Genome Editing

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Genome-Editing Technologies: Principles

and Applications
Thomas Gaj,1,4 Shannon J. Sirk,2 Sai-lan Shui,3 and Jia Liu3,4

1
Department of Bioengineering, University of California, Berkeley, California 94720
2
Department of Chemical Engineering, Stanford University, Stanford, California 94305
3
Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai, China
Correspondence: gaj@berkeley.edu; liujia@shanghaitech.edu.cn
Targeted nucleases have provided researchers with the ability to manipulate virtually any
genomic sequence, enabling the facile creation of isogenic cell lines and animal models for
the study of human disease, and promoting exciting new possibilities for human gene
therapy. Here we review three foundational technologies—clustered regularly interspaced
short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9), transcription acti-
vator-like effector nucleases (TALENs), and zinc-finger nucleases (ZFNs). We discuss the
engineering advances that facilitated their development and highlight several achievements
in genome engineering that were made possible by these tools. We also consider artificial
transcription factors, illustrating how this technology can complement targeted nucleases for
synthetic biology and gene therapy.
n recent years, the emergence of highly versa- nize new genomic sequences has driven a revo-
I tile genome-editing technologies has pro-
vided investigators with the ability to rapidly
lution in genome editing that has accelerated
scientific breakthroughs and discoveries in dis-
and economically introduce sequence-specific ciplines as diverse as synthetic biology, human
modifications into the genomes of a broad spec- gene therapy, disease modeling, drug discovery,
trum of cell types and organisms. The core tech- neuroscience, and the agricultural sciences.
nologies now most commonly used to facilitate The diverse array of genetic outcomes made
genome editing, shown in Figure 1, are (1) clus- possible by these technologies is the result, in
tered regularly interspaced short palindromic large part, of their ability to efficiently induce
repeats (CRISPR)-CRISPR-associated protein targeted DNA double-strand breaks (DSBs).
9 (Cas9), (2) transcription activator-like effec- These DNA breaks then drive activation of cel-
tor nucleases (TALENs), (3) zinc-finger nucle- lular DNA repair pathways and facilitate the in-
ases (ZFNs), and (4) homing endonucleases or troduction of site-specific genomic modifica-
meganucleases. tions (Rouet et al. 1994; Choulika et al. 1995).
In particular, the ease with which CRISPR- This process is most often used to achieve gene
Cas9 and TALENs can be configured to recog- knockout via random base insertions and/or
4
These authors contributed equally to this work.
Editors: Daniel G. Gibson, Clyde A. Hutchison III, Hamilton O. Smith, and J. Craig Venter
Additional Perspectives on Synthetic Biology available at www.cshperspectives.org
Copyright # 2016 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a023754
Cite this article as Cold Spring Harb Perspect Biol 2016;8:a023754
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T. Gaj et al.
5′- -3′
Homing endonuclease
3′- -5′
Zinc-finger
DNA-binding domain 5′-ANN-3′
1F 2F 3F 4F 5′-GNN-3′
Fokl
5′- -3′
ZFN
3′- Fokl -5′
4F 3F 2F 1F
5′-GNN-GNN-3′
TAL effector
Repeat variable DNA-binding domain TALEN
diresidues (RVDs)
NG: T Fokl
NI: A 5′- -3′
HD: C 3′- Fokl -5′
NH: G Amino-terminal
domain
Carboxy-terminal
domain
Cas9
gRNA
-5′
PAM
5′- -3′
3′- -5′
Target
Figure 1. Genome-editing technologies. Cartoons illustrating the mechanisms of targeted nucleases. From top to
bottom: homing endonucleases, zinc-finger nucleases (ZFNs), transcription activator-like effector (TALE) nu-
cleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated
protein 9 (Cas9). Homing endonucleases generally cleave their DNA substrates as dimers, and do not have
distinct binding and cleavage domains. ZFNs recognize target sites that consist of two zinc-finger binding sites
that flank a 5- to 7-base pair (bp) spacer sequence recognized by the FokI cleavage domain. TALENs recognize
target sites that consist of two TALE DNA-binding sites that flank a 12- to 20-bp spacer sequence recognized by
the FokI cleavage domain. The Cas9 nuclease is targeted to DNA sequences complementary to the targeting
sequence within the single guide RNA (gRNA) located immediately upstream of a compatible protospacer
adjacent motif (PAM). DNA and protein are not drawn to scale.
deletions that can be introduced by nonhomol- comes) (Bibikova et al. 2001, 2003; Porteus and
ogous end joining (NHEJ) (Fig. 2A) (Bibikova Baltimore 2003; Urnov et al. 2005). Indeed, the
et al. 2002). Alternatively, in the presence of a broad versatility of these genome-modifying
donor template with homology to the targeted enzymes is evidenced by the fact that they also
chromosomal site, gene integration, or base serve as the foundation for artificial transcrip-
correction via homology-directed repair (HDR) tion factors, a class of tools capable of modulat-
can occur (HDR) (Fig. 2B) (see Fig. 2 for an ing the expression of nearly any gene within a
overview of other possible genome-editing out- genome.
2 Cite this article as Cold Spring Harb Perspect Biol 2016;8:a023754

Genome-Editing Technologies
A Gene knockout Gene deletion Gene inversion
B Gene integration Gene correction
Homology arm Homology arm

Donor DNA
Genomic
target
Figure 2. Genome-editing outcomes. Targeted nucleases induce DNA double-strand breaks (DSBs) that are
repaired by nonhomologous end joining (NHEJ) or, in the presence of donor template, homology-directed
repair (HDR). (A) In the absence of a donor template, NHEJ introduces small base insertions or deletions that
can result in gene disruption. When two DSBs are induced simultaneously, the intervening genomic sequence
can be deleted or inverted. (B) In the presence of donor DNA ( plasmid or single-stranded oligonucleotide),
recombination between homologous DNA sequences present on the donor template and a specific chromo-
somal site can facilitate targeted integration. Lightning bolts indicate DSBs.
Here we review key principles of genome and Carroll 2005; Urnov et al. 2010). ZFNs
editing, emphasizing many of the engineering function as dimers, with each monomer recog-
advances that have laid the groundwork for the nizing a specific “half site” sequence—typically
creation, refinement, and implementation of nine to 18 base pairs (bps) of DNA—via the
the current suite of genome-modifying tools. zinc-finger DNA-binding domain (Fig. 1). Di-
We also provide an overview of the achieve- merization of the ZFN proteins is mediated by
ments made possible by genome editing, illus- the FokI cleavage domain, which cuts DNA
trating how this technology can enable advances within a five- to seven-bp spacer sequence that
throughout the life sciences. separates two flanking zinc-finger binding sites
(Smith et al. 2000). Each ZFN is typically com-
posed of three or four zinc-finger domains, with
TARGETED NUCLEASES each individual domain composed of 30 ami-
no acid residues that are organized in a bba
Zinc-Finger Nucleases
motif (Pavletich and Pabo 1991). The residues
ZFNs, which are fusions between a custom- that facilitate DNA recognition are located
designed Cys2-His2 zinc-finger protein and the within the a-helical domain and typically inter-
cleavage domain of the FokI restriction endo- act with three bps of DNA, with occasional over-
nuclease (Kim et al. 1996), were the first target- lap from an adjacent domain (Wolfe et al. 2000).
ed nuclease to achieve widespread use (Porteus Using methods such as phage display (Choo
Cite this article as Cold Spring Harb Perspect Biol 2016;8:a023754 3

T. Gaj et al.
and Klug 1994; Jamieson et al. 1994; Wu et al. to when expressed within cells from nucleic ac-
1995), a large number of zinc-finger domains ids (Gaj et al. 2012). Modified ZFN proteins
recognizing distinct DNA triplets have been endowed with improved cell-penetrating activ-
identified (Segal et al. 1999; Dreier et al. 2001, ity have since been described (Liu et al. 2015a).
2005; Bae et al. 2003). These domains can be ZFNickases can also facilitate gene correction in
fused together in tandem using a canonical the absence of a DSB (Kim et al. 2012; Ramirez
linker peptide (Liu et al. 1997) to generate poly- et al. 2012; Wang et al. 2012). These enzymes,
dactyl zinc-finger proteins that can target a wide which consist of one catalytically inactivated
range of possible DNA sequences (Beerli et al. ZFN monomer in combination with a second
1998, 2000a; Kim et al. 2009). In addition to this native ZFN monomer, can stimulate HDR by
“modular assembly” approach to zinc-finger nicking or cleaving one strand of DNA and are
construction, selection-based methods for con- derived from a concept first illustrated by Stod-
structing zinc-finger proteins have also been re- dard and colleagues using homing endonucle-
ported (Greisman and Pabo 1997; Isalan et al. ases (McConnell Smith et al. 2009).
2001; Hurt et al. 2003; Magnenat et al. 2004), Unlike TALENs and CRISPR-Cas9, the dif-
including those that consider context-depen- ficulty associated with constructing zinc-finger
dent interactions between adjacent zinc-finger arrays has hindered their widespread adoption
domains, such as oligomerized pool engineer- in unspecialized laboratories. In particular, it
ing (OPEN) (Maeder et al. 2008). In addition, remains challenging to create zinc-finger do-
specialized sets of validated two-finger, zinc-fin- mains that can effectively recognize all DNA
ger modules have been used to assemble zinc- triplets, especially those of the 50 -CNN-30 and
finger arrays (Kim et al. 2009; Bhakta et al. 50 -TNN-30 variety. As a result, ZFNs lack the
2013), including those that take context-depen- target flexibility inherent to more recent ge-
dent effects into account (Sander et al. 2011b; nome-editing platforms. Nevertheless, the po-
Gupta et al. 2012). tential for ZFNs to mediate specific and efficient
One major concern associated with the use genome editing is evidenced by ongoing clinical
of ZFNs for genome editing (in addition to all trials based on ZFN-mediated knockout of the
targeted nucleases) is off-target mutations (Ga- human immunodeficiency virus (HIV)-1 co-
briel et al. 2011; Pattanayak et al. 2011). As a receptor CCR5 for treatment of HIV/acquired
result, several approaches have been undertaken immune deficiency syndrome (AIDS) (Tebas
to enhance their specificity. Among the most et al. 2014) and a planned clinical trial based
successful of these has been the creation of on site-specific integration of the factor IX
obligate heterodimeric ZFN architectures that gene into the albumin locus to treat hemophilia
rely on charge – charge repulsion to prevent un- B (Clinical Trial ID: NCT02695160) (Sharma
wanted homodimerization of the FokI cleavage et al. 2015).
domain (Miller et al. 2007; Doyon et al. 2011),
thereby minimizing the potential for ZFNs to
TALE Nucleases
dimerize at off-target sites. Additionally, pro-
tein-engineering methods have been used to TALE proteins are bacterial effectors. In 2009,
enhance the cleavage efficiency of the FokI the code used by TALE proteins to recognize
cleavage domain (Guo et al. 2010). One par- DNA was uncovered (Boch et al. 2009; Moscou
ticularly promising approach for improving and Bogdanove 2009). In a matter of months,
ZFN specificity is to deliver them into cells as this discovery enabled the creation of custom
protein. Because of the intrinsic cell-penetrat- TALENs capable of modifying nearly any gene.
ing activity of zinc-finger domains (Gaj et al. Like ZFNs, TALENs are modular in form and
2014a), ZFN proteins themselves are inherently function, comprised of an amino-terminal
cell-permeable and can facilitate gene editing TALE DNA-binding domain fused to a car-
with fewer off-target effects when applied di- boxy-terminal FokI cleavage domain (Christian
rectly onto cells as purified protein compared et al. 2010; Miller et al. 2011). Also like ZFNs,

dimerization of TALEN proteins is mediated by amount of time and experience needed to as-
the FokI cleavage domain, which cuts within a semble a functional nuclease. Second, TALENs
12- to 19-bp spacer sequence that separates each have been reported to show improved specificity
TALE binding site (Fig. 1) (Miller et al. 2011). and reduced toxicity compared to some ZFNs
TALEs are typically assembled to recognize (Mussolino et al. 2014), potentially because of
between 12- to 20-bps of DNA, with more bases their increased affinity for target DNA (Meckler
typically leading to higher genome-editing et al. 2013) or perhaps a greater energetic pen-
specificity (Guilinger et al. 2014a). The TALE- alty for associating with base mismatches.
binding domain consists of a series of repeat However, TALENs are substantially larger than
domains, each 34 residues in length. Each re- ZFNs, and have a highly repetitive structure,
peat contacts DNA via the amino acid residues making their efficient delivery into cells through
at positions 12 and 13, known as the repeat the use of lentivirus (Holkers et al. 2013) or a
variable diresidues (RVDs) (Boch et al. 2009; single adeno-associated virus (AAV) particle
Moscou and Bogdanove 2009). Unlike zinc fin- challenging. Methods for overcoming these lim-
gers, which recognize DNA triplets, each TALE itations have emerged as TALENs can be readily
repeat recognizes only a single bp, with little to delivered into cells as mRNA (Mahiny et al.
no target site overlap from adjacent domains 2015; Mock et al. 2015) and even protein (Cai
(Deng et al. 2012; Mak et al. 2012). The most et al. 2014; Liu et al. 2014a), although alterna-
commonly used RVDs for assembling synthetic tive codon usage and amino acid degeneracy
TALE arrays are: NI for adenine, HD for cyto- can also be leveraged to express RVD arrays
sine, NG for thymine, and NN or HN for gua- that might be less susceptible to recombina-
nine or adenine (Boch et al. 2009; Moscou and tion (Kim et al. 2013a). In addition, adenoviral
Bogdanove 2009; Cong et al. 2012; Streubel vectors have also proven particularly useful for
et al. 2012). TALE DNA-binding domains can mediating TALEN delivery to hard-to-transfect
be constructed using a variety of methods, cell types (Holkers et al. 2014; Maggio et al.
with the most straightforward approach being 2016).
Golden Gate assembly (Cermak et al. 2011).
However, high-throughput TALE assembly
CRISPR-Cas9
methods have also been developed, including
FLASH assembly (Reyon et al. 2012), iterative The CRISPR-Cas9 system, which has a role in
capped assembly (Briggs et al. 2012), and liga- adaptive immunity in bacteria (Horvath and
tion independent cloning (Schmid-Burgk et al. Barrangou 2010; Marraffini and Sontheimer
2013), among others. More recent advances 2010), is the most recent addition to the ge-
in TALEN assembly, though, have focused on nome-editing toolbox. In bacteria, the type-II
the development of methods that can enhance CRISPR system provides protection against
their performance, including specificity profil- DNA from invading viruses and plasmids via
ing to uncover nonconventional RVDs that RNA-guided DNA cleavage by Cas proteins
improve TALEN activity (Guilinger et al. 2014a; (Wiedenheft et al. 2012; Sorek et al. 2013). Short
Yang et al. 2014; Juillerat et al. 2015; Miller segments of foreign DNA are integrated within
et al. 2015), directed evolution as means to re- the CRISPR locus and transcribed into CRISPR
fine TALE specificity (Hubbard et al. 2015), and RNA (crRNA), which then anneal to trans-ac-
even fusing TALE domains to homing endonu- tivating crRNA (tracrRNA) to direct sequence-
clease variants to generate chimeric nucleases specific degradation of pathogenic DNA by the
with extended targeting specificity (discussed Cas9 protein (Jinek et al. 2012). In 2012, Char-
in more detail below) (Boissel et al. 2014). pentier, Doudna, and co-workers reported that
Compared to ZFNs, TALENs offer two dis- target recognition by the Cas9 protein only re-
tinct advantages for genome editing. First, no quires a seed sequence within the crRNA and a
selection or directed evolution is necessary to conserved protospacer-adjacent motif (PAM)
engineer TALE arrays, dramatically reducing the upstream of the crRNA binding site (Jinek

T. Gaj et al.
et al. 2012). This system has since been simpli- cificity of this system, including using paired
fied for genome engineering (Cho et al. 2013; Cas9 nickases (Mali et al. 2013a; Ran et al.
Cong et al. 2013; Jinek et al. 2013; Mali et al. 2013), which increase gene-editing specificity
2013b) and now consists of only the Cas9 nu- by requiring the induction of two sequential
clease and a single guide RNA (gRNA) contain- and adjacent nicking events for DSB formation,
ing the essential crRNA and tracrRNA elements or truncated gRNA that are more sensitive to
(Fig. 1). Because target site recognition is me- mismatches at the genomic target site than a
diated entirely by the gRNA, CRISPR-Cas9 has full-length gRNA (Fu et al. 2014). Off-target
emerged as the most flexible and user-friendly cleavage has also been reduced by controlling
platform for genome editing, eliminating the the dosage of either the Cas9 protein or gRNA
need for engineering new proteins to recognize within the cell (Hsu et al. 2013), or even by
each new target site. The only major restriction using Cas9 variants configured to enable con-
for Cas9 target site recognition is that the PAM ditional genome editing, such as a rapamycin-
motif—which is recognized by the Cas9 nucle- inducible split-Cas9 architecture (Zetsche et al.
ase and is essential for DNA cleavage—be locat- 2015b) or a Cas9 variant that contains a strate-
ed immediately downstream of the gRNA target gically placed small-molecule-responsive intein
site. The PAM sequence for the Streptococcus domain (Davis et al. 2015). Nucleofection (Kim
pyogenes Cas9, for example, is 50 -NGG-30 (al- et al. 2014) or transient transfection (Zuris et al.
though in some cases 50 -NAG-30 can be tolerat- 2015) of a preformed Cas9 ribonucleoprotein
ed) (Hsu et al. 2013; Jiang et al. 2013; Mali et al. complex has also been shown to reduce off-
2013a). Several studies have now shed light on target effects, enabling DNA-free gene editing
the structural basis of DNA recognition by Cas9, in primary human T cells (Schumann et al.
revealing that the heteroduplex formed by the 2015), embryonic stem cells (Liu et al. 2015b),
gRNA and its complementary strand of DNA is Caenorhabditis elegans gonads (Paix et al. 2015),
housed in a positively charged groove between mouse (Menoret et al. 2015; Wang et al. 2015a)
the two nuclease domains (RuvC and HNH) and zebrafish embryos (Sung et al. 2014), and
within the Cas9 protein (Nishimasu et al. even plant protoplasts (Woo et al. 2015). The
2014), and that PAM recognition is mediated incorporation of specific chemical modifica-
by an arginine-rich motif present in Cas9 (An- tions known to protect RNA from nuclease deg-
ders et al. 2014). Doudna and colleagues have radation and stabilize secondary structure can
since proposed that DNA strand displacement further enhance Cas9 ribonucleoprotein activi-
induces a structural rearrangement within the ty (Hendel et al. 2015; Rahdar et al. 2015). In a
Cas9 protein that directs the nontarget DNA clever marriage of genome-editing platforms,
strand into the RuvC active site, which then the FokI cleavage domain has even been fused
positions the HNH domain near target DNA to an inactivated Cas9 variant to generate hy-
(Jiang et al. 2016), enabling Cas9-mediated brid nucleases that require protein dimerization
cleavage of both DNA strands. for DNA cleavage (Guilinger et al. 2014b; Tsai
The Cas9 nuclease and its gRNA can be de- et al. 2014), theoretically increasing CRISPR-
livered into cells for genome editing on the same Cas9 specificity. Similarly, fusing Cas9 to
or separate plasmids, and numerous resources DNA-binding domains has also proven effective
have been developed to facilitate target site at improving its specificity (Bolukbasi et al.
selection and gRNA construction, including 2015). Finally, several studies have recently
E-CRISP (Heigwer et al. 2014), among others. showed that protein engineering can broadly
Although Cas9 boasts the highest ease of use enhance Cas9 specificity (Kleinstiver et al.
among the targeted nuclease platforms, several 2016; Slaymaker et al. 2016) and even alter its
reports have indicated that it could be prone to PAM requirements (Kleinstiver et al. 2015), the
inducing off-target mutations (Cradick et al. latter having the potential to enable creation of
2013; Fu et al. 2013). To this end, considerable customized variants of Cas9 for allele-specific
effort has been devoted to improving the spe- gene editing, although Cas9 orthologs (Cong

et al. 2013; Esvelt et al. 2013; Hou et al. 2013; lines carrying nearly any genomic modification
Ran et al. 2015) or alternative CRISPR systems can now be generated in simply a matter of
(Zetsche et al. 2015a) with unique PAM speci- weeks. Examples of some of the outcomes that
ficities have been uncovered in nature. have become routine because of the emergence
of targeted nucleases include gene knockout
Homing Endonucleases (Santiago et al. 2008; Mali et al. 2013b), gene
deletion (Lee et al. 2010), gene inversion (Xiao
Homing endonucleases, also known as mega- et al. 2013), gene correction (Urnov et al. 2005;
nucleases, represent the final member of the Ran et al. 2013), gene addition (Moehle et al.
targeted nuclease family. These enzymes have 2007; Hockemeyer et al. 2011; Hou et al. 2013),
been reviewed at length elsewhere (Silva et al. and even chromosomal translocation (Fig. 2)
2011; Stoddard 2014) but, briefly, members of (Torres et al. 2014). In addition to cell line en-
the LAGLIDADG family of endonucleases— gineering, targeted nucleases have also expedit-
so named for the conserved amino acid motif ed the generation of genetically modified or-
present within these enzymes that interacts with ganisms, facilitating the rapid creation of
DNA—are a collection of naturally occurring transgenic zebrafish (Doyon et al. 2008; Sander
enzymes that recognize and cleave long DNA et al. 2011a; Hwang et al. 2013), mice (Cui et al.
sequences (14 – 40 bps) (Fig. 1). These enzymes 2011; Wang et al. 2013; Wu et al. 2013), rats
make extensive sequence-specific contacts with (Geurts et al. 2009; Tesson et al. 2011; Li et al.
their DNA substrate (Stoddard 2011), and thus 2013), monkeys (Liu et al. 2014c), and livestock
typically show exquisite specificity. However, (Hauschild et al. 2011; Carlson et al. 2012),
unlike ZFNs and TALENs, the binding and which together have the capacity to accelerate
cleavage domains in homing endonucleases human disease modeling and the discovery of
are not modular. This overlap in form and func- new therapeutics.
tion make their repurposing challenging, and Targeted nucleases have also emerged as
limits their utility for more routine applications powerful tools for plant engineering (Baltes
of genome editing. More recently megaTALs— and Voytas 2015). Both TALENs and CRISPR-
fusions of a rare-cleaving homing endonuclease Cas9 have been used to modify multiple alleles
to a TALE-binding domain—have been report- within hexaploid bread wheat to confer herita-
ed to induce highly specific gene modifications ble resistance to powdery mildew (Wang et al.
(Boissel et al. 2014; Lin et al. 2015a). These en- 2014b). In another study, TALENs were used to
zymes have enabled integration of antitumor knock out nonessential genes in the fatty acid
and anti-HIV factors into the human CCR5 metabolic pathway in soybean to generate a sim-
gene in both primary T cells and hematopoietic plified plant cell with reduced metabolic com-
stem/progenitor cells (Sather et al. 2015), as ponents (Haun et al. 2014). Of special note, two
well as disruption of endogenous T-cell receptor recent reports showed that purified nuclease
elements in T cells (Osborn et al. 2016), indi- proteins can be introduced directly into plant
cating their potential for enabling and enhanc- protoplasts, enabling the introduction of germ-
ing immunotherapies. line-transmissible modifications that are virtu-
ally indistinguishable from naturally occurring
GENOME-EDITING APPLICATIONS (Luo et al. 2015; Woo et al. 2015). This technical
advance could help to overcome certain regula-
Engineering Cell Lines and Organisms tory hurdles associated with the use of transgen-
Before the emergence of engineered nucleases, ic crops. Finally, targeted nucleases have also
genetically modifying mammalian cell lines was been used to inactivate pathogenic genes to pre-
labor intensive, costly, and often times limited vent viral (Lin et al. 2014) or parasitic (Ghorbal
to laboratories with specialized expertise. How- et al. 2014) infection, as well as to introduce
ever, with the advent of cost-effective and user- knockin-specific factors capable of imparting
friendly gene-editing technologies, custom cell pathogen resistance (Wu et al. 2015).

T. Gaj et al.
Intriguingly, targeted nucleases could also for functional genomics (Hilton and Gersbach
serve as conduits for curbing mosquito- or in- 2015), having facilitated the discovery of geno-
sect-borne diseases through a technique known mic loci that confer drug resistance to cells
as gene drive (Burt 2003; Sinkins and Gould (Koike-Yusa et al. 2014; Shalem et al. 2014;
2006), which harnesses genome editing to facil- Wang et al. 2014a; Zhou et al. 2014), uncovered
itate the introduction of a specific gene or mu- how cells can control induction of the host
tation that can then confer a particular pheno- immune response (Parnas et al. 2015), provided
type into a host and also be transmitted to its new insights into the genetic basis of cellular
progeny (Windbichler et al. 2011). Gene drives fitness (Hart et al. 2015; Wang et al. 2015b),
have now been tested in the malaria vector mos- and even shed light on how certain viruses in-
quitos Anopheles stephensi (Gantz et al. 2015) duce cell death (Ma et al. 2015). Genome-wide
and Anopheles gambiae (Hammond et al. 2016) CRISPR screens can also facilitate the discovery
as a means for achieving population control of functional noncoding elements (Kim et al.
and to prevent disease transmission, respective- 2013b; Korkmaz et al. 2016), and provide a
ly. However, owing to the ease with which means for studying the structure and evolution
CRISPR-Cas9 can be programmed (Gantz and of the human genome. In a remarkable example
Bier 2015), debate has ignited on the potential of the latter, Shendure and colleagues used Cas9
societal and environmental impact of this tech- to mediate integration of short randomized
nology (Esvelt et al. 2014; Akbari et al. 2015), DNA sequences into the BRCA1 and DBR1
spurring the development of safeguard ele- genes (Findlay et al. 2014). They then measured
ments that could help to minimize the risk of the functional consequences of these mutations
gene-edited organisms escaping from the labo- on fitness, achieving an unprecedented look at
ratory (DiCarlo et al. 2015). some of the factors driving genome and disease
evolution. Finally, CRISPR screens have even
proven effective in vivo, enabling the identifica-
Synthetic Biology and Genome-Scale
tion of factors involved in zebrafish develop-
Engineering
ment (Shah et al. 2015) and disease progression
Targeted nucleases also offer a facile means for in mice (Chen et al. 2015).
generating modified bacterial and yeast strains
for synthetic biology, including metabolic path-
Therapeutic Genome Editing
way engineering. Bacterial species of the order
Actinomycetales, for instance, are one of the Genome editing itself also holds tremendous
most important sources of industrially relevant potential for treating the underlying genetic
secondary metabolites. However, many Actino- causes of certain diseases (Cox et al. 2015; Por-
mycetales species are recalcitrant to genetic teus 2015; Maeder and Gersbach 2016). In one
manipulation, a fact that has severely hampered of the most successful examples of this to date,
their use for metabolic engineering. CRISPR- ZFN-mediated disruption of the HIV corecep-
Cas9 has been used to inactivate multiple genes tor CCR5 was used to engineer HIV resistance
in actinomycetes (Tong et al. 2015), indicating into both CD4þ T cells (Perez et al. 2008) and
its ability to enable the creation of designer CD34þ hematopoietic stem/progenitor cells
bacterial strains with enhanced metabolite (HSPCs) (Holt et al. 2010), proving safe and
production capabilities. CRISPR has also facil- well-tolerated in a phase I clinical trial that in-
itated multiplexed metabolic pathway engi- fused these gene-modified T cells into individ-
neering in yeast at high efficiencies (Jakociunas uals with HIV/AIDS (Tebas et al. 2014). In ad-
et al. 2015a,b), as well as random mutagenesis dition to enabling the introduction of gene
of yeast chromosomal DNA for phenotypic modification that can enhance autologous cell
screening of desired mutants (Ryan et al. therapies, targeted nucleases can also be com-
2014). Indeed, genome-wide CRISPR-based bined with viral vectors—including AAV—to
knockout screens hold tremendous potential mediate genome editing in situ (Gaj et al.

2016). For instance, delivery of an AAV vector domains. Among the first fully synthetic tran-
encoding a ZFN pair designed to target a defec- scriptional effector proteins to be generated
tive copy of the factor IX gene, along with its (Beerli et al. 1998) were those based on the fu-
repair template, led to efficient gene correction sion of engineered zinc-finger proteins with
in mouse liver, increasing factor IX protein pro- either the Herpes simplex– derived transactiva-
duction in both neonatal (Li et al. 2011) and tion domain (Sadowski et al. 1988) or the Krüp-
adult (Anguela et al. 2013) models of the dis- pel-associated box (KRAB) repression protein
ease. In vivo genome editing also recently en- (Margolin et al. 1994). Over the course of the
abled the restoration of dystrophin gene expres- next 15 years, zinc-finger-based transcriptional
sion and the rescue of muscle function in mouse modulators were expanded and featured sever-
models of Duchenne muscular dystrophy (Long al other types of effector domains (Beerli and
et al. 2015; Nelson et al. 2015; Tabebordbar et al. Barbas 2002), including, for example, the
2015). Therapeutic gene editing in a mouse Dnmt3a methyltransferase domain (Rivenbark
model of human hereditary tyrosinemia has et al. 2012; Siddique et al. 2013) and the ten-elev-
also been reported using both hydrodynamic en translocation methylcytosine dioxygenase 1
injection of plasmid DNA encoding CRISPR- (TET1) (Chen et al. 2014), which can modulate
Cas9 (Yin et al. 2014) and by combining nano- transcription via targeted methylation or de-
particle-mediated delivery of Cas9-encoding methylation, respectively. As a natural extension
mRNA with AAV-mediated delivery of the of zinc-finger transcription factors, and further
DNA template for gene correction (Yin et al. drawing on the parallels with zinc-finger pro-
2016). More recently, a dual particle AAV sys- teins, TALE transcription factors have also
tem, wherein one AAV vector carried the Cas9 emerged as an especially effective platform for
nuclease and a second harbored the gRNA and achieving targeted transcriptional modulation
donor repair template, was able to mediate cor- (Miller et al. 2011; Zhang et al. 2011). Effector
rection of a disease-causing mutation in the or- domains are generally fused to the carboxyl ter-
nithine transcarbamylase gene in the liver of a minus of the synthetic TALE array and, contrary
neonatal model of the disease (Yang et al. 2016). to the longer sequence typically required for
This work, in particular, showed that therapeu- efficient modulation by zinc-finger transcrip-
tic levels of gene correction could be achieved in tion factors, TALEs have been reported to regu-
a regenerating tissue even when using multiple late gene expression with as few as 10.5 repeats
AAV particles. Although highly promising, nu- (Boch et al. 2009). Like zinc fingers, TALEs are
merous hurdles still need to be overcome for in also compatible with numerous epigenetic
vivo applications of genome editing to reach its modifiers, including the TET1 hydroxylase cat-
full potential. Chief among these are methods alytic domain (Maeder et al. 2013b) and the
that can facilitate nuclease delivery or expression lysine-specific histone demethylase 1 (LSD1)
to only diseased cells or tissues, and the devel- (Mendenhall et al. 2013) domains, which have
opment of new strategies that can enhance HDR been used for targeted CpG demethylation and
in disease-associated postmitotic cells in vivo. histone demethylation, respectively. In particu-
lar, the ease with which a large number of TALEs
TARGETED TRANSCRIPTION FACTORS can be constructed has enabled the discovery
that tiling a promoter sequence with combina-
Tools for Modulating Gene Expression tions of synthetic transcription factors can lead
The modular qualities of zinc-finger and TALE to a synergistic increase in gene expression
proteins, in addition to the highly flexible DNA (Maeder et al. 2013b; Perez-Pinera et al. 2013).
recognition ability of CRISPR-Cas9, also pro- And, like zinc fingers (Beerli et al. 2000b; Pol-
vide investigators with the ability to modulate lock et al. 2002; Magnenat et al. 2008; Polstein
the expression of nearly any gene from its pro- and Gersbach 2012), TALE activators have also
moter or enhancer sequences via their fusion to been successfully engineered to regulate gene
transcriptional activator and repressor protein expression in response to external (Mercer

T. Gaj et al.
et al. 2014) or endogenous (Li et al. 2012) chem- velopment of second-generation CRISPR acti-
ical stimuli, optical signals (Konermann et al. vators quickly emerged as a highly active area of
2013), and even proteolytic cues (Copeland research. One particularly elegant approach for
et al. 2016; Lonzaric et al. 2016). overcoming the low activation thresholds inher-
Because of the exquisite ease with which it ent within first-generation systems was by stra-
can be programmed, the CRISPR-Cas9 system tegically inserting an RNA aptamer within a
has also been adapted for transcriptional mod- functionally inert region of the gRNA. This ap-
ulation through fusion of specific effector do- tamer recruits specific activation helper pro-
mains to a catalytically inactivated variant of the teins that work in concert with a dCas9 activator
Cas9 protein (Qi et al. 2013). Deactivation is to enhance transcription (Konermann et al.
achieved by introducing two amino acid substi- 2015; Zalatan et al. 2015). Other strategies based
tutions, D10A and H840A, into the RuvC and on directly fusing additional helper activation
NHN endonuclease domains of Cas9, respec- domains to dCas9 have also been shown to en-
tively. Although unable to cleave DNA, this mu- hance transcription (Chavez et al. 2015). Target-
tant, referred to as dCas9, retains its ability to ed acetylation of histone proteins within a pro-
bind DNA in an RNA-directed manner. Effector moter or enhancer sequence via epigenome
domains are fused to the carboxyl terminus of editing using the catalytic core of the human
the dCas9 protein and can modulate gene ex- acetyltransferase p300 fused to dCas9 can also
pression from either strand of the targeted DNA lead to robust levels of gene activation (Hilton
sequence (Farzadfard et al. 2013; Maeder et al. et al. 2015). Similarly, dCas9 repressor proteins
2013a; Perez-Pinera et al. 2013). Genome-scale targeted to distal regulatory elements have been
activation studies have indicated that the most found to facilitate chromatin remodeling and
robust levels of activation are generally observed gene repression via epigenomic modification
when dCas9 activators are targeted to -400 to (Thakore et al. 2015). Finally, by simply reduc-
-50 bp upstream from the transcriptional start ing the length of the gRNA, catalytically active
site (Gilbert et al. 2014; Hu et al. 2014). Addi- variants of Cas9 can stimulate transcription
tionally, dCas9 can inhibit gene expression by without inducing DNA breaks (Dahlman et al.
simply blocking transcriptional initiation or 2015; Kiani et al. 2015), enabling orthogonal
elongation through a process known as CRISPR gene knockout and activation with the same
interference (Qi et al. 2013), although fusing Cas9 variant in a single cell.
dCas9 to transcriptional repressor domains
can also lead to efficient silencing from the pro-
Applications of Targeted Transcriptional
moter (Gilbert et al. 2013; Zalatan et al. 2015).
Regulation
Much like zinc fingers and TALEs, methods for
achieving conditional gene modulation using Early work on the use of engineered zinc-finger
dCas9 have also been reported, including the transcription factors revealed that synthetic
fusion of a dihydrofolate reductase destabiliza- transcriptional modulators are effective tools
tion domain to dCas9, which can provide for a broad range of applications, enabling
chemical control over activation, enabling cel- such tasks as inhibiting viral replication (Pap-
lular reprogramming or differentiation (Balboa worth et al. 2003; Reynolds et al. 2003; Segal
et al. 2015). Light-inducible dCas9-based sys- et al. 2004; Eberhardy et al. 2006), modulating
tems capable of providing optical control of the expression of disease-associated loci (Gras-
gene expression provide another means for lund et al. 2005; Wilber et al. 2010), inducing
achieving conditional control of gene expres- angiogenesis for accelerated wound healing (Re-
sion (Nihongaki et al. 2015; Polstein and Gers- bar et al. 2002), and genomic screening of cel-
bach 2015). lular targets for cancer progression and drug
Although flexible, first-generation dCas9 resistance (Park et al. 2003; Blancafort et al.
activators were routinely found to display sub- 2005, 2008). Facilitated by many of the insights
optimal levels of activation. As a result, the de- gained from zinc-finger transcription factor

technology, both TALEs and CRISPR-Cas9 have DNA sequences (Gaj et al. 2013; Sirk et al. 2014;
now further expanded the possibilities of engi- Wallen et al. 2015) and even integrate therapeu-
neered transcriptional activators and repressors. tic factors into the human genome (Gaj et al.
For example, TALEs and CRISPR-Cas9 have 2014b), are one such option. More recent work
enabled rapid construction of custom genetic has indicated that single-base editing without
circuits and logic gates (Gaber et al. 2014; Lebar DNA breaks can be achieved using an engi-
et al. 2014; Liu et al. 2014b), complex gene neered Cas9 nickase complex (Komor et al.
regulation networks (Nielsen and Voigt 2014; 2016), although it remains unknown how effec-
Nissim et al. 2014), and even facilitated cellular tive this technology is in therapeutically rele-
reprogramming (Gao et al. 2013) and the dif- vant settings. By linking genomic modifications
ferentiation of mouse embryonic fibroblasts to induced by targeted nucleases to their own self-
skeletal myocytes (Chakraborty et al. 2014). degradation, self-inactivating vectors are also
dCas9 transcriptional effectors have even been poised to improve the specificity of genome
used to efficiently mediate repression and acti- editing, especially because the frequency of
vation of endogenous genes in Drosophila (Lin off-target modifications can be directly propor-
et al. 2015b) and in plant cells (Piatek et al. tional to the duration of cellular exposure to a
2015). Both TALE and Cas9 activators have nuclease (Pruett-Miller et al. 2009). In addition,
also been configured to stimulate transcription much of the knowledge behind genome engi-
of latent HIV (Zhang et al. 2015; Ji et al. 2016; neering has been obtained in immortalized cell
Limsirichai et al. 2016; Perdigao et al. 2016; lines. However, in the case of regenerative med-
Saayman et al. 2016), indicating their potential icine, it is highly desirable to genetically manip-
to work in concert with antiretroviral therapy ulate progenitor or stem-cell populations, both
for eradicating HIV infection. Importantly, of which can differ markedly from transformed
because of the ease with which the CRISPR- cell lines with respect to their epigenome or
Cas9 system can be used, genome-wide screens three-dimensional organization of their geno-
using Cas9 transcriptional activators (Gilbert mic DNA. These differences can have profound
et al. 2014; Konermann et al. 2015) and re- effects on the ability of genome-editing tools to
pressors (Gilbert et al. 2014) can be easily either modify a specific sequence or regulate
implemented to discover genes involved in a gene expression. Although the union between
number of diverse processes, including drug genome engineering and regenerative medicine
resistance and cancer metastasis. In particular, is still in its infancy, realizing the full potential of
CRISPR-based genome-scale screening meth- these technologies in stem/progenitor cells re-
ods have the potential to overcome many of quires that their functional landscape be fully
the technical hurdles associated with other explored in these genetic backgrounds. Only
contemporary screening technologies, such as then will genome editing technologies truly be
cDNA libraries and RNAi, indicating its poten- able to reprogram cell fate and behavior for the
tial for facilitating drug discovery and basic next generation of advances in synthetic biology
biological research. and gene therapy.
CONCLUSIONS ACKNOWLEDGMENTS
Despite the successes already achieved, many We gratefully acknowledge the support and
challenges remain before the full potential of mentorship of the late Carlos F. Barbas, III
genome editing can be realized. First and fore- (1964– 2014). This work is supported by the
most are the development of new tools cap- National Institutes of Health (F32GM113446
able of introducing genomic modifications in to T.G.) and ShanghaiTech University (to J.L.).
the absence of DNA breaks. Targeted recombi- We apologize to those whose important contri-
nases (Akopian et al. 2003; Mercer et al. 2012), butions were not cited because of space con-
which can be programmed to recognize specific straints.

T. Gaj et al.
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Genome-Editing Technologies: Principles and Applications

Thomas Gaj, Shannon J. Sirk, Sai-lan Shui and Jia Liu
Cold Spring Harb Perspect Biol 2016; doi: 10.1101/cshperspect.a023754 originally published online
October 6, 2016
Subject Collection Synthetic Biology
Minimal Cells−−Real and Imagined Synthetic DNA Synthesis and Assembly: Putting
John I. Glass, Chuck Merryman, Kim S. Wise, et al. the Synthetic in Synthetic Biology
Randall A. Hughes and Andrew D. Ellington
Synthetic Botany Design Automation in Synthetic Biology
Christian R. Boehm, Bernardo Pollak, Nuri Evan Appleton, Curtis Madsen, Nicholas Roehner,
Purswani, et al. et al.
Synthetic Biology in Cell and Organ Cell-Free Synthetic Biology: Engineering Beyond
Transplantation the Cell
Sean Stevens Jessica G. Perez, Jessica C. Stark and Michael C.
Jewett
Genome-Editing Technologies: Principles and The Need for Integrated Approaches in Metabolic
Applications Engineering
Thomas Gaj, Shannon J. Sirk, Sai-lan Shui, et al. Anna Lechner, Elizabeth Brunk and Jay D. Keasling
Alternative Watson−Crick Synthetic Genetic Synthetic Biology of Natural Products
Systems Rainer Breitling and Eriko Takano
Steven A. Benner, Nilesh B. Karalkar, Shuichi
Hoshika, et al.
Phage Therapy in the Era of Synthetic Biology At the Interface of Chemical and Biological
E. Magda Barbu, Kyle C. Cady and Bolyn Hubby Synthesis: An Expanded Genetic Code
Han Xiao and Peter G. Schultz
Synthetic Morphogenesis Building Spatial Synthetic Biology with
Brian P. Teague, Patrick Guye and Ron Weiss Compartments, Scaffolds, and Communities
Jessica K. Polka, Stephanie G. Hays and Pamela
A. Silver
Engineering Gene Circuits for Mammalian Cell−
Based Applications
Simon Ausländer and Martin Fussenegger
For additional articles in this collection, see http://cshperspectives.cshlp.org/cgi/collection/
Copyright © 2016 Cold Spring Harbor Laboratory Press; all rights reserved

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Genome-Editing Technologies: Principles

Thomas Gaj,1,4 Shannon J. Sirk,2 Sai-lan Shui,3 and Jia Liu3,4

2 Cite this article as Cold Spring Harb Perspect Biol 2016;8:a023754

A Gene knockout Gene deletion Gene inversion

B Gene integration Gene correction

Homology arm Homology arm

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4 Cite this article as Cold Spring Harb Perspect Biol 2016;8:a023754

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6 Cite this article as Cold Spring Harb Perspect Biol 2016;8:a023754

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8 Cite this article as Cold Spring Harb Perspect Biol 2016;8:a023754

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10 Cite this article as Cold Spring Harb Perspect Biol 2016;8:a023754

Cite this article as Cold Spring Harb Perspect Biol 2016;8:a023754 11

REFERENCES Blancafort P, Tschan MP, Bergquist S, Guthy D, Brachat A,

12 Cite this article as Cold Spring Harb Perspect Biol 2016;8:a023754

Cite this article as Cold Spring Harb Perspect Biol 2016;8:a023754 13

14 Cite this article as Cold Spring Harb Perspect Biol 2016;8:a023754

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16 Cite this article as Cold Spring Harb Perspect Biol 2016;8:a023754

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18 Cite this article as Cold Spring Harb Perspect Biol 2016;8:a023754

Cite this article as Cold Spring Harb Perspect Biol 2016;8:a023754 19

20 Cite this article as Cold Spring Harb Perspect Biol 2016;8:a023754

Genome-Editing Technologies: Principles and Applications

Subject Collection Synthetic Biology

For additional articles in this collection, see http://cshperspectives.cshlp.org/cgi/collection/

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