OMICS A Journal of Integrative Biology

Volume 19, Number 5, 2015

Review Article
ª Mary Ann Liebert, Inc.
DOI: 10.1089/omi.2015.0023

CRISPR-Cas9 Based Genome Engineering:

Opportunities in Agri-Food-Nutrition and Healthcare

Subin Raj Cheri Kunnumal Rajendran,1 Yuan-Yeu Yau,2 Dinesh Pandey,1 and Anil Kumar1

Abstract

Recently developed strategies and techniques that make use of the vast amount of genetic information to perform
targeted perturbations in the genome of living organisms are collectively referred to as genome engineering.
The wide array of applications made possible by the use of this technology range from agriculture to healthcare.
This, along with the applications involving basic biological research, has made it a very dynamic and active
field of research. This review focuses on the CRISPR system from its discovery and role in bacterial adaptive
immunity to the most recent developments, and its possible applications in agriculture and modern medicine.

Introduction exogenous genes into the genome of a cell based on homol-

ogy between target sites and donor sequences and has been

T he significance of the post-genomic era lies in un-

raveling the immense amount of available biological
data. Elucidation of genome information and manipulation
used to create knock-out and knock-in mice with the germ
line stem cells, although the frequency of recombination was
very low (1 in 106–9) (Capecchi, 1989). Gene targeting for
of human, plant, and animal genomes hold the key for knockout, modification, or replacement of genes have been
advancement in basic biology, medicine, and agricultural performed in a number of plants and is reviewed extensively
research. Recent developments in genome editing and engi- (Chen and Gao, 2014; Lozano-Juste and Cutler, 2014; Voytas,
neering are revolutionizing biological research; their potential, 2013; Voytas and Gao, 2014).
especially in the field of health care and agriculture, is seen Researchers over 20 years ago demonstrated that induc-
as vast (Carlson et al., 2012; Voytas and Gao, 2014). Also, tion of double-strand breaks (DSBs) in the genome at target
genome editing technology facilitates a reverse genetics-based sites could stimulate genome editing through HR mediated
approach for identification of genomic resources that are repair (Rudin et al., 1989). Programmable sequence-specific
available for a wide range of organisms (Urnov et al., 2010). nucleases can be used to introduce these DSBs, leading to
These technologies provide us an opportunity to manipulate modification at cleavage sites that can be directed or random
genomes and edit genes within living systems. (Bibikova et al., 2001). Cells have two major repair pathways
Genome engineering is a considerably broader term that in response to DSB: non-homologous end joining (NHEJ)
encompasses technologies to make targeted modifications and homology directed repair (HDR) pathways. The HDR
to the genome, its contexts (epigenetic marks) and function repair pathway is activated if a designed donor template is
(transcripts) (Hsu et al., 2014). Besides the applications in available at the DSB. This mediates precise substitution of
functional genomics and understanding organization at a sys- the donor template at the DSB site replacing the earlier se-
tems level, this technology provides a platform to make tar- quence (Bibikova et al., 2001, 2003; Rudin et al., 1989). In
geted mutagenesis and transgene insertion for gene therapy and the absence of template at the site, the DSBs are repaired
for crop and livestock improvement. Thus, genome engineering through the NHEJ which is error prone, mostly resulting in
enables the cell’s DNA to be precisely manipulated for a wide addition or deletion of nucleotides at the site of repair (Bi-
array of applications in the fields of basic biology, agriculture, bikova et al., 2001, 2003; Rudin et al., 1989).
medicine, and biotechnology. Different genome-editing systems have been developed
Genome editing is performed by triggering the cell’s DNA rapidly in the past few years. CRISPR-Cas9 technology is
repair mechanisms. Development of gene targeting through based on the RNA guided endonuclease (RGE) Cas9, which
homologous recombination (HR) was the first major break- can be used to target specific location of choice in the genome
through in genome modification. This mechanism integrates of virtually any organism with the help of a short RNA guide.

1
Department of Molecular Biology and Genetic Engineering, G.B. Pant University of Agriculture and Technology, Pantnagar, U.S.
Nagar, Uttarakhand, India.
2
Department of Natural Sciences, Northeastern State University, Broken Arrow, Oklahoma, USA.

1
2 RAJENDRAN ET AL.

A number of good reviews have already been published de- TALEN specificity is provided by amino acid residues acting
scribing the CRISPR-Cas9 system, its history, and develop- in pairs, each of which binds to a specific nucleotide (Moscou
ment into a powerful molecular tool for genome-engineering and Bogdanove, 2009). TALENs being highly modular are
applications in the fields of applied and basic biology easily designed as compared to ZFN. Systems utilizing ZFN
(Doudna and Charpentier, 2014; Hsu et al., 2014; Mali et al., suffers from context-dependent specificity (Carlson et al.,
2013b; Voytas and Gao, 2014). Here we will elaborate the 2012) (i.e., the design of ZFN must take into consideration
scope, history, mechanism, protocols, bioinformatic tools, various parameters such as the flanking region and epigenetic
applications, and prospects of the CRISPR-Cas9 genome state of the DNA). Both ZFN and TALEN technologies de-
engineering technology in all aspects of biological, agricul- mand elaborate design, assembly, and screening of each in-
tural, and biomedical research. dividual DNA-binding protein for a particular target site (Hsu
et al., 2014).
TALEN and ZFN are both designer nucleases requiring re-
Genome Perturbation: Engineered to Programmable
engineering for each different intended target (Carlson et al.,
Nucleases
2012). However, in CRISPR-Cas9 system, the Cas9 enzymes
As mentioned earlier, DSBs trigger either the NHEJ repair can be specified to an array of targets simply using a group of
resulting in indels (Bibikova et al., 2002) or HDR directing guide RNA (gRNA), which makes the use of CRISPR-Cas9
precise template-dependant substitution at the DSB (Bibi- system easier. In this strategy, only the 20-nt targeting se-
kova et al., 2001, 2003; Hsu et al., 2014; Rudin et al., 1989). quence lying within the guide RNA needs to be changed in
Research in this field is focused on engineering sequence- order to target different genes. This makes the RNA-guided
specific nucleases (SSNs) to cleave at target locations in the CRISPR-Cas9 system relatively robust, affordable, and eas-
genome, generating DSBs that can be manipulated using iest to engineer among the four designer nucleases. The short
these repair pathways. The NHEJ triggered indels are useful guide RNA recognizes the target sequence via Watson-Crick
for generating targeted gene knockouts, whereas the HDR- base pairing.
based repair mechanism can be used to insert a gene, fix or In prokaryotes, the CRISPR-Cas9 system provides a nat-
replace a mutated allele for performing targeted genetic ural defense mechanism against bacteriophages by using the
modification or gene therapy. Thus DSB induced HDR and guide RNAs to direct RNA-dependent Cas9 to corresponding
NHEJ pathways are established as precise mechanisms of phage sequences that are targeted and cleaved. In order to
targeted genome editing in eukaryotes. reprogram Cas9 enzyme to target a specified location in
So far, four types of engineered SSNs have been used for the genome, a sequence of interest can be used to replace the
targeted genetic alternations (Fig. 1). Meganucleases, also phage sequence in the guide RNA. In this way, Cas9 can be
termed homing endonucleases, were the first to be intro- used as a guided missile to target any location in the genome
duced. These are derived from microbial mobile genetic el- through gRNA. Instead of using a number of bulky engi-
ements and recognize long specific sequences ( > 14 nt) for neered proteins such as TALENs or ZFNs for simultaneous
cleavage (Smith et al., 2006). Next, the zinc finger nucleases targeting of multiple sites, a library of gRNA along with a
(ZFN) are obtained by fusing eukaryotic transcription factors single Cas9 can efficiently perform multiplex genome editing
with the cleavage domain of Fok I restriction endonuclease (Cong et al., 2013). A study conducted in maize showed
(Miller et al., 2007; Urnov et al., 2005). The third, tran- that TALEN and Cas9 have similar mutagenesis efficiency
scription activator-like effector nucleases (TALENs) are for intended target sites (Liang et al., 2014). Cas9 and
generated by fusing the Fok I cleavage domain to the binding TALEN are also shown to have comparable efficiency
domain of TALE effector proteins of the plant pathogen for HDR-directed modifications in the Drosophila genome
Xanthomonas (Christian et al., 2010; Miller et al., 2011; (Yu et al., 2014).
Moscou and Bogdanove, 2009). The binding domain binds to
a specific DNA sequence that gets cleaved by the Fok I nu-
Development of the CRISPR-Cas9 System
clease domain. The most recent among the designer nuclease
technology is the RNA-guided CRISPR-Cas9 system (type The CRISPR-Cas9 story began in 1987 with the discovery
II), which is derived from the prokaryotic adaptive immune of a mysterious locus in the E. coli genome adjacent to the iap
system involved in bacterial defense via recognition and gene (Ishino et al., 1987). It was described as a series of direct
cleavage of phage DNA (Hsu et al., 2014; Mali et al., 2013b). repeating DNA sequences (29 nt) intervened by similar sized,
This technology revolves around the Cas9 protein that serves nonrepetitive DNA (32 nt). Further studies and in silico
as a RNA-guided nuclease to cleave a specific DNA sequence. analysis identified these motifs (27–37 nt) in many sequenced
Among the four systems mentioned above, Meganucleases, bacteria (40%) and archaea (90%) (Mojica et al., 2000).
ZFN, and TALENs function through specific DNA–protein These repetitive elements were initially called short regu-
interactions, while CRISPR-Cas9 action is mediated through larly spaced repeats (SRSR), but were later changed to
DNA–RNA–protein interaction. Re-engineering the mega- clustered regularly interspersed short palindromic repeats
nuclease DNA recognition domain has proven to be more (CRISPR) ( Jansen et al., 2002; Mojica et al., 2000). Inter-
difficult compared to those of ZFN and TALENs because it spersed sequences within the repeats called ‘spacers’ are
has integrated DNA binding and nuclease domains, which generally of similar size. A search for the identities of these
lack clarity in terms of causal relationship between the amino CRISPR spacers led researchers to know about their ho-
acid sequence and target DNA specificity (Pauwels et al., mology to extra-chromosomal (conjugative plasmids) and
2014). In ZFN that functions as a dimer, DNA binding is bacteriophage DNA, which is known to be related to spacer-
facilitated by an array of zinc fingers in which each individual containing prokaryote (Bolotin et al., 2005; Mojica et al.,
module specifies a 3-nt target (Voytas, 2013). In contrast, the 2005; Pourcel et al., 2005).
CRISPR-CAS9 BASED GENOME ENGINEERING 3

The phage-resistant bacterial strains expressed the CRISPR production of phage-resistant microbial cultures for dairy
loci (Tang et al., 2002), suggesting its involvement in pro- industry (Quiberoni et al., 2010).
karyotic adaptive immune memory and response (Mojica et al.,
2005). It was observed that phage-resistant strains became sus-
Functional Organization, Diversity, and Activity of Cas9
ceptible upon modification of corresponding spacer, and gained
resistance upon addition of novel spacers to the CRISPR loci The structure and diversity of the CRISPR-Cas9 system
(Barrangou et al., 2007; Datsenko et al., 2012). Genes flanking have already been extensively reviewed (Chylinski et al.,
CRISPRs were identified and shown to be conserved, and were 2014; Hsu et al., 2014; Mali et al., 2013b). The Cas9 enzyme
named CRISPR-associated (cas) genes ( Jansen et al., 2002). has a recognition (REC) and nuclease (NUC) lobe along with
Computational analysis demonstrated genes encoding Cas pro- a PAM interacting (PI) C-terminal domain (CTD) (Nishi-
teins have nuclease, helicase, polymerase, and various DNA and masu et al., 2014). The NUC lobe consists of two nuclease
RNA binding domains ( Jansen et al., 2002). While cas genes subdomains, RuvC and HNH. The noncomplimentary and
are translated into proteins, the CRISPR sequences are tran- complimentary strand of the target are bound and cleaved by
scribed as long RNA molecules that are later processed into RuvC and HNH subdomains, respectively, creating a DSB in
shorter CRISPR RNA (crRNA). the DNA during ‘interference’. If either of the nuclease do-
The mechanism of prokaryotic CRISPR-based acquired mains is dysfunctional, the Cas9 enzymes produce targeted
immunity has been described in three steps: (1) spacer ac- nicks instead of DSBs and such enzymes are referred as
quisition, (2) crRNA processing, and (3) interference (Ma- nickases (Mali et al., 2013a). The a-helix rich REC lobe
karova et al., 2006). In the first step, spacer integration into recognizes and facilitates target binding.
CRISPR locus is mediated by Cas1-Cas2 complex (Nuñez Basically, the Cas9 structure folds and re-organizes itself
et al., 2014). In the incoming foreign phage, Cas1 recognizes around the gRNA in a manner that facilitates target DNA
a unique protospacer adjacent motif (PAM), near to which a binding (Nishimasu et al., 2014). It is inferred that Cas9 ac-
sequence of defined length (protospacer) is excised by it tivity is triggered upon gRNA binding to target DNA forming
(Makarova et al., 2011b). The excised sequence, along with the RNA–DNA duplex. When Cas9 does not bind to gRNA
a single nucleotide of PAM, is integrated into the CRISPR and target DNA, it remains in an auto-inhibited conformation
locus of the host genome and completes the acquisition ( Jinek et al., 2014). It is understood that certain defined re-
(Swarts et al., 2012). gions of the gRNA are necessary for its transferability as well
Based on machinery of crRNA processing and interfer- as activity of Cas9 in different systems (Briner et al., 2014).
ence that are variable in prokaryotes, there are three types PI-CTD binding to PAM is an absolute requirement for en-
of CRISPR systems (reviewed in Chylinski et al., 2014; zyme function. PI-CTD confers PAM specificity and also
Makarova et al., 2011b). crRNA processing and interference governs Cas9 activity through steric hindrance (Nishimasu
in type I and type III CRISPR systems have been shown to et al., 2014). PAM recognition by the PI-CTD is a prereq-
involve multi-ribonucleoprotein complexes such as CAS- uisite for ATP-independent-strand-separation of target DNA,
CADE proteins (type I) and RAMP module (Type III) (Brouns followed by DNA (target)–gRNA heteroduplex formation
2009; Hale et al., 2013). In comparison, type II CRISPR-Cas (Anders et al., 2014).
system is less complex, making it suitable for development Homology-based searches in sequence databases have
into a powerful genome-editing tool. During the second step, identified more than thousand Cas9 nucleases from different
pre-crRNA processing is guided by the binding of trans- bacteria (Chylinski et al., 2014; Makarova et al., 2011b). All
encoded small RNA (tracrRNA) (Deltcheva et al., 2011), of these are exclusively associated with the type II CRISPR
which directs RNase III catalysis, leading to cleavage of system and are further classified as subtypes (IIA–IIC) based
hybridized pre-crRNA-tracrRNA in the presence of Cas9. on the structural organization of the CRISPR locus (Makar-
The cleaved mature crRNA remains associated with tracrR- ova et al., 2011a). Cas9 proteins can also be categorized into
NA and Cas9 (Deltcheva et al., 2011; Xing et al., 2001). The groups on the basis of size (1100, 1350, or1500 amino acids).
spacer sequence in the crRNA makes complementary base Despite all the variations in length, all Cas9 proteins have the
pairing with the target sequence and guides Cas9 (previously same domain architecture and organization [NUC (RuvC and
named as Cas5, Csn1 or Csx12), the nuclease enzyme in HNH domains) and REC nodes along with the PI-CTD].
type II CRISPR, (Garneau et al., 2010). The tracrRNA Variations in size lie within the REC node that is responsible
also hybridizes to crRNA and plays an important role for the association with gRNA and target sequence ( Jinek
in facilitating RNA-guided targeting of Cas9 (Deltcheva et al., 2014). Sequence coding for REC node is poorly con-
et al., 2011). served and it can be easily modified by means of recombi-
The target-cleaving mechanism must also distinguish itself nation and truncation. Such modifications on the REC node
(CRISPR locus in the genome) from non-self (i.e., invading holds promise for re-engineering Cas9 for optimizing pa-
phage). This is possible by PAM, which is required along rameters such as DNA binding, cleavage and protein size
with protospacer for target cleavage (Gasiunas et al., 2012). (Hsu et al., 2014). Unlike the Type I and Type III CRISPR
PAM acts as a targeting component that differentiates self systems which are found in both bacteria and archeae, Type II
from non-self and prevents CRISPR from targeting its own CRISPR system is only found in bacteria (Chylinski et al.,
locus. PAM also dictates the target search mechanism of the 2014; Hsu et al., 2014).
Cas9 enzyme that is elaborated in the later sections. Following Cas9 associates with gRNA or the crRNA-tracrRNA hy-
its discovery and characterization, there arose the possibil- brid forming a ribonucleoprotein complex that initiates the
ity of developing the RNA-guided interference mechanism target search mechanism. The interaction between PI-CTD of
of Cas9 into a programmable genome editing tool. Various Cas9 and PAM sequence at the 3’ flanking region of the target
groups also started to use the natural CRISPR array for site facilitates strand separation via the action of phosphate
4 RAJENDRAN ET AL.

lock loop of the CTD stabilizing unwound DNA (Anders Target Range and Specificity of Cas9
et al., 2014). This indicates that Cas9-gRNA complexes
initially associate with the PAM sequence, thereby initiating The PAM sequence recognition determines a lot of pa-
separation of the target strands for the binding mecha- rameters of the Cas9 activity; these include, self vs. nonself
nism. PAM recognition followed by gRNA complementarily determination, target search mechanism, target strand sepa-
binding to the target sequence creates conformational ration, and the transition between target binding and cleavage
changes in HNH and RuvC domains and activates nuclease conformations (Anders et al., 2014; Gasiunas et al., 2012;
activity (Nishimasu et al., 2014). Hsu et al., 2013; Nishimasu et al., 2014). In addition to these
indispensable functions, PAM sequences are also impor-
tant in determining targeting space of Cas9. For example,
CRISPR-Cas9: Advancement as a Versatile
SpCas9, whose PAM sequence is NGG, allows it to target
Genome Editing Tool
roughly every 8 bp within the human genome (Cong et al.,
Successful interference against exogenous plasmid and 2013; Hsu et al., 2013, 2014). The PAM sequence is specific
phage infection by CRISPR II system following transfer to a particular Cas9 ortholog, as in the case of S. thermo-
from S. thermophilus to E. coli effectively demonstrated the philus, which has two Cas9 homologs, each having a differ-
transferability of Cas9 system for the first time (Sapra- ent PAM sequence specificity (Hsu et al., 2014). Cas9
nauskas et al., 2011). Also, purified Cas9 (derived from S. equipped with additional PAM sequences will expand tar-
thermophilus and S. pyogenes) has been shown to target geting range of the technology. Such additions of new Cas9
desired DNA sequence in vitro (Gasiunas et al., 2012; Jinek homologs are possible either through computer-aided meta-
et al., 2012). The SpCas9 derived from S. pyogenes cuts 3 genomic analysis of prokaryotes (Chylinski et al., 2014) or by
bp upstream of the PAM making a blunt DSB ( Jinek et al., manipulating established PI domains to recognize new PAM
2012). As mentioned earlier, Cas9 activity is dependent sequences (Nishimasu et al., 2014). By manipulating PI-
on crRNA to specify the target sequence and tracrRNA to CTD, one can reduce and modify PAM size to improve
facilitate the cleavage. To reduce complexity, a chimeric flexibility of Cas9 for PAM recognition. Orthogonal genome
short guide RNA (sgRNA or simply gRNA) is constructed engineering is benefited by the delivery Cas9 proteins with
by fusing tracrRNA and crRNA. This chimeric gRNA has different PAM specificity.
been shown to facilitate DNA cleavage in vitro ( Jinek et al., Simultaneous functional manipulations to various loca-
2012). tions within the same host genome are possible with the help
Genome editing was successfully demonstrated in mam- of different Cas9 enzymes with varying PAM specificity.
malian cells using Cas9 from S. thermophilus and S. pyogenes For example, Cas9 enzymes with independent nuclease and
with appropriate gRNA (Cong et al., 2013; Mali et al., transcriptional repression, actively function inside a host
2013b). The gRNA or crRNA-tracrRNA hybrid directed simultaneously performing different functions in different
Cas9 cleavage in the mammalian cell genome to stimulate loci (Esvelt et al., 2013). Cas9 activity in terms of chro-
either NHEJ or HDR-based editing. Cas9 (SpCas9) has al- matin accessibility is still not well defined at this point. The
ready been used for genome editing in a wide variety of functionality of Cas9 in the heterochromatic regions needs
organisms and cell types ranging from bacteria, yeast, zeb- validation. dCas9, the nuclease inactivated form of Cas9,
rafish, roundworm, fruitfly, crop plants, mouse, goat, mon- functions as a fusion protein with effectors such as tran-
key, to human cell lines (Sander and Joung, 2014). The scriptional activators tethered to it. In fact, dCas9 (Cas9
number of experimental organisms in which Cas9-mediated nuclease deficient) fusion protein has been shown to induce
genome editing has been demonstrated is ever increasing. expression from loci found within the inaccessible chro-
The CRISPR system in prokaryotes encodes multiple matin (Hsu et al., 2014; Perez-Pinera et al., 2013). DNA
spacers in crRNA that are processed to act as guides indi- methylation states extend negative influence on TALEN
vidually specifying cleavage of each target. The ability to activity (Valton et al., 2012). However, DNA cleavage by
individually specify target cleavage is the major advantage of Cas9 either in vitro or in vivo is unaffected by methylation
the CRISPR system in genome editing. The Cas9 enzyme can (Hsu et al., 2013, 2014).
be incorporated along with a library of gRNAs to target Off-target cleavage is a major cause of concern especially
multiple locations in the genome. This process is referred to in health applications since the modifications made are per-
as multiplexing and it has been successfully demonstrated in manent. Therefore, Cas9 specificity has been extensively
mammalian and plant [Bread wheat (Triticum aestivum)] tested and analyzed by using a library of mismatched gRNA,
cells with an array of spacers in a battery of gRNA (Cong in vitro selection, and reporter assays (Fu et al., 2013; Hsu
et al., 2013; Mali et al., 2013b; Wang et al., 2014). However, et al., 2013; Pattanayak et al., 2013). The study headed by
it is seen that multiple gRNA stacked in expression cassettes Joung and Sander demonstrated that SpCas9 can tolerate up
exceeds size limitations for cloning, thereby lowering the to five mismatches between gRNA and target DNA for mu-
overall efficiency. A novel strategy to overcome this problem tagenesis in an off-target site (Fu et al., 2013). It is easy to
involves repeats of gRNA and tRNA integrated into a syn- evaluate the influence of mismatches in gRNA on Cas9 ac-
thetic gene. The expressed transcript is recognized by tRNA tivity because the latter is dependent on complementarity
processing enzymes which cleave at the junctions, precisely with target DNA. This evaluation is not possible with ZFNs
releasing the multiple gRNAs that is targeted simultaneously and TALENs because of the difficulties involved in preparing
to multiple sites. The universality of the tRNA processing DNA binding protein libraries having variable specificity for
enzymes in life forms enables this multiplex genome engi- target sequences.
neering technology to be applied to virtually any organism Cas9 specificity studies illustrate that mismatch toler-
(Xie et al., 2015). ance depends on the number, position, and distribution of the
CRISPR-CAS9 BASED GENOME ENGINEERING 5

mismatches (Hsu et al., 2013, 2014; Mali et al., 2013a). et al., 2012; Nishimasu et al., 2014). Properly spaced nicks in
Researchers infer mismatches closer to PAM sequence are the genome can be created by Cas9 nickase specified with a
more significant, and the mismatches farther from PAM pair of gRNA. Such appropriately targeted nicks can dupli-
have lower impact on Cas9 specificity. It has been observed cate the effects of DSB (Mali et al., 2013a; Ran et al., 2013).
that Cas9 temporarily binds to several off-target sites in the Off-target nicks are repaired efficiently by the base excision
genome without cleaving them (Wu et al., 2014). PAM rec- repair machinery in the cell, as these will not have the as-
ognition triggers transient Cas9 binding, but extensive in- sociated, cooperative nick (Dianov and Hübscher 2013; Hsu
teraction between PAM distal sequences and the 5’ end of et al., 2014). However, further investigations are needed to
gRNA is necessary for mediating cleavage (Cencic et al., compare Cas9 nickases and wild-type Cas9 in the activation
2014). Concerns about off-target activity vary depending on of HDR for precise substitution of sequences.
application of Cas9 and tend to be less when Cas9 is intended The second strategy to increase target specificity of Cas9 is
as a sequence specific nuclease. by the use of truncated gRNA (Fu et al., 2014). Binding
Higher levels of concerns arise when Cas9 is used as a affinity is lowered when mismatches exist between target and
targeted DNA binding device, also referred as programmable gRNA, but these are mostly tolerated during off-target
homing device, which sometimes binds at several unspecified cleavage of Cas9 due to excessive binding energy. Truncated
sites throughout the genome. Enzymatic concentration is also gRNA limits excessive binding affinity with target DNA and
an important parameter for off-target activity of Cas9 with reduces Cas9 enzyme’s mismatch tolerance. These strategies
more errors seen at higher concentrations of enzyme. Studies can be combined to reduce off-target editing (Fu et al., 2014).
by Hsu et al. (2013) demonstrated that the specificity in- The third strategy developed to improve cleavage speci-
creased drastically when the concentration of sgRNA and ficity is the use of fCas9, which was generated by fusing
Cas9 used in transfection was reduced from 400 to 10 ng of the Fok I nuclease domain to a catalytically inactive Cas9
Cas9-gRNA plasmid. Similarly, four-fold and seven-fold (Guilinger et al.2014). Two fCas9 monomers are needed
increase in specificity was observed when amounts of plas- to make a DSB, analogous to the functioning of nickases.
mid transfected were reduced from 400 ng to 50 ng and 50 ng Results of this study showed fCas9 has a four-fold higher
to 10 ng, respectively. specificity than paired nickases at loci with similar off-tar-
Three strategies have been devised to improve targeting get sites and > 140-fold higher specificity than wild-type
fidelity of Cas9. The first strategy is to employ nickases Cas9. The gRNA modules necessary for Cas9 activity and
making targeted single stranded breaks in DNA under the orthogonality have been identified recently (Briner et al.,
direction of suitable gRNA. As mentioned earlier, in- 2014), and these findings promote better design of gRNA.
activating either of the nuclease domains (HNH or RuvC) Several tools for target selection and gRNA design along
results in Cas9 becoming a nickase, which makes nicks in- with prediction of off-target sites are already available for a
stead of DSB (Gasiunas et al., 2012; Hsu et al., 2014; Jinek wide range of organisms, and these are described in Table 1.

Table 1. Tools Available for Various Aspects of Design and Use of the CRISPR System
Sl. no. CRISPR tool Application Reference
1. http://crispr.mit.edu gRNA design and off-target (Hsu et al., 2013)
analysis
2. http://arep.med.harvard.edu/CasFinder Human and mouse exome-wide (Aach et al., 2014)
catalogs of specific sites for the
three types of Cas9.
3. https://chopchop.rc.fas.harvard.edu Optimum target site selection tool (Montague et al., 2014)
4. http://zifit.partners.org Identification of potential target (Sander et al., 2010)
sites in DNA sequences.
5. http://tools.flycrispr.molbio.wisc.edu/targetFinder CRISPR target site identification (Gratz et al., 2014)
and off target activity in
Drosophila genome.
6. http://www.e-crisp.org Assessing genomic context and (Heigwer et al., 2014)
gRNA design of target sequence.
7. http://www.genome.arizona.edu/crispr Plant exclusive portal for reagent (Xie et al., 2014a)
suggestion and gRNA design.
8. http://www.rgenome.net/cas-offinder Online and offline tool for (Bae et al., 2014)
identifying potential off target
sites and microhomology based
target sites in a number of
genomes.
9. http://crispr-ga.net Analysis of sequence data to (Güell et al., 2014)
determine the quality of genome
editing experiments.
10. Off target software package gRNA design and off-target (Xie et al., 2014b)
analysis
11. CRISPR-seek software package Optimum target site selection (Zhu et al., 2014)
software package
6 RAJENDRAN ET AL.

The nonprofit plasmid repository, Addgene, facilitates ex- transcriptional activation was demonstrated in HEK293T cells
change of CRISPR plasmids and protocols between labs for by tethering light inducible heterodimerizing proteins to dCas9
scientific and medical research applications (http://www (Light-activated Cas9 effector (LACE)) (Polstein and Gers-
.addgene.org/CRISPR). bach, 2015). Inducible transcriptional activation and genome
editing are accomplished using split-Cas9 fragments. These
Genome Engineering: Expanding the Usage fragments are rendered active via rapamycin binding dimer-
of CRISPR System ization domains, which enable controlled reassembly of Cas9
to mediate transcriptional modulation and genome editing
Wild-type Cas9 or Cas9 nickases can be used to make
(Zetsche et al., 2015).
multiple targeted genome modifications in several organ-
isms. They can be orthogonally multiplexed to probe gene
function (loss-of-function screen) and genetic variations in a Epigenetic modification
large scale (Hsu et al., 2014). Cas9 also mediates knock-in of The epigenetic modifications on the genome tune the state of
genes via the HDR pathway as mentioned earlier. Recently an differentiation and govern the cell fate (Victor et al., 2014).
alternative strategy (CRIS-PITCh) was introduced to knock- Transcription activator like effectors (TALE) bound with TET1
in genes based on microhomology-mediated-end-joining can induce demethylation at the CpG sites, which is also pre-
(MMEJ) (Nakade et al., 2014), which circumvents the dicted to be possible with dCas9 to activate genes repressed by
problems associated with organisms or cells having low HR CpG methylation (Eguchi et al., 2014; Konermann et al., 2013).
efficiency. Another strategy involves the use of small mole- Potentially, artificial epigenetic modulators (Cas9, TALE, and
cules to positively modulate HDR efficiency (three-fold) of ZF) can function independently of cellular states and signals,
Cas9 system (Yu et al., 2015). Cas9 can be developed into a making them promising tools for modifying cell fate (Eguchi
targeted homing device (dCas9) on the genome by in- et al., 2014). Orthogonal genome engineering is functionally
activating its nuclease domains; this technology has been variant manipulation of the genome simultaneously at dif-
named CRISPRi (see below). In addition, the applications of ferent loci within the same cell or in cell population. Ortho-
effectors fused with dCas9 expanded Cas9 use by enabling it gonal epigenetic and transcriptional regulatory systems for
to perform a wide range of genome-engineering applications. simultaneously activating, repressing, or modifying specific
Individual effectors such as transcriptional activators, re- loci in the genome could be made by using multiple Cas9
pressors, and chromatin remodelers can be selectively re- proteins of varying PAM requirements (Hsu et al., 2014).
cruited to a desired location in the genome by attaching them Potential crosstalk between endogenous regulatory elements
with the homing device-dCas9 (Mali et al., 2013b). Applying and effector domains needs to be assessed. Hsu et al. (2014)
different modified forms of Cas9 expands possibilities of suggested the application of prokaryotic epigenetic enzymes
genome engineering that can be defined as targeted pertur- to minimize such crosstalk.
bations to manipulate the structure (sequence), context (epi-
genetics), and function of the genome. Below are extended enChIP and imaging
applications made possible through the modified Cas9:
Specific sequences of DNA can be purified with the help of
Transcriptional modulation dCas9 used with an epitope or affinity-based tag. This puri-
fication process is called enChIP (engineered Chromatin
In CRISPRi, the dCas9 binds to a particular location in the immunoprecipitation) and when used in combination with
genome and represses transcription by hindering RNA mass spectrometry (enChIP-MS) can be used to identify
polymerase activity. This approach is particularly useful and proteins associated with genomic loci (Fujita and Fujii,
effective for repressing expression in prokaryotes, but has 2013). This method has been employed to identify telomere-
been less effective in eukaryotes (Gilbert et al., 2013; Hsu binding molecules with engineered TALE (Fujita et al., 2013).
et al., 2014). In eukaryotes, this is enforced by tethering dCas9 as a homing device when fused with a fluorescent
dCas9 with repressor domains such as SID4X and KRAB, marker like GFP can also be used in imaging. However, tiling
which silence genomic loci through chromatin modification must be done to obtain a detectable signal. Techniques such as
(Gilbert et al., 2013; Konermann et al., 2013). Similarly, in situ hybridization require chemical fixation and cannot be
transcriptional activator domains VP16/VP64 derived from performed in live cells, whereas live cell imaging is possible
viral trans-acting proteins, when fused to dCas9, have been with dCas9 (Chen et al., 2013; Hsu et al., 2014). Additionally,
shown to enhance the expression of targeted genomic loci orthogonal Cas9 proteins tagged with different colored re-
(Hsu et al., 2014; Konermann et al., 2013; Mali et al., 2013b). porters along with multiple gRNA can perform multicolor and
However, it is necessary to utilize several gRNA coinciding multi-locus tagging for nuclear localization and chromosomal
to a single genomic locus to achieve effective transcriptional architectural studies (Hsu et al., 2014).
activation. This process of targeting a single locus with
multiple gRNA is called tiling (Maeder et al., 2013; Perez-
Manipulation of ssDNA and ssRNA
Pinera et al., 2013).
Based on the crystal structure of the Cas9–gRNA–DNA Cas9 has been shown to target, modify, and substitute
tertiary complex (Nishimasu et al., 2014), a recent study single-stranded oligodeoxyribonuceotides (ssoDNA) (Hwang
demonstrated gRNA stem loop 2 and tetraloop tolerates the et al., 2013). In addition, a recent study conducted in the
addition of protein interacting aptamers. It provides a bet- Doudna lab demonstrated successful use of RCas9 to cleave
ter anchoring position for the activation domain which is ssRNA (O’Connell et al., 2014). The ability of RCas9 to
recruited via aptamers, enhancing expression (12-fold) com- modify RNA has the potential to transform the study of
pared to dCas9-VP64 (Konermann et al., 2014). Light-induced RNA function through direct (tag-less) detection, analysis,
CRISPR-CAS9 BASED GENOME ENGINEERING 7

and manipulation of transcripts. This RCas9 system consists approach to disrupt the reverse transcriptase. In doing so, they
of a programmable gRNA-guided Cas9 along with compli- demonstrated transient expression of SSN in mammalian
mentary PAMmer (PAM presenting oligonucleotides), cells through delivery of its mRNA through lentiviral vectors
RCas9 can recognize and bind specific ssRNA corresponding with modified RT (Mock et al., 2014). In order to realize the
to the gRNA (O’Connell et al., 2014). potential of protein-based therapeutics, intracellular delivery of
Thus, Cas9 and its modified versions can dramatically proteins is needed (Zuris et al., 2014). Recently, a novel method
increase the array of tools available for genome engineering. has been developed to directly deliver proteins involved in
It is an easy, reprogrammable and versatile tool for engi- genome engineering (Cre, Cas9, dCas9, and TALEN) into
neering living organisms by editing the genome sequences, cells. These proteins involved in genome engineering are
altering transcriptional states of genomic loci, changing fused to highly anionic supercharged GFP, which are deliv-
chromatin states, and rearranging 3D organization of the ered using cationic liposomes to human cell lines and mice
genome (Hsu et al., 2014). enabling efficient genome engineering (Zuris et al., 2014).
Timed delivery of the Cas9-gRNA ribonucleoprotein com-
plexes in HEK293T cells also resulted in higher efficiency
Delivery and Expression System for CRISPR-Cas9
HDR (Lin et al., 2014).
Generally, the CRISPR-Cas9 system is delivered to In plants, Voytas and colleagues reported the use of
mammalian cells via direct plasmid transfection (Long et al., Gemini Virus Replicons (GVR) for the amenable delivery
2014; Wang et al., 2013). In plants, PEG-mediated protoplast and expression of CRISPR-Cas system or TALEN for ge-
transformation or Agrobacterium-mediated transformation nome engineering. GVR can be combined with other meth-
has been employed ( Jiang et al., 2013). In recent years, ods for delivery of sequence specific nucleases, such as
vectors derived from retrovirus and lentiviruses (a complex T-DNA (Baltes et al., 2014; Voytas and Gao, 2014). Unlike
form of retrovirus) have become essential tools for delivering Agrobacterium-mediated transformation, Gemini virus can
nucleic acid to different cell types. Mechanisms involve the infect monocots and dicots, making it highly versatile as a
reverse transcriptase, providing the ability to retrotranscibe vector and suitable for engineering many crops (Mach,
their RNA genome into cDNA, which viruses insert into host 2014). The CRISPR-Cas system (along with a donor tem-
genomes (Daya and Berns, 2008). Lack of pathogenicity and plate for HDR) can be co-delivered into plant cells tran-
ability to infect both dividing and nondividing cells also siently either as DNA through viral vectors, Agrobacterium,
make Adeno-associated virus (AAV) a safe and frequently particle bombardment (Baltes et al., 2014) or as mRNA and
used viral vector for gene transfer, especially in animal proteins (Voytas and Gao, 2014). Ideally, SSN are tran-
models (Daya and Berns, 2008). Non-integrating viral vec- siently introduced to carry out the desired inherited change
tors are also available for transient delivery and expression in the genome without becoming integrated into the host
(Bobis-Wozowicz et al., 2014). genome and are degraded eventually. Even if integration
A disadvantage of commonly used lentiviral and AAV occurs, the construct can be segregated from the genome by
vectors is an insufficient capacity to contain the entire Cas9 crossing to obtain a transgene free modification in the plant
system (Cas9 with gRNA and other elements such as pro- (Voytas and Gao, 2014).
moters, reporters, and polyadenylation sequences) for de-
livery (Ding et al., 2014; Wu et al., 2010). Viral vectors with
Genome Engineering: Developments to Possibilities
higher-carrying capacity, such as the adenovirus also have
in Agriculture
disadvantages in terms of higher immunogenicity and limited
cell-type specificity (Ding et al., 2014). Cas9 and gRNA The potential of the CRISPR-Cas system in crop im-
expression cassettes in separate plasmids are generally co- provement has yet to be realized in its entirety. Several proof
transfected, although larger capacity pX330 vectors (from of concept studies have demonstrated the possibility of ge-
Feng Zhang lab) (Cong et al., 2013) can be used to accom- nome editing in plants, and most of the published studies
modate both gRNA and Cas9-encoding sequences simulta- describe gene knockouts in various plants. This approach of
neously. The all-in-one vector system containing six gRNA making knockouts is essential for elucidating gene function.
with a nickase-encoding sequence, constructed using Golden The Cas9 system is especially useful as a tool to knock out the
Gate cloning method into a single vector, has been success- diverse array of redundant genes and gene families in plants
fully employed for multiplex genome editing in HEK293T that resulted from gene duplication and polyploidization
cells (Sakuma et al., 2014). In order to overcome the delivery (Voytas and Gao, 2014). Cas9 can be harnessed to target
challenges associated with the large Cas9 protein ( > 4 kb) individual genes or multiplexed for targeting several gene
and for efficiently performing in vivo modeling, Cas9 knock- family members. This versatility enables study of the genetic
in mice expressing Cas9 in a Cre-driven manner, has been basis of many previously untraceable and complex traits in
generated (Platt et al., 2014). It is available as a resource for plants. A handful of model plants and crops have been tar-
in vivo and in vitro genome editing which is specified by the geted with the CRISPR system. These include Arabidopsis
gRNA delivered (Platt et al., 2014). (Li et al., 2013), tobacco (Li et al., 2013), rice (Shan et al.,
Hydrodynamic injection has also been employed for 2013), wheat (Wang et al., 2014), sorghum ( Jiang et al.,
in vivo genome editing in mouse liver (Platt et al., 2014; Yin 2013b), maize (Liang et al., 2014), tomato (Brooks et al.,
et al., 2014). Lentiviral vector delivery is hindered by tem- 2014), and sweet orange ( Jia and Wang, 2014) (Table 2).
plate switch-mediated recombination occurring between the Gene knockouts have significant impact on new trait de-
repetitive elements during reverse transcription in case of velopment, as can be seen from the vast array of commer-
TALEN delivery. To improve the efficient transfer of mRNA cialized natural and induced mutant genotypes that have
using lentiviral vectors, Mock et al. (2014) proceeded with an changed the agricultural landscape as a whole (Ahloowalia
8 RAJENDRAN ET AL.

Table 2. Highlights of CRISPR Based Genome Editing in Various Organisms

Species Delivery Application of Cas9 Reference
Pseudorabies Transfection 100% viral gene disrupting efficiency in (Xu et al., 2015)
virus (PRV) PK15 cell line (Poor transfection
efficiency)
E. coli Bacteriophage Removal of antibiotic resistance and (Citorik et al., 2014)
(FRGN) virulence determinants
Plasmodium Plasmid Targeted knockout of genes encoding native (Wagner et al., 2014)
falciparum transfection knob-associated histidine-rich protein
(kahrp) and erythrocyte binding antigen
Xenopus tropicalis Co-injection Multiplex genome engineering in embryos (Guo et al., 2014)
of gRNA and
mRNA of Cas9
Drosophila Co-injection of Method for rapid production of targeted (Bassett and Liu, 2014)
embryos mutants in Drosophila.
Bombyx mori Transfection ku70 knockout which enabled higher (Ma et al., 2014)
frequency of HR.
Zebrafish Co-injection Gal4 reporter knock-in transgenic fish at four (Kimura et al., 2014)
of RNA and different genomic loci
donor plasmid
Mouse AAV, Lentiviral In vivo and ex vivo editing of neurons, (Platt et al., 2014)
and particle immune cells and endothelial cells in Cas9
mediated knockin mouse. Modeling of Lung
delivery adenocarcinoma
of gRNA
Lentiviral vector Cataloguing somatic mutations to oncogenes (Sánchez-Rivera
and tumor suppressor genes, relating them et al., 2014)
to tumorigenesis.
Co-injection of Cataract causing dominant mutation in the (Wu et al., 2013)
RNA to zygote Crygc gene corrected.
AAV vector In vivo editing of brain cells, genes Mecp2, (Wang et al., 2013)
Dnmt1, Dnmt3a, Dnmt3b targeted.
Hydrodynamic Wild type Fah proteins made in mice having (Long et al., 2014)
injection tyrosinemic mutation through gene
editing.
Plasmid Muscular dystrophy model mice corrected (Yin et al., 2014)
transfection of DMD mutation.
Cationic Inner ear hair population modified (Zuris et al., 2014)
liposomes
Goat Transfection Live born goats harboring biallelic mutations (Ni et al., 2014)
resulting from somatic cell nuclear
transfer of genome edited fibroblasts.
Sheep, The NucleofectorTM Sex reversed sheep through genetic (Fan et al., 2014)
Tibetian Argali transfection inactivation of Sry gene in cell lines which
(Ovis ammonhodgsoni) can be used in the future for somatic cell
and Romney nuclear transfer.
(Ovis aries)
Pig Transfection Model for studying colon cancer and for (Tan et al., 2013)
Cattle development of therapeutics.
Model for studies in restoring human
fertility.
Monkey(Macaca Microinjection Simultaneous targeting of multiple genes in (Niu et al., 2014)
fascicularis) monkey embryos.
Human Transfection CCR5 and B2M gene disruption in human (Mandal et al., 2014)
hematopoietic stem and effector cells has
clinical potential against HIV.
Lipofectamine Repair of CFTR locus in intestinal stem cells (Schwank et al., 2013)
mediated restores clonally expanded organoids
transfection Patient derived fibroblasts used as model to (Osborn et al., 2014b)
correct Fanconi Anemia
Wheat Biolistic Targeted knockout of mloalleles using Cas9 (Wang et al., 2014)
transformation and TALEN
(continued)
CRISPR-CAS9 BASED GENOME ENGINEERING 9

Table 2. (Continued)
Species Delivery Application of Cas9 Reference
Rice Agrobacterium bel mutants with knocked out herbicide (Xu et al., 2014)
mediated tolerance gene used for production of
transformation hybrids.
Protoplast Targeting bacterial blight susceptibility ( Jiang et al., 2013)
transformation locus (promoter of OsSWEET14 and
OsSWEET11)
Sorghum Agrobacterium Immature embryo transformation with Cas9 ( Jiang et al., 2013)
mediated and gRNA
transformation
Maize Protoplast Comparable genome editing efficiencies of (Liang et al., 2014)
transformation Cas9 and TALEN
Tomato Agrobacterium Recessive mutations targeted that would (Brooks et al., 2014)
mediated result in needle-like and wiry leaves that
transformation are distinct.
Sweet Orange Agroinfiltration Promising for studying gene functions in ( Jia and Wang, 2014)
citrus

et al., 2004). Application of SSN for targeted gene knockout

is a great improvement over the random process of chemi-
cally induced nonspecific mutations. Before SSN, RNAi
technology has long been employed to knock down targeted
genes in a spatial and temporal manner to study gene func-
tion. However, sustained expression of the RNAi construct is
needed for disrupting gene function in a particular tissue,
making it undesirable in the agriculture setting (Voytas and
Gao, 2014). Another advantage of SSN over RNAi technol-
ogy is that it generates null allele while low level of tran-
scripts of the gene-of-interest still can be present in case of
RNAi, which can confound the interpretation of data.
The CRISPR-Cas9 system offers several advantages over
RNAi, because it targets duplicated genes in the genome,
modifying several alleles. For example, powdery mildew
disease is one of the most destructive wheat diseases. It
causes up to 40% damage during severe infection with op-
timal environmental conditions (Gil-Humanes and Voytas,
2014). Powdery mildew is caused by the fungus Blumeria
graminis f. sp. tritici. Recently, the Gao–Qiu groups have
used Cas9 technology to target and mutate three mildew-
resistance locus (MLO) homoeoalleles in bread wheat (Tri-
ticum aestivum L.), a hexaploid crop (Wang et al., 2014).
Self-fertilized T0 plants resulted in progeny with all the six
alleles knocked out, which conferred resistance to the pow-
dery mildew disease. Broad spectrum resistance to powdery
mildew can be incorporated into both monocot and dicot
crops through this approach, which otherwise depends on
heavy use of fungicides (Gil-Humanes and Voytas, 2014). A
similar strategy using Cas9 has been demonstrated to increase FIG. 1. The four major categories of sequence specific
rice resistance to Xanthomonas oryzae by disrupting the locus nucleases: (a) Meganucleases have the base recognition and
where pathogen effectors bind, thereby reducing pathogen nuclease domains fused together; (b) Zinc finger nucleases
virulence ( Jiang et al., 2013). acting as a dimer for making DSB, a monomer consists of
Selectable marker genes (SMG) are used to select genetic three zinc fingers, each making base specific interactions.
transformants, preferentially during transgenesis. SMGs en- The Fok I nuclease domain is tethered to the array; (c)
code proteins to help transformants detoxify antibiotics or TALE array consists of repeat variable di-residues that can
make base specific contact with the target DNA sequence. It
herbicides during selection. However, presence of SMG in also has Fok I endonuclease domain fused to it. (d) The
transgenic foods and feed products is a major concern in Cas9 endonuclease is guided by the chimeric gRNA to
GMO production. Regulatory agencies and consumers are target sequence based on complementary base pairing and
concerned regarding SMG that may negatively impact the the appropriate PAM sequence. Adapted from previous
environment and affect the health of humans and animals. It publications (Mali et al., 2013; Voytas, 2013).
10 RAJENDRAN ET AL.

is desirable to eliminate the SMGs after their use. SSNs such providing the ability to target insertion of transgene to desired
as ZFNs have been used in gene deletion (Yau and Stewart locations within the genome. This breakthrough capacity
2013), and a similar approach can be used for SMG deletion results in uniform transgenesis events by directing transgenes
using CRISPR-Cas9. to ‘safe spots’ that avoid integration into essential loci which
Although the use of HDR-mediated genome editing can disrupt important gene functions. These suitable regions
through CRISPR-Cas9 to improve crop quality is still at in genomes, denoted as safe harbor loci, have already been
its infancy, the technology is especially promising in the investigated and identified in rice (Cantos et al., 2014).
area of developing herbicide tolerance in crops. The en- In 1995, the bel (bentazon sensitive lethal) mutation in rice
zyme involved in biosynthesis of aromatic amino acids, was first created through radiation mutagenesis (350 Rad
5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), is Co60-c-ray). The bentazon-lethal rice mutants are susceptible
inhibited by the herbicide, glyphosate. Similarly, acetolactate to bentazon and sulfonylureas herbicides. These mutants are
synthase (ALS) involved in branched chain amino acid bio- used for production of hybrid rice, to prevent contamination
synthesis is also a target for several herbicides (imidazoli- of selfed seeds within hybrid seed lot. Spraying specific
nones and sulfonylureas). Modifying the domains in the plant herbicide during the seedling stage will eliminate progeny
enzymes EPSPS and ALS, which interact with correspond- from selfed plants. Xu et al. used CRISPR-Cas9 technology
ing herbicides, would confer herbicide tolerance in the plant to knockout the BEL gene through Agrobacterium- mediated
(Voytas and Gao, 2014). A proof of concept study with ZFN transformation in rice with mutagenesis efficiency between
has successfully demonstrated modification in the ALS gene 2% and 16%. The bi-allelic BEL knock-out lines showed
resulting in plant herbicide tolerance (Townsend et al., 2009). sensitivity to bentazon (Xu et al., 2014).
Computational methods predict precise amino acid substitu- SSNs can also be an essential tool for biofortification. Trait
tion within the glutathione S-transferase (GST) would direct development for nutritional enhancement follows two ap-
its activity against a diverse set of herbicides (Govindarajan proaches: (1) Enhancing the amount of nutrients in the
et al., 2015). Although bacterial transgenes have been used foodstuff, and (2) eliminating the anti-nutrients that reduces
in commercially available herbicide tolerant crops, the use of the food quality or nutrient bioavailability. In plants, major
genome engineering would provide the distinct advantage of essential amino acids such as lysine, methionine, and tryp-
circumventing the use of transgene. Transiently expressed tophan have their own biosynthetic pathways, which are
Cas9 system can be employed to generate genome engineered regulated by feedback inhibited enzymes. Transgenic crops
crops having such precise amino acid substitutions. with increased essential amino acid content are generated by
Orthologous multiplex genome engineering platform of the incorporating bacterial biosynthetic enzymes that are free of
CRISPR system is a boon for plant biologists studying meta- feedback inhibition (Ufaz and Galili, 2008). GM crops de-
bolic pathways, transport, and polygenic traits. It has been veloped by this method must have to undergo the extensive
suggested that the multiplex genome editing approach holds regulations, which reduce their commercial viability. Gen-
promise in efforts to increase understanding of the mechanisms ome editing holds promise in this regard for metabolic en-
of auxin transport in plants (Balzan et al., 2014). Chemically- gineering of essential amino acid either by (1) modifying
induced mutations in the florigen-associated plant flowering domains responsible for regulating feedback inhibition, or by
pathway offers the possibility of customizing plant architec- (2) knocking out enzymes involved in catabolism of specific
ture that translates to enhanced yield, as can be seen in the amino acids, thereby increasing amino acid content (Ufaz and
study demonstrating improvement in productivity of to- Galili, 2008).
mato (Park et al., 2014). Another possibility of the use of Eliminating anti-nutrients enhances bioavailability of nu-
SSN is in the directed mutagenesis of the genes in the trients for absorption into our system. Phytic acid is a key
florigen pathway pinpointing an optimal balance of florigen anti-nutrient that forms strong complexes with minerals such
phytohormone. This has immense promise in terms of crop as iron, making it unavailable for absorption. Biosynthetic
yield enhancement. enzymes involved in phytic acid synthesis have been targeted
Most genotypes of rice exhibit complete resistance to rust, with various approaches for increasing nutritional quality.
directing non-host resistance to rust pathogens (Ayliffe et al., Maize lines knocked out for the gene 1,3,4,5,6-pentaki-
2011). Identification and transfer of these resistance (R) sphosphate 2-kinase (IPKI) coding for an enzyme involved
genes to other cereal crops is a very promising area of re- with the final step of phytate biosynthesis, has been generated
search. Analyzing and mining data concerned with seed ge- through ZFN mutagenesis (Shukla et al., 2009). A similar
nomics is considered a herculean task for plant biologists. approach could be followed for increasing the bioavailable
Multiplex genome engineering is predicted to play an im- calcium in plants by the reduction of oxalate biosynthesis.
portant role in the future for dissecting the various regulatory Cyanogenic glucosides, linamarin and lotaustralin in tapi-
networks involved in seed development (Becker et al., 2014). oca can be reduced by suppression of their biosynthetic
Similar approaches can be directed at elucidating the diverse genes ( Jørgensen et al., 2005). Similarly, gliadins could be
plant signaling and response cascades involved with abiotic knocked-out with CRISPR-Cas9 system, as a strategy for
and biotic stresses, which can be modulated by genome en- developing gluten-free diet for celiac disease patients (Gil-
gineering for stress management, toughening up crop plants Humanes et al., 2014).
against a wide array of stresses. Cas9-based genome editing has already been successfully
In conventional plant transformation, each transgenic employed in goat (Ni et al., 2014), sheep (Fan et al., 2014),
event results from a random and unpredictable insertion of a pig (Tan et al., 2013), and cattle (Tan et al., 2013) (Table 2).
transgene into the genome. As a result, each event is pre- There is promise for improving livestock quality in the future
disposed to independent evaluation of performance. HDR by the use of this technology. Biofortification of milk through
mediated by Cas9 gives transgenic technology a new edge by the knockout of allergenic gene such as b-lactoglobulin in
CRISPR-CAS9 BASED GENOME ENGINEERING 11

genome-engineered dairy animals might be possible in the ders such as retinitis pigmentosum and transthyretin-related
future. Although distant, potential of this technology for hereditary amyloidosis (Hsu et al., 2014). Variations in the
improving the quality of agricultural produce as well as non-genic (enhancer) regions have been shown to underlie
livestock remains massive. autoimmune diseases, making DNA manipulations through
Use of Cas9 sequence specific nucleases is especially ex- genome editing a promising therapeutic intervention for their
citing in the field of agriculture because these targeted mitigation (Farh et al., 2014).
modifications in crops from transient delivery of SSN are Besides repairing disorder associated genes, genome
indistinguishable from naturally occurring mutants or those editing-based regenerative medicine can also be used to
generated from chemical and physical mutagenesis (Voytas protect individuals from disease risk by disrupting certain
and Gao, 2014). Transient delivery of SSNs through genes. Proof of concept studies in mice has shown that dis-
agroinfiltration, viral vectors, or physical methods such as rupting the Pcsk9 gene (PCSK9 is an antagonist to the LDL
biolistics, may also be employed. NHEJ-induced knockouts receptor) in vivo (liver) has therapeutic promise against car-
of genes are indistinguishable from chemical or physical diovascular disease in humans (Ding et al., 2014). Successful
mutagenesis, resulting in nontransgenic-like plants contain- clinical trials against HIV infection have been reported
ing stably inherited desired sequence changes ( Jiang et al., through the use of SSNs (ZFN) for disruption of the CCR5
2014). This potential will necessitate a different regulatory encoding gene required by HIV-1 for entry into host. Viral
framework for covering genome engineered crops. The infection is crippled because of disrupted receptor activity in
USDA has discreetly stated that genome modified crops with the CD4 + cells. These results were derived from genome
ZFN fall outside their regulatory authority. Dow Agros- edited stem cells that were transplanted into patients. Similar
ciences were assured by the USDA of not requiring regula- results have been obtained in HIV-inoculated cell cultures as
tory oversight for the corn developed through ZFN based well (Tebas et al., 2014; Ye et al., 2014). Engrafting Cas9
genome editing (Waltz, 2012). However, HDR-mediated modified CCR5 - human hematopoietic stem and progenitor
modification through engineered nucleases in plants is con- cells is a promising approach in combating AIDS (Mandal
sidered as GMOs because the nucleic acids are delivered and et al., 2014).
incorporated into the genome. The HDR modifications would Targeted homing devices for manipulation of transcrip-
come under the same category as GMOs for biosafety con- tional networks and epigenetic landscapes is more favorable
siderations in countries that adhere to process-based bio- for regulating cell fate than naturally occurring activators,
safety regulation. since targeted homing devices are free from endogenous
regulatory and signaling elements (Eguchi et al., 2014).
Current strategies employ generating iPS from patient-
CRISPR-Cas9: Biomedical Advancements
derived adult cells and differentiating them into cell type of
and Opportunities
interest using regulatory elements like miRNA and TFs
Genome editing enables the rapid generation of cellular (Chanda et al., 2014; Victor et al., 2014). The advent of tar-
and animal models. Disease mutations are studied by using geted genome engineering allows DNA homing devices
models or by making a phenocopy of a particular disorder fused with regulators to perform tasks with an ease and ef-
(Sander and Joung, 2014). Genome-wide association studies ficiency which can improve regenerative medicine by leaps
(GWAS) have found several regions in the genome that and bounds.
harbor potential risks for polygenic diseases such as diabetes, The Cas9 system has also been demonstrated to have po-
Alzheimer’s, schizophrenia, and autism. Cas9 based multi- tential in antimicrobial therapies that re-sensitize bacteria to
plex genome engineering holds promise in assessing the roles antibiotics. This can be used to modulate virulence of bac-
of these loci, both individually and simultaneously. Effects terial populations. Phages and conjugative plasmids enabled
of genome modifications can be tracked by genome editing of delivery of SSNs to microbial populations, using Cas9 pro-
stem cells, followed by their differentiation into cell type of grammed to target specific sequences underlying antibiotic
interest (Hsu et al., 2014). Cas9 mouse lines to express Cas9 resistance and virulence in bacteria (Citorik et al., 2014). A
in a constitutive or tissue specific manner have been gener- study has revealed the possibility of engineering heritable
ated by crossing a Cre-depended Cas9 mouse with specific resistance in cattle towards Mycobacterium bovis (tubercu-
Cre-driver strains. Delivery of specific gRNA to the Cas9 losis) by HDR-mediated knock-in of mouse gene SP110
mice enabled both ex vivo and in vivo genome editing of using TALEN nickases (Wu et al., 2015). Malarial para-
neurons, immune cells, and endothelial cells. Simultaneous site Plasmodium falciparum has been notoriously resistant
modeling of lung adenocarcinoma through multiplexing has to efforts of the research community to elucidate its intra-
also been demonstrated (Platt et al., 2014). erythrocytic developmental genetics, slowing down the de-
Genome engineering holds great promise for regenerative velopment of novel drugs and vaccines. CRISPR-Cas9 has
medicine-based therapeutics. Direct genome editing in tis- emerged as a fast and efficient tool that has been successfully
sues can be a primary route for treatment. Several proof of applied in this regard to manipulate or knockout malaria
concept studies have proposed such methods for correcting genes (Ghorbal et al., 2014; Wagner et al., 2014), which had
monogenic recessive genetic disorders such as hemophilia taken extensive time previously.
(Li et al., 2011), cystic fibrosis (Schwank et al., 2013), Du-
chenne muscular dystrophy (Ousterout et al., 2013), tyr-
Conclusion
osinemia (Yin et al., 2014), Fanconi anemia (Osborn et al.,
2014), and sickle cell anemia (Sun and Zhao, 2014). In- Signifying the importance of translational research, CRISPR-
activation of a mutant allele via genome editing has been Cas9 technology has witnessed a greatly accelerated de-
proposed for correcting dominant negative genetic disor- velopment from the role in bacterial immunity to therapeutics
12 RAJENDRAN ET AL.

and crop improvement. Its speedy adoption has been made Briner AE, Donohoue PD, Gomaa AA, et al. (2014). Guide
possible by the online user platforms and open source dis- RNA functional modules direct Cas9 activity and orthogo-
tribution. Prior to clinical translation of Cas9, its safety and nality. Mol Cell 56, 333–339.
physiological effects still need to be thoroughly assessed and Brooks C, Nekrasov V, Lippman Z, and Van Eck J. (2014).
characterized. Concerned authorities must determine whe- Efficient gene editing in tomato in the first generation using
ther CRISPR-Cas9 based genome engineering technology the CRISPR/Cas9 system. Plant Physiol 166, 1292–1297.
applied to crops merits an overall exemption from the regu- Brouns SJJ. (2009). Antiviral defense in prokaryotes. Science
latory process or a less rigorous regulation. 321, 560–564.
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Acknowledgment cent motif (PAM)-distal sequences engage CRISPR Cas9
We thank Mona Easterling for her critical proofreading of DNA target cleavage. PLoS One 9, e109213.
the manuscript. Chanda S, Ang CE, Davila J, et al. (2014). Generation of in-
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Author Disclosure Statement Chen B, Gilbert LA, Cimini BA, et al. (2013). Dynamic im-
The authors declare that they have no competing interests. aging of genomic loci in living human cells by an optimized
CRISPR/Cas system. Cell 155, 1479–1491.
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tomato PR and wound-related genes by a mutagenized tomato E-mail: [email protected]