Neuroscience and Biobehavioral Reviews 108 (2020) 732–748

Contents lists available at ScienceDirect

Neuroscience and Biobehavioral Reviews

journal homepage: www.elsevier.com/locate/neubiorev

Review article

A neuroscientist’s guide to transgenic mice and other genetic tools T

a b,c, a,d,e,
Shaghayegh Navabpour , Janine L. Kwapis **, Timothy J. Jarome *
a
Fralin Biomedical Research Institute, Translational Biology, Medicine and Health, Roanoke, VA, USA
b
Department of Biology, Pennsylvania State University, College Park, PA, USA
c
Center for the Molecular Investigation of Neurological Disorders (CMIND), Pennsylvania State University, College Park, PA, USA
d
Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA
e
School of Neuroscience, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA

A R T I C LE I N FO A B S T R A C T

Keywords: The past decade has produced an explosion in the number and variety of genetic tools available to neu-
Transgenic mice roscientists, resulting in an unprecedented ability to precisely manipulate the genome and epigenome in be-
Knockouts having animals. However, no single resource exists that describes all of the tools available to neuroscientists.
Cre Here, we review the genetic, transgenic, and viral techniques that are currently available to probe the complex
DREADDs
relationship between genes and cognition. Topics covered include types of traditional transgenic mouse models
CRISPR
Brain
(knockout, knock-in, reporter lines), inducible systems (Cre-loxP, Tet-On, Tet-Off) and cell- and circuit-specific
Behavior systems (TetTag, TRAP, DIO-DREADD). Additionally, we provide details on virus-mediated and siRNA/shRNA
approaches, as well as a comprehensive discussion of the myriad manipulations that can be made using the
CRISPR-Cas9 system, including single base pair editing and spatially- and temporally-regulated gene-specific
transcriptional control. Collectively, this review will serve as a guide to assist neuroscientists in identifying and
choosing the appropriate genetic tools available to study the complex relationship between the brain and be-
havior.

1. Introduction methodology, continue to be a useful tool today (Rocha-Martins et al.,

2015).
The development of transgenic mice dates back to the early twenty After the establishment of early knockout mice, several important
century when it was first discovered that homologous genes could cross developments and modifications have widened its use in different fields
over and recombine (Morgan, 1911). More than 60 years later, it be- of study. For example, today neuroscientists can choose from global
came known that during meiosis eukaryotes recruit similar machinery knockout lines, gene knock-ins and a variety of reporter lines.
to exchange segments of DNA between homologous chromosomes. Additionally, site-specific recombinases (SSR) have been developed to
Subsequent work by Richard Palmiter, Gali Martin and Nobel lauretes modify the DNA with temporal and cell type specificity. The use of
Richard Axel (2004), Mario Capecchi, Oliver Smithies, and Martin these systems and others have enabled researchers to conditionally
Evans (2007) led to the development of procedures for inactivating a induce or suppress the expression of a gene of interest in a temporally-
targeted gene in the mouse genome using ES cells (Brinster et al., 1981, controlled and/or cell type-specific manner (Bouabe and Okkenhaug,
1982; Capecchi, 1989; Martin, 1981; Thomas and Capecchi, 1987; 2013). In this review, we will discuss the different transgenic mouse
Thomas et al., 1986; Wigler et al., 1977). These efforts ultimately led a lines available to neuroscientists, citing specific examples of how these
number of labs to independently generate the first gene knockout mice diverse tools have been used in ways that have significantly advanced
(Joyner et al., 1989; Koller et al., 1989, 1990; Schwartzberg et al., our current understanding of the nervous system. The emphasis here is
1989; Zijlstra et al., 1989). This “knockout” technology has let scientists on the lines available as opposed to methods on how to develop
study the function of specific genes in physiology, development and transgenic mice, which has been discussed in detail previously (Doyle
pathology. The knockout mice generated using this technology became et al., 2012). Additionally, we will discuss other genetic tools available
an essential tool that helped fuel many of the initial discoveries in to neuroscientists, such as siRNAs, viral-mediated approaches, and
neuroscience and, while their use has declined due to more advanced variations of the CRISPR-Cas9 system.

⁎
Corresponding author at: 175 West Campus Dr., 2150 Litton-Reaves Hall, Blacksburg, VA 24061, USA.
⁎⁎
Corresponding author at: 208 Mueller Laboratory, University Park, PA 16802, USA.
E-mail addresses: [email protected] (J.L. Kwapis), [email protected] (T.J. Jarome).

https://doi.org/10.1016/j.neubiorev.2019.12.013
Received 12 July 2019; Received in revised form 5 November 2019; Accepted 9 December 2019
Available online 13 December 2019
0149-7634/ © 2019 Elsevier Ltd. All rights reserved.
S. Navabpour, et al. Neuroscience and Biobehavioral Reviews 108 (2020) 732–748

2. Knockouts, knock-ins and reporter lines synaptic plasticity and memory formation (Gao et al., 2015; Kang et al.,
2001; Kwapis et al., 2018; Malleret et al., 2001; Vogel-Ciernia et al.,
2.1. Knockouts 2013; White et al., 2016). Additionally, knock-in approaches can be
used to create constitutively active forms of a protein (Bach et al., 1995;
Generation of knockout mice is a common procedure in neu- Mayford et al., 1995; Serita et al., 2017; Suzuki et al., 2011). Other
roscience research. An engineered DNA sequence is introduced into ES knock-in lines consist of point mutations that can prevent DNA binding
cells that are isolated from a mouse blastocyst. This plasmid contains (Oitzl et al., 2001) or in which a single codon is substituted, resulting in
mutations in the target DNA sequence, resulting in a partial or complete a change in the translated amino acid. Such a method is particularly
loss of the coding gene. ES cells that incorporated the plasmid or knock- useful when trying to assess the function of a specific protein phos-
out gene are then isolated and inserted into a mouse blastocyst, which is phorylation site in a given biological function (Briand et al., 2015; Giese
then implanted into the uterus of a female mouse. The offspring will be et al., 1998; Lee et al., 2003). However, if the knock-in mutations are
chimeras, which are then crossbred with other wildtype mice to pro- not spatially and temporally controlled, then the transgenic mouse lines
duce a heterozygous line (F1). Mice obtained in the F1 line are then developed using this approach can suffer from many of the same lim-
interbred to result in an F2 line in which some of the offspring will be itations as the knockout mouse.
homozygous for the knockout gene. Importantly, wild-type littermates Another widely used form of knock-in includes “humanized” mu-
from the F2 generation can then be used as the proper control group for tations in which a mouse has a human gene inserted into its genome.
the homozygous knockouts. This approach has been very useful in studying neurodevelopmental
Global knockouts are the most basic type of genetically modified and neurodegenerative disorders. For example, one well-known trans-
mice and, while they have a number of important limitations (Eisener- genic mouse line contains a knock-in of the mutated human beta-
Dorman et al., 2009), have been critical in initially evaluating the im- amyloid precursor protein (APP), which has been widely used in the
portance of a specific gene to nervous system function. For example, study of Alzheimer’s disease (Hsiao et al., 1996; Sasaguri et al., 2017),
knockout mouse lines were the first to implicate a number of tran- while another carries a Shank3 mutation associated with human autism
scription factors and immediate early genes in synaptic plasticity and spectrum disorders (Yoo et al., 2019). Additionally, humanized knock-
memory formation in the brain (Ahn et al., 2008; Bourtchuladze et al., in mouse lines have also been used extensively in other preclinical
1994; Gupta et al., 2010; Jarome et al., 2015; Kogan et al., 1997; models, such as understanding tumor growth, infectous disease and
Migaud et al., 1998; Plath et al., 2006; Ramamoorthi et al., 2011; immune system function (Walsh et al., 2017). Consequently, while
Selcher et al., 2001; Silva et al., 1992; Zhou et al., 2016). However, due dominant-negative and other loss- or gain-of-function knock-in mice
to the limitations of these knockouts, many conflicting results have may be useful in determining the role of a specific gene in a given
sometimes been obtained using the same transgenic line, as in the study biological process, humanized knock-in lines are better suited for
of the Nuclear Factor Kappa B protein p50 in synaptic plasticity and translational research relevant to human disease.
spatial memory (Denis-Donini et al., 2008; Kassed et al., 2002;
Lehmann et al., 2010; Oikawa et al., 2012). Some studies have shown 2.3. Reporter lines
that p50 deletion results in deficits in synaptic plasticity and memory
formation, while others have reported that the same deletion enhances While knockout and knock-in mice provide a means of controlling
learning and memory. These conflicting results are likely due to com- the expression and function of a particular gene, in some cases it may be
pensation effects triggered by the permanent gene deletion, an effect more useful to track the subcellular localization of a specific gene or
that is especially likely in cases in which a number of different proteins protein or monitor cellular activity. In these cases, reporter mouse lines
have redundant functions within a specific cellular signaling pathway are ideal. One of the most robust tools to visualize and trace the cells
or molecular process. Additionally, in some instances, global knockouts and their behaviors in living animals is using fluorescent proteins.
are lethal or lead to gross abnormalities in physiology and brain de- Initially, beta-galactosidase of Escherichia coli (LacZ) was the tool often
velopment (Gangloff et al., 2004; Li et al., 1992; Yamashita et al., used as a reporter in fixed samples (Abe and Fujimori, 2013). However,
2005), preventing the characterization of a specific gene in a given GFP, the gene encoding green fluorescent protein, was cloned (Prasher
neurological process. While global knockouts serve an initial purpose as et al., 1992) and subsequently used as a fluorescent label in vivo
a “screener” of potential gene candidates for a given biological process, (Chalfie et al., 1994). This led to the generation of the first mouse line
conditional mutations that better control the regional, temporal and that expresses green fluorescent protein (Okabe et al., 1997), which is
cell-type characteristics of the knockout are usually needed to fully now widely used to study the biological characteristics of living cells,
understand the importance of the identified candidate gene to nervous including the localization of subcellular structures, gene transcription
system function. and translation, and cell cycle progression (Abe and Fujimori, 2013). To
date, a wide variety of transgenic reporter lines are available, including
2.2. Knock-ins expression patterns that are native (EGFP, mCherry, etc.) or localized to
the nucleus (H2B-GFP, H2B-mCherry), membrane (m-tdTomato/m-
While the development of a knock-out mice line can be a useful EGFP), microtubules (Tau-EGFP), Golgi apparatus (Golgi-GFP), mi-
means of determining the role of a particular gene in a given biological tochondria (Mito-EGFP) or actin cytoskeleton (Venus-Actin) (Abe et al.,
process, in other cases it is best to leave the DNA sequence present in 2011; Kawamoto et al., 2000; Kurotaki et al., 2007; Muzumdar et al.,
the genome but instead alter the function of the coding gene. Knock-in 2007; Pratt et al., 2000). While these are just a few examples, we refer
mice provide a way of doing this by altering the function of a particular the reader to an excellent review by Abe and Fujimori (2013) that
gene through the replacement of the original DNA sequence with a provides an exhaustive list of available reporter mouse lines.
modified version. This technique has enabled researchers to introduce a Linkage of fluorescent reporters to specific genes has allowed
mutated gene to a host mouse genome and investigate the roles of that tracking of receptor insertion and immediate-early gene expression in
mutation in a variety of biological processes (Harper, 2010). Knock-in the brain during memory formation (Mitsushima et al., 2011; Xie et al.,
mice have been used to examine a wide range of hypotheses in neu- 2014), though the major advantage in these reporter lines has been
roscience by generating loss-of-function and gain-of-function mice for mapping circuits and systems involved in specific neurological pro-
numerous gene targets. For example, dominant-negative mutations in cesses (Barth, 2007). GFP or LacZ under control of immediate-early
which a catalytically impaired mutant out-competes the endogenous gene (IEG) promoters such as fos and arc have allowed neuroscientists
gene, ultimately impairing that gene’s function, have been used to as- to track neuronal activity in a specific population of cells in response to
sess the importance of protein function for a variety of genes during a variety of stimulations or events in vivo (Barth et al., 2004; Clem and

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Barth, 2006; Wang et al., 2006). These IEG-based reporter lines recently expressed throughout the developing and adult telencephalon, in-
have been integrated with inducible technology, allowing un- cluding the cerebral cortex, olfactory bulbs, and hippocampus, and
precedented mapping of network-wide neuronal activity and will be begins around embryonic day 9.5 (E9.5). As expression of cortical Emx1
discussed in more detail in a later section. is primarily restricted to projection neurons and has not been detected
In addition to fluorescent reporters, neuronal activity and protein in glia, it is often used to drive neuron-specific transgene expression
expression can also be monitored in transgenic mice using luciferase- (Jin et al., 2000). This approach has been particularly useful in iden-
based reporters (Ishimoto et al., 2015). Luciferases are a class of oxi- tifying the role of specific genes in hippocampus-dependent memory
dative enzymes that use a chemical reaction to convert energy into formation, including CaMKII and Intraflagellar Transport 88
light. Promoter regions or whole transgenes can be inserted upstream of (Achterberg et al., 2014; Berbari et al., 2014; Haettig et al., 2013).
the luciferase gene to allow the researcher to accurately measure en-
zymatic activity by quantitating the emission of light. Thus, in a luci- 3.3. Examples of other neuron- and region-specific promoters
ferase-based reporter mouse line, the gene of interest (or its promoter
region) is fused to a bioluminescent reporter, allowing for quantifica- CaMKIIα and EMX1 are not the only promoters that specifically
tion of the resulting luciferase expression. The luciferase gene can be target neurons and provide a degree of spatial and temporal control
under the control of almost any promoter and, unlike fluorescent pro- over transgene expression. Other neuron-specific promoters include the
teins, it is not subject to high background from tissue autofluorescence human synapsin 1 (hSyn) (Kugler et al., 2003) promoter, the neuron-
(Contag and Bachmann, 2002; Serganova and Blasberg, 2005), re- specific enolase (NSE) promoter, and the platelet-derived growth factor
sulting in a superior signal-to-noise ratio. However, if the goal is to beta chain promoter. Of these, hSyn has the highest specificity for
visually track the expression of a gene or protein, then luciferase is not a neuronal expression (Hioki et al., 2007) and has been used for suc-
good option as it is strictly quantitative and fluorescent reporters should cessful transgene expression and altered memory formation (Jaitner
be used. et al., 2016). To further improve specificity, distinct populations of
neurons can be targeted using promoters that selectively target distinct
3. Spatially- and temporally-specific promoter lines cell types. For example, the choline acetyltransferase (ChAT) promoter
can be used to express a transgene in cholinergic neurons (Bloem et al.,
As mentioned above, a major limitation to the use of global gene 2014; Lopez et al., 2019). Similarly, glutamate acid decarboxylase 67
knockouts or knock-ins is their lack of specificity in both time and (GAD67) and glutamate receptor 1 (GluR1) promoters can selectively
space. One way to overcome these issues is to put the targeted mutation target GABAergic neurons. To target dopaminergic neurons, a re-
under the control of a cell-type specific promoter. Using a promoter to searcher can use either the dopamine receptor D1a (Drd1a) or the
drive expression can provide some spatial control by targeting selective preprotachykinin 1 (Tac1) promoter (Delzor et al., 2012). Some pro-
cell types that populate distinct brain regions. Some promoters can also moters even allow for region-specific control; gonadotropin-releasing
provide a degree of temporal control, restricting transgene expression hormone (mGnRH) promoter, for example, specifically targets the hy-
to a specific developmental time window. The AllenBrain Institute pothalamus (Kim et al., 2002). Conversely, gene mutations can be
(http://connectivity.brain-map.org/transgenic/) has some great re- targeted to astrocytes using the glial fibrillary acidic protein (GFAP)
sources for identifying appropriate cell-type-specific promoters for promoter, which has been used to limit LacZ reporter gene expression
neurons and other brain cell types to help researchers choose the cor- to only astrocytes throughout the central nervous system (Brenner
rect promoter. In this section we will focus on two of the most widely et al., 1994).
used promoters in neuroscience (CaMKIIα and EMX1), but will also list It is important to note that the right promoter depends on the ex-
others that have been developed and used with some frequency. perimental conditions, including the known expression pattern of the
However, it should be noted that this is not meant to be an exhaustive gene of interest, the intended cell- and region-specific targets, and the
list of the available transgene promoters. desired onset of the transgene (e.g. immediately versus later in devel-
opment). For instance, CaMKIIα start to express around postnatal day 1
3.1. CaMKIIα (P1; Kool et al., 2016), while EMX1 mRNA is detected at E9.5 in mouse
brains (Chan et al., 2001). Syn1 expression in rats starts at E13 (Ye and
One of the most widely used transgenic promoter lines is the 1.3 Kb Marth, 2004), ChAT at E11 in mouse neurons (Huber and Ernsberger,
promoter derived from the calcium/calmodulin-dependent kinase II 2006) and GAD67 at E17 in rats (Popp et al., 2009). In many cases there
alpha (CaMKIIα) gene, which allows the mutation to be restricted to may be more than one promoter which can achieve the desired trans-
excitatory neurons in the neocortex and hippocampus with an expres- gene expression profile, so pilot studies may be needed to determine
sion pattern that starts during early development. This promoter has which promoter works best under your specific experimental condi-
been widely used as a method to spatially control gene deletions and tions.
dominant negative and constitutively active mutations in a neuron-
specific manner during complex physiological states (Hasegawa et al., 3.4. Limitations
2009; Mansuy et al., 1998; Mayford et al., 1996; Winder et al., 1998;
Zhou et al., 2016). Typically, CaMKIIα is used when the researcher aims Although promoter lines can improve temporal and spatial specifi-
to manipulate the gene of interest in forebrain excitatory neurons after city, they also have limitations. Many of these promoters (including
development. For example, some researchers have used this promoter both the EMX and CaMKII promoters) express early in development
to overexpress a truncated form of the protein phosphatase calcineurin, although their use is often intended to manipulate gene expression in
which allowed localization of the transgene to the forebrain and hip- adult animals. This could lead to gross changes in neuroanatomy or
pocampus, and found significant effects on long-term potentiation cellular physiology. Further, the persistent nature of these manipula-
(LTP) and memory formation (Mansuy et al., 1998; Winder et al., tions might trigger compensatory mechanisms which could confound
1998). the effects of the gene mutations. Finally, while these promoters can be
effective at limiting the number and type of affected cells, some leaki-
3.2. EMX1 ness may occur, affecting unintended cell types, albeit at lower levels.
As a result, while these transgenic lines are widely used and are largely
EMX1 is a second promoter that enables improved spatial and an effective means of determining a specific gene’s function in the
temporal control of the intended manipulation. EMX1 is a mouse brain, often they need to be combined with more sophisticated ap-
homologue of the Drosophila homeobox gene empty spiracles that is proaches to improve specificity.

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Table 1
Viruses.
Vector Type Capacity Onset Duration Advantages Disadvantages

Adeno dsDNA ∼8 kb Days Weeks High packaging capacity High immune response
Transient
Integrates into genome BSL2
AAV ssDNA ∼5kb Weeks Years Safe (BS1) Limited packaging compacity
Easy production Does not integrate into genome
Long lasting
Low immune response
Good penetration
Anterograde or retrograde delivery
Lentivirus RNA ∼8 kb Weeks Years High packaging capacity BSL2
Integrates into genome Expression is spatially limited
Easy production
Long lasting
Low immune response
Rabies Virus RNA ∼5kb Days Weeks Rapid expression BSL2
Cytotoxicity by 2 weeks
HSV-1 dsDNA ∼30-50kb Hours 8-10 days Largest packaging capacity BSL2
Neurotropic More difficult to produce
Transient
LT-HSV-1 dsDNA ∼30-50kb Weeks Indefinite Largest packaging capacity BSL2
Retrograde transfer properties More difficult to produce
Neurotropic
Long lasting

AAV: Adenoassociated Virus; HSV-1: Herpes Simplex Virus.

4. Viruses dividing cells, albeit with a limited DNA capacity (< 5 kb) and without
integration into the genome. (Deyle and Russell, 2009; Robbins and
In the following sections 4.7 we will discuss several genetic and Ghivizzani, 1998). A major advantage of AAVs are that they produce a
post-transcriptional manipulations that that have a high level of tem- low immune response and provide long-term transgene expression,
poral and spatial specificity. Because most cell types in the brain are lasting years in some animals (Lundstrom, 2018; Wojno et al., 2013).
difficult to transfect, the foreign material (DNA) designed to induce This relatively safe viral vector (BSL1) has both retrograde (like AAV6)
these manipulations is often packaged into a virus for efficient delivery and anterograde (like AAV2) serotypes, and could therefore be used to
into cells. There are several different types of viral vectors that can be answer circuit-specific questions. Recently, a group of researchers de-
used for this purpose, each with its own advantages and disadvantages veloped a series of AAVs that can be injected into the tail vein and cross
(Table 1). In this section, we will provide details on the different viral the blood-brain barrier to infect most areas of the brain (Deverman
approaches that can be used for transgene delivery. et al., 2016), allowing the researcher to avoid using invasive and
technical stereotaxic surgeries to deliver the virus. Other uses of AAV
4.1. Adenoviruses include, but are not limited to, injecting AAV vectors containing Cre
recombinase into brain areas of flox mice (discussed in Section 5), faster
Adenoviruses infect a wide range of cells, including dividing and and more efficient gene knock out animals (Wang et al., 2018b), op-
non-dividing cells, with a high DNA packaging capacity (∼8 kb), but togenetics (Parr-Brownlie et al., 2015), and gene therapy, such as Rett
the strong immunogenicity of adenoviruses poses a significant limita- syndrome preclinical research (Sinnett and Gray, 2017) and phase I/II
tion to their use (Robbins and Ghivizzani, 1998). Recent improvements clinical trials for Parkinson’s disease (Sun and Schaffer, 2018).
in adenoviral vectors have drastically reduced this adversive immune
response, however, making them more attractive as delivery vehicles
for transgenic DNA or shRNA (discussed in the next section). As ade- 4.3. Lentiviruses
noviruses express in a short-term, episomal manner, they are particu-
larly effective for conditional transgene expression as the manipulation Lentiviruses belong to the ssRNA family of retroviruses that are
will be transient (Lundstrom, 2018), though this would not be case for usually based on HIV-1. The lentiviral particle has the reverse tran-
gene editing studies since DNA recombination would have already oc- scriptase, integrase, and proteinase needed for the replication, allowing
curred. Importantly, this limited expression pattern can be a limitation lentiviruses to infect both dividing and non-dividing cells (Escors and
if long-term transgene expression is desired. Adenoviral vectors have Breckpot, 2010). They can infect a broad range of hosts with low cy-
many reported uses including carrying reporter genes to the target cells totoxicity, making them appropriate for a wide range of model organ-
(Hermens et al., 1997), CRISPR-Cas9 gene editing (Cheng et al., 2014), isms. Further, like AAVs (Lundstrom, 2018), lentiviruses produce per-
local delivery of Cre recombinase and cancer gene therapy (Wold and sistent expression, in part because they permanently integrate into the
Toth, 2013). An important consideration when using adenoviruses is host genome and can even be transduced to the next generation of
that they pose a risk to humans, requiring operation with biosafety level mitotic cells (Chen and Gonçalves, 2016). Through pseudotyping (ex-
2 (BSL2), which can also be a limitation depending on the facilities pressing glycoproteins originating from a different virus), lentiviruses
available. can be used to achieve a high level of spatial specificity with limited
spread (Parr-Brownlie et al., 2015). These vectors are used in zinc-
finger nucleases (Lombardo et al., 2007), CRISPR-mediated multiple
4.2. Adeno-associated viruses (AAV)
gene editing (Zetsche et al., 2016), delivery of shRNAs (Li et al., 2014)
or “decoys” (Dias et al., 2014) and have been used in clinical trails
Adeno-associated virus (AAV) is a non-human pathogen and a
(Escors and Breckpot, 2010). As with adenoviruses, lentiviruses need to
member of the parvovirus family that is the most widely used viral
be handled at BSL2, as there is a small risk to humans.
delivery system in neuroscience. AAV can infect both dividing and non-

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4.4. Herpes simplex virus (HSV) 5.1. siRNAs

Herpes simplex virus (HSV) is a large enveloped dsDNA with a re- In RNAi, a complementary RNA molecule binds to a target mRNA
latively wide host range that also requires handling at BSL2. HSVs are transcript to silence its expression. This evolutionary conserved process
naturally neurotropic, making them ideal for specifically targeting is believed to be a mechanism that host cells use to protect against
neurons without the additional requirement of a neuron-specific pro- invading pathogens. This defense system has been repurposed to con-
moter. Further, they do not integrate into the host DNA but rather stay trol gene expression using small interfering RNAs (siRNA) to silence
episomal as a circular molecule, minimizing off-target effects that might expression of the target gene (Lovett-Racke et al., 2005). siRNAs get
occur during viral integration into the host genome. As HSV is also a incorporated into the RNA-induced silencing complex (RISC), resulting
retrograde virus, it will be transferred to the CNS even after injection in siRNA strand separation and binding of one strand of siRNA to the
into the peripheral nervous system (Artusi et al., 2018; Lundstrom, target mRNA. This binding allows RISC to cleave the mRNA and de-
2018). Newer generations of replication-deficient HSV vectors cause a grade it (Hannon, 2002). This method is transient, as the targeted gene
low immune response with an extremely large insert capacity (> 30 kb) continues to be transcribed as the siRNA molecules are degraded, ul-
(Lundstrom, 2018). Short-term HSVs are expressed rapidly (peak at timately resulting in a loss gene silencing (Morris, 2011). Despite this, a
∼3d post-injection) and transiently (gone by ∼10d) at the site of in- large number of studies have successfully used siRNA technology to
jection (Neve et al., 2005). Modified long-term retrograde HSV vectors, investigate the role(s) of specific genes in memory formation, including
in comparsion, are retroactively transported from the axon terminal to transcription factors, protein phosphatases and epigenetic modifiers
the cell body and are indefinitely expressed following injection, making (Kremer et al., 2018; Peters et al., 2009).
it possible to do circuit-specific manipulations (Fenno et al., 2014;
Sarno and Robison, 2018). Many researchers use HSV in gene therapy, 5.2. shRNAs
CRISPR technology (Wang et al., 2018a) or optogenetic methods (Li
et al., 2019). To address this transient expression limitation of siRNAs, re-
searchers have designed a variety of vectors that are capable of in-
tegrating into DNA by encoding a long inverted repeat, which when
4.5. Limitations transcribed in vivo forms a short hairpin RNA (shRNA). The difference
here is that while siRNAs are delivered directly to the cytosol, shRNA
Viral manipulations of gene expression have many benefits over integrates into the host cell DNA and gets transcribed via RNA poly-
breeding-based approaches, including the ability to spatially and tem- merase II or III. Subsecuently, it is transferred into the cytoplasm,
porally restrict the manipulation through stereotaxic delivery of the cleaved by Dicer, and processed into siRNA (Jacob, 2006; Moore et al.,
viruses. Further, as many viruses are intended to manipulate gene ex- 2010). Similar to siRNAs, a number of studies have successfully ma-
pression in wild type animals, it is unnecessary to maintain a genetic nipulated a variety of biological processes and memory formation
mouse line for these manipulations, avoiding expensive and time-con- through brain-region specific expression of shRNAs (i.e., Li et al., 2014;
suming colony maintenance. Nonetheless, viruses also have limitations. Neuner et al., 2015). The choice between shRNA and siRNA depends on
For example, although stereotaxically delivering the viruses can pro- factors such as time demands and the need for a more or less stable
duce spatial specificity, stereotaxic surgery can have adverse affects and knock-down of the gene of interest. There are also limitations to
requires specialized equipment and training not necessary for breeding- siRNAs, including toxicity that can occur as a result of the transfection
based manipulations. Further, as mentioned above, viral vectors require reagents needed to get siRNA into cells in vivo, the possibility of off-
safety precautions and handling under BSL1 or BSL2 conditions (Collins target effects, and a reduction in siRNA concentration following cell
et al., 2017). Packaging size can also be a limiting factor for the use of division. siRNA may require multiple injections to maintain an optimal
some viruses, such as AAV, and an adverse immune response can limit level of gene silencing whereas shRNA is continuously transcribed, even
the utility of other viral vectors, especially adenoviruses. Finally, the in daughter cells, and significantly increases the reproducibility of re-
genetic manipulation is affected by the targeting and spread of the sults (Moore et al., 2010).
virus, which can change across batches, making viral manipulations
inherently more variable than traditional breeding methods. This con- 5.3. Accell siRNAs
cern, however, can be mitigated by normalizing the titer before injec-
tion and verifying viral expression and spread in each animal to ensure The simplest way to deliver RNAi is through cytosolic introduction
that only animals with the desired manipulation are used in analyses. of si/shRNA plasmids. This method, however, is primarily used for
Viral approaches therefore have unique advantages and disadvantages transient in vitro studies and cell types amenable to transfection. As
and the most appropriate virus will be dictated by experimental factors, transfection is difficult to achieve in brain cells in vivo, other methods
including the size of the desired transgene, the spatial precision ne- have been developed to introduce RNAi into neurons in animals, in-
cessary, and the temporal requirements of the task. cluding the use of viral vectors (e.g. adenovirus or lentivirus) to deliver
siRNA or shRNA into the brain. However, this can be an expensive and
time-consuming process. Recently, the development of novel siRNA
5. Spatially controlled manipulations via siRNA and shRNAs technology has circumvented this problem. Accell siRNA is a func-
tionally validated commercial product in which a chemical modifica-
Knockout and knock-in approaches aim to alter gene expression by tion allows for siRNA delivery into difficult-to-transfect cells without
inserting DNA to either delete/modify, overexpress or outcompete en- the need for viral vectors or other transfection reagents (Nakajima
dogenous genes. Another strategy to manipulate gene expression while et al., 2012). These Accell siRNAs are neuron-preferring and have a
gaining some temporal and spatial control is post-transcriptional gene rapid induction profile in which expression typically peaks in less than
silencing through the use of RNA interference (RNAi) techniques in the 48 h post-injection. We have used these siRNAs extensively in the ro-
cytoplasm (Langlois et al., 2005) and nucleus (Robb et al., 2005). RNAi dent brain and have consistently achieved successful, rapid gene
technology has a number of potential uses in neuroscience (Miller et al., knockdown for a variety of different targets in both rats and mice
2005) and has been used to study the role of specific genes in synaptic (Alaghband et al., 2018; Jarome et al., 2015, 2018; Kwapis et al., 2018;
plasticity, memory formation and disease. In this section we will discuss Webb et al., 2017). As a result, Accell siRNA can be especially ad-
RNAi technology, focusing on siRNA, shRNA and recently developed vantegous if the researcher desires rapid, transient knockdown with no
Accell siRNA. biosafety concern. Compared to viral approaches, however,

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knockdowns made with Accell siRNA are relatively difficult to verify

following a behavioral experiment, as the knockdown is transient and
the siRNA does not contain an easily visualized epitope tag. As with
other manipulations, the specific siRNA delivery method (virus vs Ac-
cell) should be chosen based on the specific requirements of the ex-
periment.

6. Spatially- and temporally-specific conditional genetic

manipulations

Although much information has been learned using knockout,

knock-in, or specific promoter-based approaches to manipulate genetic
information, these methods lack both the spatial and temporal specifi-
city needed to answer basic neuroscience questions. Promoter lines are
not typically capable of restricting the genetic modification to a specific
brain structure, such as the basolateral amygdala or the dorsal hippo-
campus. Further, many promoter lines used to temporally restrict ge-
netic manipulations to the post-development period, such as CaMKIIα,
still express relatively early in development (∼3 weeks postnatally),
making it difficult to determine whether effects observed in the adult or
aged mouse are due to the long-term manipulation of this gene
throughout the animal’s lifespan (Kojima et al., 1997). Furthermore,
while siRNAs and shRNAs are a powerful mechanism of controlling
post-transcriptional processing, they are often transient, can only target
a small number of cells and a single brain region at a time and are
limited only to gene silencing. To address these limitations, conditional
knockout approaches were developed to improve both spatial- and
temporal-specificity. There are a number of methods used to create
conditional genetic manipulations, including the Cre/lox system, the
FLP/FRT system, and the Dre/rox system. More recently, these re-
combination systems have been modified to be ligand-sensitive, re-
sponsive to either tetracycline (e.g. Tet-On and Tet-Off systems) or ta-
moxifen (e.g. CreERT2) to enable precise temporal control over when
the recombination event (and thus the desired genetic manipulation)
occurs. All of these approaches can selectively alter gene expression in a
tissue-specific manner to control when and where the genetic manip-
ulation occurs, and each system has benefits and limitations that should
be considered when designing an experiment, as discussed below.

6.1. Recombination-based approaches: Cre, FLP, and Dre

The development of site-specific in vivo DNA recombination sys-

Fig. 1. Cre-loxP system. Cre-induced recombination can be used to induce ei-
tems in the early 1990s allowed neuroscientists to ask more precise
ther deletion or expression of a target gene. (A) Schematic depicting Cre-in-
questions about where individual genes function in the brain and when duced deletion. Cre cuts at LoxP sites flanking the gene of interest, removing the
these genes are needed across the lifespan. The first system to be de- gene in any region expressing Cre. (B) Schematic depicting a typical Cre-in-
veloped was the Cre/lox recombination system (Orban et al., 1992), duced expression system. LoxP sites flank a stop sequence that prevents ex-
which remains the most widely-used conditional approach to date. In pression of the gene. Exposure to Cre excises the stop sequence, allowing the
the Cre/lox system, Cre recombinase recognizes loxP sequences and target gene to be expressed. (C) Schematic of the DIO (Double Inverted
drives their recombination, removing any genetic material between Orientation) system (also called the Flip-Excision (FLEx) system). The vector
loxP sites that share the same orientation (Sauer, 1998). Typically, expresses the gene of interest in an antisense orientation flanked by two unique
conditional knockout mice are bred with loxP sites flanking the gene of lox sites (loxP and lox2272). The addition of Cre catalyzes recombination of
interest, so that exposure to Cre recombinase drives the selective re- these two sites that permantly flips the gene into the sense orientation, allowing
for expression.
moval of that gene only in tissues where Cre is expressed (Fig. 1A). For
example, Tsien and colleagues crossed NR1 floxed mice (the NR1 sub-
unit of the NMDA receptor is flanked with loxP sites) with mice ex- “stop” sequence is bred to a promoter line that expresses Cre re-
pressing Cre under the CaMKIIα or KA1 promoter (Tsien et al., 1996). combinase in a site- and time-specific manner, leading to selective ex-
The resulting mice had selective deletions of the NR1 subunit of the pression of the gene of interest wherever (and whenever) Cre is ex-
NMDA receptor in either area CA1 (CaMKII promoter) or CA3 (KA1 pressed (Fig. 1B).
promoter) of the dorsal hippocampus, allowing the researchers to de- The Cre/lox system has been used to create a wide range of genetic
termine that NR1-containing NMDARs are critical for spatial learning in deletions using a variety of promoter lines that selectively express Cre
area CA1 and for pattern completion in CA3 (Cui et al., 2004). within specific cell types (e.g. excitatory forebrain neurons) during
The Cre/lox system can also be used to drive expression of a target specific points in development (e.g. early postnatal development). For a
gene (such as a fluorescent reporter or a dominant-negative transgene) comprehensive review of the different Cre promoter lines available to
by removing a floxed transcription termination sequence (or “stop” drive tissue- and time-specific deletion of LoxP-flanked genes, the
sequence) inserted between the promoter and the transgene (Madisen reader is referred to Kim et al. (2018) as well as The Jackson Laboratory
et al., 2010). In this case, a mutant mouse carrying the LoxP-flanked (the JAX Cre repository: https://www.jax.org/research-and-faculty/

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resources/cre-repository). Thus, in this Cre/lox system, the spatial and 2018). Cre can be delivered through a modified virus (Table 1, see
temporal precision of the genetic deletion is limited by the specificity of Section 4 for an in-depth comparison of viral delivery systems) directly
the promoter line used to express Cre. into the brain structure of interest to provide tight spatial and temporal
In addition to the widely-used Cre/lox system, other recombination- control of the genetic manipulation. As Cre is only expressed where the
based systems have been developed to manipulate gene expression in a virus is delivered, this method can be used to achieve site-specific
site- and time-specific manner, including the FLP/FRT system and the manipulation of the target gene. Within the injected region, cell-type
Dre/rox system. In the FLP/FRT system, the yeast-derived flippase specificity can be achieved through both the viral capsid protein (e.g.,
(FLP) recombinase recognizes FRT (Flippase Recognition Target) sites AAV 2/1 is packaged in the capsid from serotype 1, which pre-
and drives their recombination, removing any genetic material between ferentially infects neurons (Burger et al., 2004)) and the promoter
FRT sites. Although the FLP/FRT system was first identified around the driving Cre expression (e.g., the CaMKIIα promoter primarily drives
same time as the Cre/lox system, it was initially less efficient than Cre expression in forebrain excitatory neurons (Mayford et al., 1996)). Fi-
(Buchholz et al., 1998), leading to a slower adoption in the field. Since nally, as the floxed animal expresses the target gene normally until
its initial discovery, FLP has been modified to become more efficient at delivery of the virus encoding Cre, it develops and ages normally until
mammalian body temperatures and the latest version of FLP, FLPo, has the moment of virus-mediated Cre delivery. This avoids the spatial and
recombination efficiency similar to that of Cre (Kranz et al., 2010). temporal limitations of promoter-line approaches to delivering Cre re-
Despite its improved efficiency, FLP/FRT is not as commonly used as combinase. For example, in a recent paper, we injected AAV-CaMKII-
Cre/loxP, which has a wider array of promoter lines and other tools Cre into the dorsal hippocampus of 18-month-old HDAC3flox/flox mice to
available that do not yet exist for FLP/FRT. show that hippocampus-specific deletion of HDAC3 improved long-
The Dre/rox system is a second, more recently described alternative term memory in aging mice (Kwapis et al., 2018). Importantly, this
to the Cre/lox approach. As with Cre/lox, the Dre/rox system consists virus-based strategy allowed the animals to develop and age with the
of a recombinase (DreO) that recognizes and recombines two identical target gene intact before the virus was injected at 18 months of age.
DNA sequences (called rox sites), deleting any genomic DNA in be- As with transgenic mouse lines, viral vectors can be used to express
tween. Like Cre/Lox, Dre/rox is extremely efficient in driving re- a wide range of genetic manipulations beyond encoding Cre re-
combination in specific cell types (Anastassiadis et al., 2009). Although combinase. For example, the virus can simply code for a gene of interest
functionally identical to the Cre/lox system, Dre/rox relies on a unique to test the effects of overexpression in the injected brain region (e.g.,
recombinase and distinct recombination sites, making it a com- Kaas et al., 2013). Alternatively, a virus can express a mutant version of
plementary system that can be used to complement and extend Cre/lox- the gene of interest that it is catalytically dead (Alaghband et al., 2017;
based manipulations. Kwapis et al., 2017) or encodes for a phosphomimic or phosphomutant
As most conditional knockout approaches rely on the Cre/lox protein of interest (Han et al., 2007; Vogel Ciernia et al., 2017). Ex-
system, the number of promoter lines and other tools available for this pression of the transgene can also be placed under the control of Cre
system far outnumber those available for either the FLP/FRT or DRE/ using either a floxed “stop” cassette, as described above (see Fig. 1B) or
rox systems. The strength in these redundant approaches is twofold. using the DIO (Double Inverted Orientation) system (also called the
First, the FLP/FRT and DRE/rox systems can be used to verify the re- Flip-Excision, or FLEx system; Fig. 1C). In the DIO/FLEx system, the
sults of a Cre/lox manipulation with an independent recombinase viral vector expresses the transgene in an antisense orientation between
system. Second, as each system uses a unique recombinase and re- two unique lox sites (loxP and lox2272) so that no functional gene
cognition site combination, these systems are complementary to each product is expressed in the absence of Cre. The lox sites are organized in
other and can be multiplexed in vivo to create relatively complex gene oppositing orientations (compared to being in the same orientation, see
programs across development (Fenno et al., 2014; Schonhuber et al., Fig. 1A, B), allowing Cre to invert the genetic material between com-
2014). By combining the three systems, researchers can begin to ad- patible lox sites. This allows two sequential recombination events to
dress questions about how different genes interact in specific brain occur: 1) Cre inverts the transgene between the loxP sites into the sense
regions during development or during complex behaviors. orientation and 2) the genetic material between the lox2272 sites (now
in the same orientation) is removed, excising one of the loxP sites. The
6.2. Narrowing the temporal window: using viruses to drive site-specific resulting product has two incompatible lox sequences, preventing fur-
recombination ther recombination. Thus, the transgene is flipped into the correct or-
ientation and expresses in any Cre-positive cells. This enables both cell
Conditional genetic manpulations typically rely on promoter lines to type-specific expression of the gene of interest and enables circuit-
express Cre (or FLP or Dre) in a site- and time-specific manner by specific expression through the use of a combination of retrograde and
placing recombinase expression under the control of a promoter or anterograde viruses, as described in the next section.
enhancer element that expresses in a specific cell type. Thus, the ma-
nipulation specificity is achieved by breeding mice carrying the re- 6.3. Inducible systems: Tet-On, Tet-Off and CreER
combination recognition (e.g. LoxP) sites in all cells with mice that only
express the recombinase (e.g. Cre) in certain cell types. Although this is Although the Cre/lox system allows the experimenter to induce the
a powerful method of controlling specific subtypes of neurons in broad manipulation at a specific time, the genetic manipulation typically has
regions of the brain, it is limited by the availability and accuracy of the a gradual onset (as it requires expression on the promoter or virus to
promoter line used to express Cre. If no promoter line exists for a drive the manipulation) and is permanent once recombination is com-
particular cell type or brain region, for example, it may not be possible plete. These limitations can be overcome with the use of ligand-sensi-
to achieve site-specific recombination through this method. Further, tive manipulations that are rapidly induced and reversible, providing
most promoter lines turn on relatively early in development, altering more precise temporal specificity over when the genetic information is
the gene early in the lifespan, potentially impacting both development expressed. There are two major inducible systems capable of this tight
and the aging process. Finally, other organisms used in neuroscience temporal control: tetracycline-based and taxmoxifen-based.
have relatively few promoter lines compared to mice, limiting the scope In the basic Tet expression system, a transcriptional activator in-
and specificity of the manipulations that can be achieved through duces expression of the transgene only in the presence (or absence) of
breeding-based methods. tetracycline or its derivative, doxycycline (Dox) (Fig. 2). Both the “Tet-
One way to avoid these issues is to use viruses to express Cre and Off” and the “Tet-On” systems contain two core components: 1) the
other transgenes in a site-specific manner in the adult brain. (Jarome doxycycline-sensitive transcriptional activator and 2) the target gene
et al., 2015; Kwapis et al., 2018; McQuown et al., 2011; Shu et al., under the control of a tetracycline-responsive promoter element (TRE),

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Fig. 2. Tet-inducible systems. The target gene is under control

of the tetracycline-responsive promoter element (TRE), which
consists of a TetO binding site fused to a short promoter se-
quence. Transcriptional control of target gene is achieved by
presence or absence of doxycycline (Tet). (A) In the Tet-Off
system, the tetracycline-controlled transactivator protein
(tTA) binds to the TRE only in the absence of Tet, which allows
active transcription of the target gene (left). When Tet is ap-
plied, tTA is unable to bind TRE and transcription of the
transgene is repressed (right). (B) In the Tet-On system, the
reverse tetracycline-controlled transactivator protein (rtTA)
only binds to the TRE in the presence of Tet, which allows
active transcription of the target gene (right). When Tet is not
present, tTA is unable to bind TRE and transcription of the
transgene is repressed (left).

consisting of the tetO binding site fused to a short promoter sequence. A more recent addition to the conditional manipulation toolbox is
In the Tet-Off system, the tetracycline-controlled transactivator protein the development of the CreER system, which combines the powerful
(tTa) binds to the TRE only in the absence of Dox, allowing the ex- Cre-based approach with ligand-dependent activation to gain temporal
perimenter to prevent expression of the transgene by maintaining the specificity over the genetic manipulation (Denny et al., 2014;
animal on Dox, typically provided in the diet. Thus, the investigator can Guenthner et al., 2013). The CreER system consists of two components:
remove Dox from the diet at a specfic timepoint in the experiment to 1) the target transgene floxed by loxP sites and 2) a tamoxifen-sensitive
restrict transgene expression to a discrete experimental window, such Cre recombinase, called CreERT2. To make the tamoxifen-dependent
as a training session. Administration of Dox after this timepoint (e.g. CreERT2, Cre is fused to a mutated human estrogen receptor that re-
immeciately after training) will close the window, preventing further sponds to tamoxifen and 4-hydroxytamoxifen (4-OHT) rather than en-
expression of the transgene. In an early demonstration of this system, dogenous estradiol. In the absence of tamoxifen, CreERT2 is sequestered
Mayford and colleagues (Mayford et al., 1996) used the Tet-Off system in the cytoplasm, but application of tamoxifen allows CreERT2 to
to conditionally express a constitutively active mutant CaMKIIα trans- translocate to the nucleus, where it drives recombination of loxP sites
gene in adult mice. Removal of dox before LTP or behavioral training flanking the transgene. The CreER system not only improves the tem-
led to expression of this calcium-independent CaMKIIα and impaired poral resolution of the Cre/loxP system (recombination occurs within a
both hippocampal LTP and spatial memory. This was a reversible effect, few days of tamoxifen exposure (Brocard et al., 1997)), it can also
as placing the animal back on Dox suppressed expression of the mutant improve the spatial specificity of the deletion, as tamoxifen can be lo-
CaMKII transgene and ameliorated these impairments in LTP and cally injected into the appropriate brain region. Combining this site-
memory. Therefore, the authors were able to demonstrate for the first and temporal precision with promoter-specific expression of CreERT2
time that CaMKIIα was critical for LTP and memory formation in the and/or the target transgene allows the experimenter to precisely con-
adult brain, independent of its role in development, which was not trol which cells express the manipulation at a specific point in time.
affected by their temporally-restricted manipulation. The advent of tamoxifen- and tetracycline-inducibule genetic ma-
To prevent expression of the transgene outside of the desired tem- nipulations has greatly expanded the questions that can be addressed
poral window, the Tet-Off system requires long-term, continuous ad- with conditional knockouts, as the fine-tuned spatial resolution can
ministration of Dox, which may cause off-target effects (Sultan et al., more carefully control when the manipulation is induced. Nonetheless,
2013). Further, expression of the target gene in the Tet-Off system there are important drawbacks to using these systems that should be
depends on the removal of Dox from the appropriate tissues, which carefully considered. First, leakiness has been reported for both sys-
limits how rapidly the transgene can be induced (Das et al., 2016). To tems, so that some transgene expression has been observed in the “off”
avoid these limitations, the Tet-On system was developed, in which a state, even before the removal of Dox (tetracycline-based system) or
reverse tetracycline-controlled transactivator (rtTA) only recognizes the addition of 4-OHT (tamoxifen-based system) (Forster et al., 1999;
TRE in the presence of doxycycline. Therefore, in the Tet-On system, the Heffner et al., 2012; Kristianto et al., 2017; Schmidt et al., 2000; Vooijs
transgene remains off until Dox is administered, allowing the researcher et al., 2001; Zhu et al., 2001). It is therefore critically important to
to rapidly drive expression by acutely administering Dox. As with the include control mice (e.g. mice with the transgene but maintained on
Tet-Off system, this expression is transient and reversible, allowing the Dox or not given 4-OHT) both when characterizing transgene expres-
investigator to control a tight window of transgene expression. sion and when studying behavior. Second, although these ligand-in-
As with the Cre/lox system, the Tet-On and Tet-Off systems can gain ducible systems avoid many of the off-target effects that can occur with
spatial specificity through the use of different promoters to drive ex- long-term Cre exposure (Loonstra et al., 2001; Pfeifer et al., 2001),
pression of the tTA or rtTA transactivator in specific cell types. Thus, injections of tamoxifen/4-OHT or tetracycline/doxycycline can also
the basic tetracycline system is limited by the specificity of the chosen cause unintended side effects, such as diarrhea, colitis, and apoptosis of
promoters. As with the Cre/lox system, viral methods can be used to gastric parietal cells (Huh et al., 2012; Riond and Riviere, 1988). As
provide additional spatial specificity by restricting one of the two these issues can alter behavior, it is again important to include appro-
components (usually the tTA) to a specific region of the brain. Notably, priate control groups that receive tamoxifen/doxycycline without the
these tetracycline systems have been elegantly redesigned to allow cells inducible transgene. Finally, there are limits to the administration
active during a discrete window of time (e.g. during a memory acqui- protocol itself, including differences in the time window of effectiveness
sition session) to be tagged for future manipulation, as discussed in the and differences in recombination efficiency across both strain and sex
next session. that can introduce variability (Abram et al., 2014; Sandlesh et al., 2018;

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Valny et al., 2016). Therefore, if tight spatial control over the genetic pattern of cells active during memory acquisition or sensory stimulation
manipulation is not necessary for a particular experimental question, a to demonstrate that cells active during fear memory acquisition are
standard, non-inducible manipulation may be more appropriate to preferentially reactivated during the retrieval of that memory (Denny
avoid some of these issues. et al., 2014; Reijmers et al., 2007; Tayler et al., 2013). Although their
initial use was purely observational, the TetTag and CreERT2 systems
7. Cell- and circuit-specific expression have since been modified to express different regulators of neural ac-
tivity that allow bidirectional control over tagged neurons, to test the
The conditional genetic systems described above have not only al- role of these cells in behavior. For example, the Hen lab bred Arc-
lowed researchers to restrict genetic manipulations in both time and CreERT2 mice with mice expressing a STOP-floxed inhibitory optoge-
space but have also laid the foundation for the ultra-precise manip- netic receptor Archaerhodopsin-3 (Arch). By injecting these mice with
ulations that have been developed in the past decade. Recent advances 4-OHT before training, they were able to express Arch selectively in
in this field have made it possible for neuronal activity and even genetic cells active during context fear acquisition. Optogenetic inhibition of
information to be altered within specific circuits during defined time these tagged cells at a later timepoint revealed that this population of
windows of activity. These technical improvements have allowed re- cells is critical for successful memory retrieval (Denny et al., 2014). The
searchers to identify the roles of discrete neuronal networks in specific ArcCreERT2 mouse line has also been used with the excitatory optoge-
phases of behavior. Further, it is now possible to “tag” a population of netic receptor channel rhodopsin (ChR2) to allow the tagged population
neurons active during a discrete time window and manipulate the ac- of neurons to be stimulated at a later timepoint, allowing for bidirec-
tivity of those cells at a later timepoint. This unprecedented precision tional control (Lacagnina et al., 2019). The TetTag and CreERT2 systems
has fundamentally changed how researchers approach questions about can also be combined with DREADDs (Designer Receptors Exclusively
the role of genetics in behavior as well as the function of active cells in Activated by Designer Drugs) to allow for chemogenetic control over
memory formation. the tagged population of cells (Garner et al., 2012). Together, these
methods have been used to interrogate the memory “engram;” by sti-
7.1. Identification of active cells: TetTag, CreERT2 mulating or inhibiting these cell populations, researchers have forced or
prevented memory recall (Cowansage et al., 2014; Denny et al., 2014;
To identify and access the subset of neurons engaged by a particular Tanaka et al., 2014), changed the information encoded in an en-
task, the Mayford lab developed the TetTag mouse, which persistently dogenous memory (Garner et al., 2012), improved extinction memory
tags neurons activated during a specific time window controlled by the (Denny et al., 2014; Lacagnina et al., 2019), and even created a false
investigator (Reijmers and Mayford, 2009; Reijmers et al., 2007). This memory (Ramirez et al., 2013).
mouse line takes advantage of the dynamic, activity-responsive im- The use of viruses with anterograde and retrograde properties (see
mediate early gene (IEG) cFos, which is rapidly (∼5 min) upregulated Section 4) has also made it possible to manipulate neural circuits with
in active neurons after a learning event (Strekalova et al., 2003; Webb incredible precision. It is now possible to selectively stimulate or inhibit
et al., 2017) or other stimulation event (Kaczmarek, 1992). The tTA populations of cells that connect two brain regions. The observation
element is placed under the control of the cFos promoter, so that tTA that optogenetic receptors get transported from the soma to the axon
expression is limited to cells activated by a sufficiently salient event terminal made it relatively simple for researchers to test the role of
(Reijmers et al., 2007). In the second part of the system, the tetO pro- specific neural circuits in behavior. By injecting an anterograde virus
moter drives bidirectional transcription of a reporter gene (LacZ) as into region A and implanting an optic fiber in region B, the cells making
well as a dox-insensitive tTA mutant (tTA*) capable of maintaining its efferent projections from region A to B can be selectively stimulated or
own expression through a transcriptional feedback loop (Fig. 3A). Thus, inhibited at the axon terminal (Fig. 4A) (Carreno et al., 2016; Carter
the removal of Dox opens a window during which cFos drives persistent and de Lecea, 2011). Similarly, retrograde viruses can also be used to
expression of tTA* and the LacZ reporter gene selectively in active determine the role of specific projection pathways; injection of a ret-
neurons. Placing the mice back on Dox prevents tagging of new neurons rograde virus that enters through axon terminals in combination with
without affecting the Dox-insensitive tTA*-mediated transcription, optical fiber implantation in an upstream structure allows for selective
which supports continued LacZ expression from the tagged neurons, activation or silencing of cells connecting these regions (Carter and de
maintaining the tag that was induced when those cells became active. Lecea, 2011).
In a similar manner, the ArcCreERT2 and TRAP (Targeted The ability to manipulate projection-specific neural pathways has
Recombination in Active Populations; Fig. 3) mouse lines both exploit been extended to DREADD systems (Fig. 4B) as well (Smith et al.,
the activity-dependent expression pattern of the IEGs Arc and cFos to 2016). Although they lack the precise temporal resolution of optoge-
selectively express the tamoxifen-dependent CreERT2 in active neural netic manipulations, DREADDs allow for remote activation or inhibi-
populations (Denny et al., 2014; Guenthner et al., 2013). Co-expressing tion of the target circuit with a systemic injection of the designer drug
the IEG-dependent CreERT2 with a cre-dependent fluorescent reporter CNO (Clozapine-n-oxide), rather than requiring an invasive optical
allows active neurons to drive permanent recombination of the reporter implant to allow light to be delivered to a specific brain region. In order
gene only when tamoxifen is present. A researcher can therefore open to target a specific population of neurons with DREADDs, Cre-based
the tagging window before a stimulation event, such as a training ses- recombination in a specific cell type (using a transgenic Cre promoter
sion, by injecting tamoxifen or 4-OHT (the major active metabolite of line or a virus expressing Cre under a cell type-specific promoter) can be
tamoxifen), to allow active neurons to persistently express the fluor- combined with a virus encoding the DREADD gene in a flipped DIO/
escent tag for future visualization. This window automatically closes as FLEx configuration (see Section 6). The DIO/FLEx system allows the
tamoxifen/4-OHT is metabolized, within approximately 12 h. This DREADD virus to remain dormant until Cre application, which flips the
CreERT2-based system has a few advantages over the TetTag system, gene into the sense orientation and allows its expression. Thus, in a
including a more precise temporal window for tagging (hours compared promoter line, the locally-injected DIO-DREADD is only expressed in a
to days), permanent recombination-based modifications, a wide array functional orientation in cell types expressing Cre. To achieve pathway-
of available Cre-based genetic tools, and the ability to delete en- specific control, a retrograde virus (e.g. CAV-2) expressing Cre can be
dogenous genes (Guenthner et al., 2013; Reijmers and Mayford, 2009). injected into target region B and an anterograde virus encoding the
DIO-DREADD can be expressed in upstream region A (Fig. 4B). Only
7.2. Manipulation of active cells: optogenetics, DREADDs, DIO/FLEx cells projecting from A to B will receive both viruses, allowing for
DREADD expression specifically in this group of cells (Boender et al.,
The TetTag and CreERT2 systems were initially used to observe the 2014). Thus, exposure to a systemic injection of CNO will selectively

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Fig. 3. TetTag and TRAP systems allow neu-

rons active during specific time windows to be
persistently tagged. (A) Tet-tag system. Two
transgenes control tagging of active neurons: 1)
tTA under the control of the activity-dependent
cFos promoter and a TetO promoter that drives
both the gene of interest (e.g. a reporter gene)
and the dox-insensitive tTA* mutant that drives
persistent expression. The presence of
Doxycyline (Dox) at rest prevents tTa-TetO-
mediated expression (left). Removing Dox
(right) opens a temporary tagging window
during which tTa binds the TetO promoter and
drives expression of the desired gene selec-
tively in cells activated during that window.
(B) The TRAP system also requires two trans-
genes: 1) The tamoxifen-dependent CreERT2
under the control of an activity-sensitive pro-
moter like cFos or Arc and 2) a transgene that
expresses the gene of interest (e.g. a reporter
gene) in a Cre-dependent manner. In the ab-
sence of tamoxifen (TM) or its metabolite 4-
OHT (left), Cre is sequestered to the cytoplasm,
preventing expression of the target gene.
Injecting TM or 4-OHT (right) opens a tagging
window in which CreERT2 recombination oc-
curs selectively in activated cells.

activate or repress this pathway-specific group of cells. components: 1) the CRISPR-associated endonuclease (Cas) protein,
which cuts the target DNA and 2) the single guide RNA (sgRNA) which
8. The CRISPR revolution directs Cas9 to the correct genomic site, although there are restrictions
to where the sgRNA can target (e.g. the sequence must be unique and
The combination of IEG-based promoters, Cre recombination, and contain a protospacer adjacent motif, or PAM (Walters et al., 2015)). In
methods of modulating neural activity (optogenetics and DREADDs) the simplest iteration of this system, CRISPR/Cas9 is used to create a
have allowed researchers to manipulate the activity of cells at specific gene knockout (Fig. 5A). To this end, the sgRNA, which is programmed
timepoints in distinct circuits. Understanding the roles of individual to target the gene of interest, forms a complex with Cas9 and directs it
genes within these neural circuits has been relatively difficult until very to the target gene, where it creates a double-strand break (DSB) of the
recently, however. The recent advent of genome editing technology, DNA at that locus. Repairing that break with the non-homologous end
such as zinc finger nucleases (ZFNs), transcription activator-like ef- joining (NHEJ) pathway typically results in insertion or deletion errors
fector nucleases (TALENs) and CRISPR/Cas9, has made it possible to at the site, causing frameshift mutations that disrupt expression of the
bidirectionally interrogate single genes in a circuit-specific manner. target gene. In instances in which the NHEJ pathway corrects the DSB
Due to the many advantages of CRISPR/Cas9, such as versatility, cost without mutating the sequence, Cas9 cuts the site again to increase the
and ease of use, we will focus specifically on it as opposed to ZFNs and likelihood of mutation. Thus, in theory, the system can be targeted to
TALENS. Although this work is at a very early stage, especially in in delete any gene of interest simply by designing the sgRNA to direct the
vivo studies of brain function, this simple and powerful technique has Cas9 to the appropriate locus.
already greatly expanded our ability to drive precise genetic manip- The CRISPR/Cas9 system can also be used to alter genomic DNA,
ulations within specific cell types. such as making a single nucleotide substitution (to make a catalytically
inactive point mutant, for example) or adding in an epitope tag for
visualization of the gene (Ran et al., 2013). To drive these knock-in
8.1. CRISPR as a tool to make site-specific genetic and epigenetic changes in the gene sequence, the researcher must provide a repair
modifications template that serves to “fix” the DSB with homology directed repair.
Although this system can produce precise modifications to endogenous
The CRISPR/Cas9 system is a flexible and powerful way to edit an genes, it is relatively inefficient and produces a heterogenous popula-
organism’s genome or bidirectionally alter the expression of en- tion of cells that are unedited or mutated with the NHEJ pathway in
dogenous target genes (Wang et al., 2016). CRISPR/Cas9 has two core

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Fig. 4. Circuit-specific use of optogenetics and DREADDs. (A) An example of

the use of optogenetics to selectively interrogate neurons projecting in a specific
circuit. Here, an anterograde optogenetic virus (e.g. AAV1-ChR2) is injected
into brain region “A” and the terminals are optically stimulated with an optical
fiber implanted into efferent region “B” to selectively activate neurons pro-
jecting from A to B. (B) An example of the use of DIO-DREADDs to selectively
interrogate a neural circuit. An anterograde DIO-DREADD is injected into re-
gion A and a retrograde Cre virus is injected into efferent region B. As the DIO-
DREADD is inactive in the absence of Cre, only cells that receive both viruses
will express DREADDS and will respond to systemic injection of CNO.
Fig. 5. Basic CRISPR-Cas9 systems. The CRISPR-Cas9 system can be used to edit
a gene out or to control gene-specific transcriptional activation or silencing. (A)
addition to cells containing the desired genetic edit. Further, as these In the traditional CRISPR-Cas9 system, the catalytically active Cas9 complex is
knock-in mutations rely on the homology-dependent repair pathway, recruited to a target DNA region via a synthetic guide RNA (sgRNA; top). The
which is not typically present in neurons (Ran et al., 2013), efforts to Cas9 will “cut” the DNA, resulting in a double-stranded break (bottom). (B) In
modify genetic information with this system in the brain have not yet the CRISPR-dCas9 system, the Cas9 is catalytically inactive, so cannot cut DNA,
but can still bind DNA as directed by the sgRNA. The dCas9 is fused to a
been successful (Walters et al., 2015). Another method of generating
transcriptional activator such as p65 (top) or repressor such as KRAB (bottom),
mutations in vivo includes the use of Cas9 nickases (nCas9) or en-
which allows endogenous transcriptional control without editing the genome.
zymatically-inactive “dead” Cas9 (dCas9) to generate single base edits.
Cas9 nickases are modified to cut only one strand of DNA, rather than
creating a double strand break like a traditional Cas9 enzyme. Thus, Therefore, despite its promise as a simple mechanism to modify genetic
two nCas9 must be combined to generate a double-strand break to information in vivo, there are a number of technical issues that need to
produce a traditional CRISPR-based knockout, which improves preci- be resolved before CRISPR/Cas9 or dCas9/nCas9-based nucleotide edits
sion. To generate a single base mutation, nCas9 or dCas9 can be fused can be used to modify genomic DNA in the brain of a behaving animal.
with a DNA deaminase to modify a single nucleotide in a target gene in In addition to deletions and knock-ins, the CRISPR/Cas9 system has
vivo (Eid et al., 2018; Molla and Yang, 2019). Although this technique been modified to transcriptionally activate or transcriptionally repress
can be used relatively efficiently to produce C→T and A→G mutations, an endogenous gene without affecting the underlying sequence
there are a number of limitations that restrict its usefulness (Eid et al., (Fig. 5B). In these systems, the Cas9 enzyme is inactivated with point
2018). For example, these deaminases cannot make other types of point mutations, making it unable to cut the DNA at the targeted site. Rather,
mutations besides the C→T and A→G conversions. Further this system the dead Cas9 serves as a scaffold for transcriptional activators, re-
lacks precision in the face of a string of Cs or As, so that all of the pressors, or even chromatin modifiers to alter the expression of a target
cytadine or adenine bases within the editing window of the CRISPR gene (Adli, 2018). A simple form of this system consists of the dCas9
system will be modified. Finally, as with homology-driven knock-ins, fused to either a transcriptional activator (e.g. VP64) or repressor (e.g.
the efficacy of single base editing in the brain has yet to be determined. KRAB) to drive or inhibit transcription of the target gene, respectively.

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To further enhance expression of the target gene, slight modifications in need to introduce multiple viruses; the researcher needs only to express
the CRISPR activation system can recruit additional transcriptional the sgRNA (or even multiple sgRNAs), which readily fit into a typical
activators to augment expression of the target gene. For example, the AAV cassette. Additionally, a Cre-sensitive version of this knock-in
dCas9-VPR activator system contains dCas9 fused to three different mouse is also available, allowing for cell- and circuit-specific expression
transcriptional activators: VP64, p65, and Rta that together increase of Cas9.
expression of the target gene more than a single activator alone (Chavez Transgenic lines encoding dCas9 are also becoming available to
et al., 2015, 2016; Gilbert et al., 2013; Mali et al., 2013). Similarly, the allow for CRISPR-based gene activation or repression. For example, Cre-
CRISPR-SAM (Synergistic Activation Mediator) system uses a modified inducible versions of the gene-activating SunTag system are now
sgRNA containing MS2 aptamers to recruit additional activators (p65 available from The Jackson Laboratory. In the SunTag system, dCas9 is
and HSF1) fused to an MS2 coat protein to the target gene (Konermann fused to a repeating polypeptide sequence that is recognized by the
et al., 2015). The modified sgRNA recruits both MS2-p65-HSF1 and the GCN4 antibody. Fusing GCN4 antibodies to transcriptional activators
dCas9-VP64 fusion protein to the target gene to synergistically drive like VP64, p65, and HSF1, allows multiple copies of these transcrip-
transcription. The CRISPR-SAM system has been successfully used in tional activators to be scaffolded to the gene locus targeted by the
vivo to drive expression of the circadian gene Per1 in the dorsal hip- sgRNA (Tanenbaum et al., 2014). A researcher can therefore drive ex-
pocampus to improve long-term memory formation in aging mice pression of a target gene in a site-specific manner by introducing both
(Kwapis et al., 2018). Finally, more by fusing chromatin modifiers to Cre and an appropriate sgRNA in the target brain region. Although
dCas9 (or driving the recruitment of a chromatin modifier through the there are very few dCas9-based transgenic lines currently available, as
use of MS2), researchers can theoretically modify the chromatin at a CRISPR-based technologies advance, it is likely that the number of
precise genomic locus. For example, targeting a dCas9-HAT (e.g. p300) available dCas9 transgenic mouse lines will continue to improve, as
to a specific gene should promote acetylation at that site, promoting an well.
open chromatin formation that is permissive to gene expression without A second method to express CRISPR/Cas9 in the brain is through the
directly activating transcription. Finally, CRISPR can also be used to use of viruses. Virus-mediated expression of the full CRISPR/Cas9
target long non-coding RNA (lncRNA) sequences to a specific genomic system has the major advantage of improving flexibility; the researcher
locus with the dCas9-based CRISPR Display (CRISPR-Disp) system, in does not need to maintain a transgenic mouse line and can apply the
which the long RNA sequence is incorporated into the sgRNA as an system to a wide range of animal models at a desired age with a loca-
“aptameric accessory” domain (Perez-Pinera et al., 2015; Shechner lized intracranial virus injection. The major disadvantage, as discussed
et al., 2015). Although this work is in an early phase and much is un- above, is that the CRISPR system is too large to be packaged in a single
known, the CRISPR/Cas9 system has already fundamentally changed AAV. In order to express Cas9/dCas9, the sgRNA, and any other re-
how researchers can study gene function in the brain. quired elements, multiple AAVs must be used. Although a basic Cas9
As CRISPR systems get more sophisticated, increasingly precise and and sgRNA with short promoter sequences could be expressed in a
complex manipulations are becoming feasible. Entire gene programs single lentivirus, which has a larger packaging limit than AAV, a dCas9
can theoretically be coordinated by multiplexing different sgRNAs with system including transcriptional activators or repressors and a fluor-
transcriptional activators and repressors. Introducing multiple sgRNAs escent protein for localization easily exceeds the ∼9.7kB limit. Multiple
to target dCas9-VPR to different regions along the promoter of a single viruses (three AAVs or two lentiviruses) expressing different dCas9-
gene can drive a larger increase in transcription than one sgRNA alone based activation systems have been successfully used to drive gene
(Savell et al., 2019), indicating that dCas9-based activation scaffolds expression in different brain regions in vivo, including the hippo-
can be targeted along a promoter to synergistically increase transcrip- campus, nucleus accumbens, and prefrontal cortex (Kwapis et al., 2018;
tion. Further, multiplexing sgRNAs targeting three different genes can Savell et al., 2019). Using multiple viruses is problematic, however, as
increase the expression of each of the three target genes in neuronal only the cells infected with all of the viruses get a functional CRISPR
culture (Savell et al., 2019), indicating that the CRISPR/dCas9 system system. An alternative solution to this problem is to use HSVs, which
can coordinately increase the expression of multiple genes at the same are neurotropic and have a much larger packaging limit (> 30kB)
time. Researchers can gain an extra layer of control by also shaping the (Sarno and Robison, 2018) that can accommodate the entire CRISPR/
chromatin landscape across the genome using multiple sgRNAs to target dCas9 system, avoiding the requirement for multiple viruses. HSV-
chromatin modifying enzymes to specific genes, promoters, or enhancer mediated expression of a target gene is more transient than expression
elements. This ability to simultaneously manipulate multiple genetic with AAVs or lentiviruses; expression peaks around 3-5d and eliminated
and epigenetic events is critical for understanding how any individual by 8-10d after infection (Sarno and Robison, 2018). Thus, if the beha-
gene contributes to behavior within the millieu of reactions happening vioral task and the research question are amenable to this shorter
within each neuron. Although there is much research on the effects of timecourse, the use of HSVs are an effective delivery method for large
individual genetic manipulations in vivo, very little is understood about CRISPR/dCas9 systems into the brain (Lorsch et al., 2019; Walters
how these genes function relative to other genetic variants, epigenetic et al., 2017).
manipulations, or compensatory mechanisms, in part due to the lack of New nanoparticle-based delivery systems like CRISPR-Gold are
appropriate tools. With the ability to mimic a relatively complex and currently being developed to avoid the use of viruses altogether, which
nuanced genetic program, CRISPR tools have the potential to broaden have limitations that restrict its use in a clinical setting. CRISPR-Gold
our understanding of the interactions between these mechaisms. uses a gold nanoparticle complex to package the Cas9 molecule and
sgRNA inside of a polymer that allows the package to cross the cell
8.2. How to express CRISPR systems in the brain membrane before allowing the Cas9 and sgRNA to be released (Lee
et al., 2017). Thus, like viruses, CRISPR-Gold allows for site-specific
CRISPR/Cas9 can be expressed in the central nervous system via expression of CRISPR but has the additional advantage of packaging the
three different methods: a transgenic mouse line, with viruses or using a entire CRISPR system in a single nanoparticle. Further, CRISPR-Gold
nanoparticle delivery system. One popular approach is to use a trans- avoids both the toxicity and the immune response induced by viral
genic mouse expressing Cas9 at the ROSA26 locus under a CAG pro- CRISPR infusions (Lee et al., 2017) and its transient expression prevents
moter, which produces constitutive, brain-wide expression of cas9 the off-target effects observed following persistent viral-induced Cas9
(Platt et al., 2014). To create a mutation, a researcher simply needs to expression. Since its initial development, CRISPR-Gold has been used to
introduce a sgRNA (or multiple sgRNAs) to target Cas9 to the appro- edit genes in the adult mouse brain, most notably the metabotropic
priate gene(s) using a virus. As Cas9 is a large protein that requires its glutamate receptor 5 (mGluR5) gene, deletion of which reduced the
own AAV due to packaging limitations, this transgenic line avoids the excessive repetitive behaviors observed in a mouse model of fragile X

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syndrome (Lee et al., 2018). As nanoparticle and viral delivery methods more fine temporal control over gene expression. In the most popular of
continue to improve, it is becoming increasingly likely that CRISPR/ these systems, light-activated CRISPR effector (LACE), the blue light-
Cas9 will be a viable therapeutic method for gene editing in patients. sensitive cryptochrome 2 (CRY2) protein is fused to an effector domain
Finally, CRISPR/Cas9 plasmids can also be delivered into the brain (VP64 or p65) that can promote transcription upon binding to CIB1,
using the PolyPlus transfection reagent in vivo Jet-PEI. This polymer- which is fused to the dCas9. In the absence of light, the CRY2-effector
based reagent acts by condensing nucleic acids into stable nanoparticle- fusion is unable to bind CIB1. When exposed to blue light, however,
like structures small enough to diffuse into tissue, which occurs via CRY2 undergoes a reversible conformational change that allows it to
endocytosis. This transfection reagent has a number of advantages over bind CIB1, scaffolding the effector domain to the target gene to induce
virus-mediated systems, including no biohazard concerns and low costs. its transcription (Nihongaki et al., 2015; Polstein and Gersbach, 2015).
However, disadvantages include the proprietary nature of the product, LACE therefore allows rapid and reversible activation of a gene during
which limits our understanding of exactly how it works to deliver for- behavior.
eign material into the cell, and the lack of cell type specificity, though As CRISPR technology continues to improve, the list of questions
this could be overcome through the use of specifc promoters in the that can be experimentally addressed also continues to expand. For
CRISPR constructs. Similar to CRISPR-Gold, few studies have used this example, until very recently it was not possible to determine whether
CRISPR delivery system to date. However, one recent study was able to epigenetic modifications at a specific gene during memory formation
express CRISPR-dCas9 constructs in the mouse hippocampus using in affected its expression and ultimate effects on memory (Heller et al.,
vivo Jet-PEI and achieved transcriptional control over their target gene 2014). Clever systems now combine dCas9 with epigenetic modifiers,
and altered memory formation (Butler et al., 2019). allowing the researcher to direct site-specific chromatin modifications
during behavior by scaffolding a chromatin-modifying enzyme to a
8.3. Using CRISPR to direct circuit-specific genetic modifications specific gene promoter. In theory, this method could be used to “open”
or “close” chromatin at a specific locus, allowing the endogenous
In addition to improving the ease of precise genetic manipulations, transcriptional machinery to produce physiologically relevant expres-
CRISPR/Cas9 can also be used to interrogate the roles of specific genes sion of a specific gene under either permissive or restrictive chromatin
within defined neural circuits in the brain. Although it is possible to conditions, respectively. Whether this system can be successfully used
overexpress a gene or knock down a gene in a specific neural circuit by to modify chromatin in vivo during behavior and, if so, whether these
combining promoter-line Cre expression with a Cre-dependent con- precise chromatin modifications are sufficient to improve or impair
struct, CRISPR/Cas9 makes it possible to create knockouts or over- memory remains to be seen. CRISPR systems will continue to grow
expression from endogenous genes in a population of cells with a spe- more sophisticated as Cas9 gets incorporated into existing systems of
cific projection target. Creating a circuit-specific manipulation using “tagging” active neurons, such as the TRAP system (Fig. 3B) and new
CRISPR/Cas9 or CRISPR/dCas9 can be accomplished with either a Cre- tools, such as light-sensitive CRISPR-based interference, are developed.
based CRISPR system or by using retrograde virus expression to express The addition of CRISPR into the neuroscientist’s toolkit has given us the
a portion of the functional CRISPR system. Transgenic models encoding ability to make precise manipulations that were once inaccessible,
Cre-sensitive versions of both Cas9 and dCas9 systems are available opening the door to answering incredibly detailed questions about the
from The Jackson Laboratory (www.jax.org) that allow the researcher causal role of genetic and epigenetic modifications in altering behavior
to limit expression of the CRISPR system to cell populations expressing in vivo.
Cre. Therefore, by crossing these mice with a promoter line expressing
Cre in a specific population of neurons or by locally injecting a virus 9. Conclusions
encoding Cre under a cell type-specific promoter, expression of the
system can be restricted to a subset of neurons with similar properties. As genetic manipulations become more common and more sophis-
Viruses with retrograde properties can also be used to restrict CRISPR- ticated, neuroscientists can answer increasingly detailed questions
based manipulations to a specific neural circuit. By splitting up the about the role of different genetic and epigenetic mechanisms in brain
components of a functional CRISPR system into two components (the function. In addition to time-tested techniques like gene knockouts and
sgRNA and the dCas9) and delivering the dCas9 component via a ret- knock-ins, more recent tools, including CRISPR-Cas9, are now available
rograde HSV into one brain region (“B”) and the sgRNA via local viral to manipulate specific genes in spatially- and temporally-specific sys-
expression in a brain region that projects to this site (“A”), CRISPR tems. Neuroscientists can now probe the function of a specific gene
could be used to selectively delete or modify the expression of a single within a desired circuit at a specific point in time, such as during a
gene in neurons projecting from brain region A to region B (Sarno and learning event. With this powerful, ever-expanding toolkit available to
Robison, 2018). us, neuroscientists are poised to drastically advance our understanding
CRISPR has also been incorporated into inducible systems to restrict of brain function.
when the gene editing occurs during behavioral experiments. Using
these systems, it is possible to induce a CRISPR-based manipulation Declaration of Competing Interest
during a specific phase of memory formation, for example. There are
two primary methods for temporally restricting Cas9: incorporation None.
into a Tet-On (or Tet-Off) system or using light-sensitive effectors. In
the Tet-On system, a transgene coding for Cas9/dCas9 and any neces- Acknowledgements
sary effector domains are placed under the control of doxycycline, so
that exposure to doxycycline (or removal of Dox in the Tet-Off system) This work was supported in part by startup funds from the College
drives transcription of the CRISPR system (Cao et al., 2016). As with of Agricultural and Life Sciences and the School of Neuroscience at
other Tet-based systems (see Fig. 2), activity-regulated promoters like Virginia Tech (TJJ). It was also supported by startup funds from the
Arc or cFos can be used to express the tTA or rtTA gene, limiting ex- Eberly College of Science and Department of Biology at Pennsylvania
pression of Cas9/dCas9 to cells activated by stimulation during the State University (JLK) and the National Institute on Aging (NIA) grant
“active” window. Thus, the CRISPR system can be used to delete, in- K99/R00 AG056596 (JLK).
hibit, or activate a gene selectively in cells activated by a specific event,
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