected] [email protected]

Dr. Hyejung Won Qiuming Yao

University of North Carolina at Chapel Hill University of Nebraska Lincoln
[email protected] [email protected]

Ms. Sharlynn Wu Ms. Tiffany Yee

Macquarie University St. Jude Children's Research Hospital
[email protected] [email protected]

Dr. Lin Wu Zhezhen Yu

Harvard University Cold Spring Harbor Laboratory
[email protected] [email protected]

Dr. Jenny Xiao Yi Yu

Illumina Beam Therapeutics
[email protected] [email protected]

Mr. Ruosen Xie Mr. Bernd Zetsche

University of Wisconsin-Madison Beam Therapeutics
[email protected] [email protected]

Ms. Chenxiao Xue Prof. Feng Zhang

Institute of Genetics and Developmental University of Minnesota
Biology [email protected]
[email protected]
Dr. Feng Zhang
Dr. Satoshi Yamamoto The Broad Institute of MIT and Harvard
Chugai Pharmaceutical Co., Ltd. [email protected]
[email protected]
Dr. Hanrui Zhang Dr. Pei Zhuang
Columbia University Medical Center Stanford University
[email protected] [email protected]

Dr. Sheng Zhang Ms. Norjin Zolboot

University of Texas Health Science Center- The Scripps Research Institute
Houston [email protected]
[email protected]
Yanfei Zou
Qiangge Zhang Thermo Fisher Scientific
MIT [email protected]
[email protected]
Dr. John Zuris
Weijia Zhang Editas Medicine
Francis Crick Institute [email protected]
[email protected]

Ms. Chunying Zhao

Pfizer
[email protected]

Mr. Kevin Zhao

Broad Institute/Harvard University
[email protected]

Dr. Bin Zhao

Stanford University
[email protected]

Dr. zheng zhou

Massachusetts Institute of Technology
[email protected]

Dr. Xingliang Zhou

Kite, A Gilead Company
[email protected]

Mr. Yan Zhou

Wellcome Trust Sanger Institute
[email protected]

Mr. Wenlong Zhu

University of St Andrews
[email protected]