CRISPR-Cas9 For Genome Engineering

Leading Edge
Review
Development and Applications of
CRISPR-Cas9 for Genome Engineering
Patrick D. Hsu,1,2,3 Eric S. Lander,1 and Feng Zhang1,2,*
1Broad
Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA 02141, USA

Institute for Brain Research, Department of Brain and Cognitive Sciences, Department of Biological Engineering,
Massachusetts Institute of Technology, Cambridge, MA 02139, USA
3Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA
*Correspondence: zhang@broadinstitute.org
http://dx.doi.org/10.1016/j.cell.2014.05.010
2McGovern
Recent advances in genome engineering technologies based on the CRISPR-associated RNAguided endonuclease Cas9 are enabling the systematic interrogation of mammalian genome
function. Analogous to the search function in modern word processors, Cas9 can be guided to
specific locations within complex genomes by a short RNA search string. Using this system,
DNA sequences within the endogenous genome and their functional outputs are now easily edited
or modulated in virtually any organism of choice. Cas9-mediated genetic perturbation is simple and
scalable, empowering researchers to elucidate the functional organization of the genome at the
systems level and establish causal linkages between genetic variations and biological phenotypes.
In this Review, we describe the development and applications of Cas9 for a variety of research or
translational applications while highlighting challenges as well as future directions. Derived from a
remarkable microbial defense system, Cas9 is driving innovative applications from basic biology to
biotechnology and medicine.
Introduction
The development of recombinant DNA technology in the 1970s
marked the beginning of a new era for biology. For the first
time, molecular biologists gained the ability to manipulate DNA
molecules, making it possible to study genes and harness
them to develop novel medicine and biotechnology. Recent
advances in genome engineering technologies are sparking a
new revolution in biological research. Rather than studying
DNA taken out of the context of the genome, researchers can
now directly edit or modulate the function of DNA sequences
in their endogenous context in virtually any organism of choice,
enabling them to elucidate the functional organization of the
genome at the systems level, as well as identify causal genetic
variations.
Broadly speaking, genome engineering refers to the process
of making targeted modifications to the genome, its contexts
(e.g., epigenetic marks), or its outputs (e.g., transcripts). The
ability to do so easily and efficiently in eukaryotic and especially
mammalian cells holds immense promise to transform basic science, biotechnology, and medicine (Figure 1).
For life sciences research, technologies that can delete, insert,
and modify the DNA sequences of cells or organisms enable dissecting the function of specific genes and regulatory elements.
Multiplexed editing could further allow the interrogation of
gene or protein networks at a larger scale. Similarly, manipulating transcriptional regulation or chromatin states at particular
loci can reveal how genetic material is organized and utilized
within a cell, illuminating relationships between the architecture
1262 Cell 157, June 5, 2014 2014 Elsevier Inc.
of the genome and its functions. In biotechnology, precise

manipulation of genetic building blocks and regulatory machinery also facilitates the reverse engineering or reconstruction of
useful biological systems, for example, by enhancing biofuel
production pathways in industrially relevant organisms or by
creating infection-resistant crops. Additionally, genome engineering is stimulating a new generation of drug development
processes and medical therapeutics. Perturbation of multiple
genes simultaneously could model the additive effects that underlie complex polygenic disorders, leading to new drug targets,
while genome editing could directly correct harmful mutations in
the context of human gene therapy (Tebas et al., 2014).
Eukaryotic genomes contain billions of DNA bases and are
difficult to manipulate. One of the breakthroughs in genome
manipulation has been the development of gene targeting by
homologous recombination (HR), which integrates exogenous
repair templates that contain sequence homology to the donor
site (Figure 2A) (Capecchi, 1989). HR-mediated targeting has
facilitated the generation of knockin and knockout animal
models via manipulation of germline competent stem cells,
dramatically advancing many areas of biological research. However, although HR-mediated gene targeting produces highly precise alterations, the desired recombination events occur
extremely infrequently (1 in 106109 cells) (Capecchi, 1989), presenting enormous challenges for large-scale applications of
gene-targeting experiments.
To overcome these challenges, a series of programmable
nuclease-based genome editing technologies have been
Figure 1. Applications of Genome Engineering

Genetic and epigenetic control of cells with genome engineering technologies
is enabling a broad range of applications from basic biology to biotechnology
and medicine. (Clockwise from top) Causal genetic mutations or epigenetic
variants associated with altered biological function or disease phenotypes can
now be rapidly and efficiently recapitulated in animal or cellular models (Animal
models, Genetic variation). Manipulating biological circuits could also facilitate
the generation of useful synthetic materials, such as algae-derived, silicabased diatoms for oral drug delivery (Materials). Additionally, precise genetic
engineering of important agricultural crops could confer resistance to environmental deprivation or pathogenic infection, improving food security while
avoiding the introduction of foreign DNA (Food). Sustainable and cost-effective biofuels are attractive sources for renewable energy, which could be
achieved by creating efficient metabolic pathways for ethanol production in
algae or corn (Fuel). Direct in vivo correction of genetic or epigenetic defects in
somatic tissue would be permanent genetic solutions that address the root
cause of genetically encoded disorders (Gene surgery). Finally, engineering
cells to optimize high yield generation of drug precursors in bacterial factories
could significantly reduce the cost and accessibility of useful therapeutics
(Drug development).
developed in recent years, enabling targeted and efficient modification of a variety of eukaryotic and particularly mammalian
species. Of the current generation of genome editing technologies, the most rapidly developing is the class of RNA-guided
endonucleases known as Cas9 from the microbial adaptive immune system CRISPR (clustered regularly interspaced short
palindromic repeats), which can be easily targeted to virtually
any genomic location of choice by a short RNA guide. Here,
we review the development and applications of the CRISPRassociated endonuclease Cas9 as a platform technology for
achieving targeted perturbation of endogenous genomic elements and also discuss challenges and future avenues for innovation.
Programmable Nucleases as Tools for Efficient and
Precise Genome Editing
A series of studies by Haber and Jasin (Rudin et al., 1989; Plessis
et al., 1992; Rouet et al., 1994; Choulika et al., 1995; Bibikova
et al., 2001; Bibikova et al., 2003) led to the realization that tar-
geted DNA double-strand breaks (DSBs) could greatly stimulate

genome editing through HR-mediated recombination events.
Subsequently, Carroll and Chandrasegaran demonstrated the
potential of designer nucleases based on zinc finger proteins
for efficient, locus-specific HR (Bibikova et al., 2001, 2003).
Moreover, it was shown in the absence of an exogenous homology repair template that localized DSBs can induce insertions or
deletion mutations (indels) via the error-prone nonhomologous
end-joining (NHEJ) repair pathway (Figure 2A) (Bibikova et al.,
2002). These early genome editing studies established DSBinduced HR and NHEJ as powerful pathways for the versatile
and precise modification of eukaryotic genomes.
To achieve effective genome editing via introduction of sitespecific DNA DSBs, four major classes of customizable DNAbinding proteins have been engineered so far: meganucleases
derived from microbial mobile genetic elements (Smith et al.,
2006), zinc finger (ZF) nucleases based on eukaryotic transcription factors (Urnov et al., 2005; Miller et al., 2007), transcription
activator-like effectors (TALEs) from Xanthomonas bacteria
(Christian et al., 2010; Miller et al., 2011; Boch et al., 2009; Moscou and Bogdanove, 2009), and most recently the RNA-guided
DNA endonuclease Cas9 from the type II bacterial adaptive immune system CRISPR (Cong et al., 2013; Mali et al., 2013a).
Meganuclease, ZF, and TALE proteins all recognize specific
DNA sequences through protein-DNA interactions. Although
meganucleases integrate its nuclease and DNA-binding
domains, ZF and TALE proteins consist of individual modules
targeting 3 or 1 nucleotides (nt) of DNA, respectively
(Figure 2B). ZFs and TALEs can be assembled in desired combinations and attached to the nuclease domain of FokI to direct
nucleolytic activity toward specific genomic loci. Each of these
platforms, however, has unique limitations.
Meganucleases have not been widely adopted as a genome
engineering platform due to lack of clear correspondence
between meganuclease protein residues and their target DNA
sequence specificity. ZF domains, on the other hand, exhibit
context-dependent binding preference due to crosstalk between
adjacent modules when assembled into a larger array (Maeder
et al., 2008). Although multiple strategies have been developed
to account for these limitations (Gonzaelz et al., 2010; Sander
et al., 2011), assembly of functional ZFPs with the desired DNA
binding specificity remains a major challenge that requires an
extensive screening process. Similarly, although TALE DNAbinding monomers are for the most part modular, they can still
suffer from context-dependent specificity (Juillerat et al., 2014),
and their repetitive sequences render construction of novel
TALE arrays labor intensive and costly.
Given the challenges associated with engineering of modular
DNA-binding proteins, new modes of recognition would significantly simplify the development of custom nucleases. The
CRISPR nuclease Cas9 is targeted by a short guide RNA that
recognizes the target DNA via Watson-Crick base pairing
(Figure 2C). The guide sequence within these CRISPR RNAs
typically corresponds to phage sequences, constituting the natural mechanism for CRISPR antiviral defense, but can be easily
replaced by a sequence of interest to retarget the Cas9
nuclease. Multiplexed targeting by Cas9 can now be achieved
at unprecedented scale by introducing a battery of short guide
Cell 157, June 5, 2014 2014 Elsevier Inc. 1263
Figure 2. Genome Editing Technologies

Exploit Endogenous DNA Repair Machinery
(A) DNA double-strand breaks (DSBs) are typically
repaired by nonhomologous end-joining (NHEJ) or
homology-directed repair (HDR). In the errorprone NHEJ pathway, Ku heterodimers bind to
DSB ends and serve as a molecular scaffold for
associated repair proteins. Indels are introduced
when the complementary strands undergo end
resection and misaligned repair due to microhomology, eventually leading to frameshift mutations and gene knockout. Alternatively, Rad51
proteins may bind DSB ends during the initial
phase of HDR, recruiting accessory factors that
direct genomic recombination with homology
arms on an exogenous repair template. Bypassing
the matching sister chromatid facilitates the
introduction of precise gene modifications.
(B) Zinc finger (ZF) proteins and transcription
activator-like effectors (TALEs) are naturally
occurring DNA-binding domains that can be
modularly assembled to target specific sequences. ZF and TALE domains each recognize 3
and 1 bp of DNA, respectively. Such DNA-binding
proteins can be fused to the FokI endonuclease to
generate programmable site-specific nucleases.
(C) The Cas9 nuclease from the microbial CRISPR
adaptive immune system is localized to specific
DNA sequences via the guide sequence on its
guide RNA (red), directly base-pairing with the
DNA target. Binding of a protospacer-adjacent
motif (PAM, blue) downstream of the target locus
helps to direct Cas9-mediated DSBs.
RNAs rather than a library of large, bulky proteins. The ease of

Cas9 targeting, its high efficiency as a site-specific nuclease,
and the possibility for highly multiplexed modifications have
opened up a broad range of biological applications across basic
research to biotechnology and medicine.
The utility of customizable DNA-binding domains extends far
beyond genome editing with site-specific endonucleases.
Fusing them to modular, sequence-agnostic functional effector
domains allows flexible recruitment of desired perturbations,
such as transcriptional activation, to a locus of interest (Xu and
Bestor, 1997; Beerli et al., 2000a; Konermann et al., 2013;
Maeder et al., 2013a; Mendenhall et al., 2013). In fact, any
modular enzymatic component can, in principle, be substituted,
allowing facile additions to the genome engineering toolbox.
Integration of genome- and epigenome-modifying enzymes
with inducible protein regulation further allows precise temporal
control of dynamic processes (Beerli et al., 2000b; Konermann
et al., 2013).
CRISPR-Cas9: From Yogurt to Genome Editing
The recent development of the Cas9 endonuclease for genome
editing draws upon more than a decade of basic research into
understanding the biological function of the mysterious repetitive
elements now known as CRISPR (Figure 3), which are found
throughout the bacterial and archaeal diversity. CRISPR loci
typically consist of a clustered set of CRISPR-associated (Cas)
genes and the signature CRISPR arraya series of repeat
sequences (direct repeats) interspaced by variable sequences
(spacers) corresponding to sequences within foreign genetic
elements (protospacers) (Figure 4). Whereas Cas genes are
translated into proteins, most CRISPR arrays are first tran1264 Cell 157, June 5, 2014 2014 Elsevier Inc.
scribed as a single RNA before subsequent processing into

shorter CRISPR RNAs (crRNAs), which direct the nucleolytic
activity of certain Cas enzymes to degrade target nucleic acids.
The CRISPR story began in 1987. While studying the iap
enzyme involved in isozyme conversion of alkaline phosphatase
in E. coli, Nakata and colleagues reported a curious set of 29 nt
repeats downstream of the iap gene (Ishino et al., 1987). Unlike
most repetitive elements, which typically take the form of tandem
repeats like TALE repeat monomers, these 29 nt repeats were
interspaced by five intervening 32 nt nonrepetitive sequences.
Over the next 10 years, as more microbial genomes were
sequenced, additional repeat elements were reported from
genomes of different bacterial and archaeal strains. Mojica and
colleagues eventually classified interspaced repeat sequences
as a unique family of clustered repeat elements present in
>40% of sequenced bacteria and 90% of archaea (Mojica
et al., 2000).
These early findings began to stimulate interest in such microbial repeat elements. By 2002, Jansen and Mojica coined the
acronym CRISPR to unify the description of microbial genomic
loci consisting of an interspaced repeat array (Jansen et al.,
2002; Barrangou and van der Oost, 2013). At the same time,
several clusters of signature CRISPR-associated (cas) genes
were identified to be well conserved and typically adjacent to
the repeat elements (Jansen et al., 2002), serving as a basis for
the eventual classification of three different types of CRISPR
systems (types IIII) (Haft et al., 2005; Makarova et al., 2011b).
Types I and III CRISPR loci contain multiple Cas proteins, now
known to form complexes with crRNA (CASCADE complex for
type I; Cmr or Csm RAMP complexes for type III) to facilitate
the recognition and destruction of target nucleic acids (Brouns
Figure 3. Key Studies Characterizing and Engineering CRISPR Systems

Cas9 has also been referred to as Cas5, Csx12, and Csn1 in literature prior to 2012. For clarity, we exclusively adopt the Cas9 nomenclature throughout this
Review. CRISPR, clustered regularly interspaced short palindromic repeats; Cas, CRISPR-associated; crRNA, CRISPR RNA; DSB, double-strand break;
tracrRNA, trans-activating CRISPR RNA.
et al., 2008; Hale et al., 2009) (Figure 4). In contrast, the type II
system has a significantly reduced number of Cas proteins.
However, despite increasingly detailed mapping and annotation
of CRISPR loci across many microbial species, their biological
significance remained elusive.
A key turning point came in 2005, when systematic analysis of
the spacer sequences separating the individual direct repeats
suggested their extrachromosomal and phage-associated origins (Mojica et al., 2005; Pourcel et al., 2005; Bolotin et al.,
2005). This insight was tremendously exciting, especially given
previous studies showing that CRISPR loci are transcribed
(Tang et al., 2002) and that viruses are unable to infect archaeal
cells carrying spacers corresponding to their own genomes
(Mojica et al., 2005). Together, these findings led to the speculation that CRISPR arrays serve as an immune memory and
defense mechanism, and individual spacers facilitate defense
against bacteriophage infection by exploiting Watson-Crick
base-pairing between nucleic acids (Mojica et al., 2005; Pourcel
et al., 2005). Despite these compelling realizations that CRISPR
loci might be involved in microbial immunity, the specific mechanism of how the spacers act to mediate viral defense remained
a challenging puzzle. Several hypotheses were raised, including
thoughts that CRISPR spacers act as small RNA guides to
degrade viral transcripts in a RNAi-like mechanism (Makarova

et al., 2006) or that CRISPR spacers direct Cas enzymes to
cleave viral DNA at spacer-matching regions (Bolotin et al.,
2005).
Working with the dairy production bacterial strain Streptococcus thermophilus at the food ingredient company Danisco,
Horvath and colleagues uncovered the first experimental
evidence for the natural role of a type II CRISPR system as an
adaptive immunity system, demonstrating a nucleic-acid-based
immune system in which CRISPR spacers dictate target specificity while Cas enzymes control spacer acquisition and phage
defense (Barrangou et al., 2007). A rapid series of studies illuminating the mechanisms of CRISPR defense followed shortly and
helped to establish the mechanism as well as function of all three
types of CRISPR loci in adaptive immunity. By studying the type I
CRISPR locus of Escherichia coli, van der Oost and colleagues
showed that CRISPR arrays are transcribed and converted into
small crRNAs containing individual spacers to guide Cas
nuclease activity (Brouns et al., 2008). In the same year,
CRISPR-mediated defense by a type III-A CRISPR system
from Staphylococcus epidermidis was demonstrated to block
plasmid conjugation, establishing the target of Cas enzyme
activity as DNA rather than RNA (Marraffini and Sontheimer,
Figure 4. Natural Mechanisms of Microbial

CRISPR Systems in Adaptive Immunity
Following invasion of the cell by foreign genetic
elements from bacteriophages or plasmids (step
1: phage infection), certain CRISPR-associated
(Cas) enzymes acquire spacers from the exogenous protospacer sequences and install them into
the CRISPR locus within the prokaryotic genome
(step 2: spacer acquisition). These spacers are
segregated between direct repeats that allow the
CRISPR system to mediate self and nonself
recognition. The CRISPR array is a noncoding
RNA transcript that is enzymatically maturated
through distinct pathways that are unique to each
type of CRISPR system (step 3: crRNA biogenesis
and processing).
In types I and III CRISPR, the pre-crRNA transcript
is cleaved within the repeats by CRISPR-associated ribonucleases, releasing multiple small
crRNAs. Type III crRNA intermediates are further
processed at the 30 end by yet-to-be-identified
RNases to produce the fully mature transcript. In
type II CRISPR, an associated trans-activating
CRISPR RNA (tracrRNA) hybridizes with the direct
repeats, forming an RNA duplex that is cleaved
and processed by endogenous RNase III and
other unknown nucleases. Maturated crRNAs
from type I and III CRISPR systems are then
loaded onto effector protein complexes for target
recognition and degradation. In type II systems,
crRNA-tracrRNA hybrids complex with Cas9 to
mediate interference.
Both type I and III CRISPR systems use multiprotein interference modules to facilitate target
recognition. In type I CRISPR, the Cascade complex is loaded with a crRNA molecule, constituting
a catalytically inert surveillance complex that recognizes target DNA. The Cas3 nuclease is then
recruited to the Cascade-bound R loop, mediating
target degradation. In type III CRISPR, crRNAs associate either with Csm or Cmr complexes that bind and cleave DNA and RNA substrates, respectively. In
contrast, the type II system requires only the Cas9 nuclease to degrade DNA matching its dual guide RNA consisting of a crRNA-tracrRNA hybrid.
2008), although later investigation of a different type III-B system

from Pyrococcus furiosus also revealed crRNA-directed RNA
cleavage activity (Hale et al., 2009, 2012).
As the pace of CRISPR research accelerated, researchers
quickly unraveled many details of each type of CRISPR system
(Figure 4). Building on an earlier speculation that protospaceradjacent motifs (PAMs) may direct the type II Cas9 nuclease to
cleave DNA (Bolotin et al., 2005), Moineau and colleagues highlighted the importance of PAM sequences by demonstrating that
PAM mutations in phage genomes circumvented CRISPR interference (Deveau et al., 2008). Additionally, for types I and II, the
lack of PAM within the direct repeat sequence within the CRISPR
array prevents self-targeting by the CRISPR system. In type III
systems, however, mismatches between the 50 end of the crRNA
and the DNA target are required for plasmid interference (Marraffini and Sontheimer, 2010).
By 2010, just 3 years after the first experimental evidence for
CRISPR in bacterial immunity, the basic function and mechanisms of CRISPR systems were becoming clear. A variety of
groups had begun to harness the natural CRISPR system for
various biotechnological applications, including the generation
of phage-resistant dairy cultures (Quiberoni et al., 2010) and
phylogenetic classification of bacterial strains (Horvath et al.,
2008, 2009). However, genome editing applications had not
yet been explored.
Around this time, two studies characterizing the functional

mechanisms of the native type II CRISPR system elucidated
the basic components that proved vital for engineering a simple
RNA-programmable DNA endonuclease for genome editing.
First, Moineau and colleagues used genetic studies in Streptococcus thermophilus to reveal that Cas9 (formerly called
Cas5, Csn1, or Csx12) is the only enzyme within the cas
gene cluster that mediates target DNA cleavage (Garneau
et al., 2010). Next, Charpentier and colleagues revealed a
key component in the biogenesis and processing of crRNA
in type II CRISPR systemsa noncoding trans-activating
crRNA (tracrRNA) that hybridizes with crRNA to facilitate
RNA-guided targeting of Cas9 (Deltcheva et al., 2011). This
dual RNA hybrid, together with Cas9 and endogenous RNase
III, is required for processing the CRISPR array transcript
into mature crRNAs (Deltcheva et al., 2011). These two studies
suggested that there are at least three components (Cas9,
the mature crRNA, and tracrRNA) that are essential for reconstituting the type II CRISPR nuclease system. Given the
increasing importance of programmable site-specific nucleases
based on ZFs and TALEs for enhancing eukaryotic genome
editing, it was tantalizing to think that perhaps Cas9 could
be developed into an RNA-guided genome editing system.
From this point, the race to harness Cas9 for genome editing
was on.
In 2011, Siksnys and colleagues first demonstrated that the

type II CRISPR system is transferrable, in that transplantation
of the type II CRISPR locus from Streptococcus thermophilus
into Escherichia coli is able to reconstitute CRISPR interference
in a different bacterial strain (Sapranauskas et al., 2011). By
2012, biochemical characterizations by the groups of Charpentier, Doudna, and Siksnys showed that purified Cas9 from Streptococcus thermophilus or Streptococcus pyogenes can be
guided by crRNAs to cleave target DNA in vitro (Jinek et al.,
2012; Gasiunas et al., 2012), in agreement with previous bacterial studies (Garneau et al., 2010; Deltcheva et al., 2011; Sapranauskas et al., 2011). Furthermore, a single guide RNA (sgRNA)
can be constructed by fusing a crRNA containing the targeting
guide sequence to a tracrRNA that facilitates DNA cleavage by
Cas9 in vitro (Jinek et al., 2012).
In 2013, a pair of studies simultaneously showed how to successfully engineer type II CRISPR systems from Streptococcus
thermophilus (Cong et al., 2013) and Streptococcus pyogenes
(Cong et al., 2013; Mali et al., 2013a) to accomplish genome
editing in mammalian cells. Heterologous expression of mature
crRNA-tracrRNA hybrids (Cong et al., 2013) as well as sgRNAs
(Cong et al., 2013; Mali et al., 2013a) directs Cas9 cleavage
within the mammalian cellular genome to stimulate NHEJ or
HDR-mediated genome editing. Multiple guide RNAs can also
be used to target several genes at once. Since these initial
studies, Cas9 has been used by thousands of laboratories for
genome editing applications in a variety of experimental model
systems (Sander and Joung, 2014). The rapid adoption of the
Cas9 technology was also greatly accelerated through a combination of open-source distributors such as Addgene, as well
as a number of online user forums such as http://www.
genome-engineering.org and http://www.egenome.org.
Structural Organization and Domain Architecture of
Cas9
The family of Cas9 proteins is characterized by two signature
nuclease domains, RuvC and HNH, each named based on
homology to known nuclease domain structures (Figure 2C).
Though HNH is a single nuclease domain, the full RuvC domain
is divided into three subdomains across the linear protein
sequence, with RuvC I near the N-terminal region of Cas9 and
RuvC II/III flanking the HNH domain near the middle of the protein. Recently, a pair of structural studies shed light on the structural mechanism of RNA-guided DNA cleavage by Cas9.
First, single-particle EM reconstructions of the Streptococcus
pyogenes Cas9 (SpCas9) revealed a large structural rearrangement between apo-Cas9 unbound to nucleic acid and Cas9 in
complex with crRNA and tracrRNA, forming a central channel
to accommodate the RNA-DNA heteroduplex (Jinek et al.,
2014). Second, a high-resolution structure of SpCas9 in complex
with sgRNA and the complementary strand of target DNA further
revealed the domain organization to comprise of an a-helical
recognition (REC) lobe and a nuclease (NUC) lobe consisting of
the HNH domain, assembled RuvC subdomains, and a PAMinteracting (PI) C-terminal region (Nishimasu et al., 2014)
(Figure 5A and Movie S1).
Together, these two studies support the model that SpCas9
unbound to target DNA or guide RNA exhibits an autoinhibited
conformation in which the HNH domain active site is blocked

by the RuvC domain and is positioned away from the REC lobe
(Jinek et al., 2014). Binding of the RNA-DNA heteroduplex would
additionally be sterically inhibited by the orientation of the C-terminal domain. As a result, apo-Cas9 likely cannot bind nor cleave
target DNA. Like many ribonucleoprotein complexes, the guide
RNA serves as a scaffold around which Cas9 can fold and organize its various domains (Nishimasu et al., 2014).
The crystal structure of SpCas9 in complex with an sgRNA and
target DNA also revealed how the REC lobe facilitates target
binding. An arginine-rich bridge helix (BH) within the REC lobe
is responsible for contacting the 30 812 nt of the RNA-DNA heteroduplex (Nishimasu et al., 2014), which correspond with the
seed sequence identified through guide sequence mutation experiments (Jinek et al., 2012; Cong et al., 2013; Fu et al., 2013;
Hsu et al., 2013; Pattanayak et al., 2013; Mali et al., 2013b).
The SpCas9 structure also provides a useful scaffold for engineering or refactoring of Cas9 and sgRNA. Because the REC2
domain of SpCas9 is poorly conserved in shorter orthologs,
domain recombination or truncation is a promising approach
for minimizing Cas9 size. SpCas9 mutants lacking REC2 retain
roughly 50% of wild-type cleavage activity, which could be partly
attributed to their weaker expression levels (Nishimasu et al.,
2014). Introducing combinations of orthologous domain recombination, truncation, and peptide linkers could facilitate the
generation of a suite of Cas9 mutant variants optimized for
different parameters such as DNA binding, DNA cleavage, or
overall protein size.
Metagenomic, Structural, and Functional Diversity of
Cas9
Cas9 is exclusively associated with the type II CRISPR locus and
serves as the signature type II gene. Based on the diversity of
associated Cas genes, type II CRISPR loci are further subdivided
into three subtypes (IIAIIC) (Figure 5B) (Makarova et al., 2011a;
Chylinski et al., 2013). Type II CRISPR loci mostly consist of the
cas9, cas1, and cas2 genes, as well as a CRISPR array and
tracrRNA. Type IIC CRISPR systems contain only this minimal
set of cas genes, whereas types IIA and IIB have an additional
signature csn2 or cas4 gene, respectively (Chylinski et al., 2013).
Subtype classification of type II CRISPR loci is based on the
architecture and organization of each CRISPR locus. For
example, type IIA and IIB loci usually consist of four cas genes,
whereas type IIC loci only contain three cas genes. However,
this classification does not reflect the structural diversity of
Cas9 proteins, which exhibit sequence homology and length
variability irrespective of the subtype classification of their
parental CRISPR locus. Of >1,000 Cas9 nucleases identified
from sequence databases (UniProt) based on homology, protein
length is rather heterogeneous, roughly ranging from 900 to 1600
amino acids (Figure 5C). The length distribution of most Cas9
proteins can be divided into two populations centered around
1,100 and 1,350 amino acids in length. It is worth noting that a
third population of large Cas9 proteins belonging to subtype
IIA, formerly called Csx12, typically contain around 1500 amino
acids.
Despite the apparent diversity of protein length, all Cas9 proteins share similar domain architecture (Makarova et al., 2011a;
Figure 5. Structural and Metagenomic Diversity of Cas9 Orthologs

(A) Crystal structure of Streptococcus pyogenes Cas9 in complex with guide RNA and target DNA.
(B) Canonical CRISPR locus organization from type II CRISPR systems, which can be classified into IIA-IIC based on their cas gene clusters. Whereas type IIC
CRISPR loci contain the minimal set of cas9, cas1, and cas2, IIA and IIB retain their signature csn2 and cas4 genes, respectively.
(C) Histogram displaying length distribution of known Cas9 orthologs as described in UniProt, HAMAP protein family profile MF_01480.
(D) Phylogenetic tree displaying the microbial origin of Cas9 nucleases from the type II CRISPR immune system. Taxonomic information was derived from
greengenes 16S rRNA gene sequence alignment, and the tree was visualized using the Interactive Tree of Life tool (iTol).
(E) Four Cas9 orthologs from families IIA, IIB, and IIC were aligned by ClustalW (BLOSUM). Domain alignment is based on the Streptococcus pyogenes Cas9,
whereas residues highlighted in red indicate highly conserved catalytic residues within the RuvC I and HNH nuclease domains.
Chylinski et al., 2013, 2014; Fonfara et al., 2014), consisting of

the RuvC and HNH nuclease domains and the REC domain, an
a-helix-rich region with an Arg-rich bridge helix. Unlike type I
and III CRISPR systems, which are found in both bacteria and
archaea, type II CRISPRs have so far only been found in bacterial
strains (Chylinski et al., 2013). The majority of Cas9 orthologs in
fact belong to the phyla of Bacteroidetes, Proteobacteria, and
Firmicutes (Figure 5D).
The length difference among Cas9 proteins largely results
from variable conservation of the REC domain (Figure 5E), which
associates with the sgRNA and target DNA. For example, the
type IIC Actinomyces naeslundii Cas9, which is more compact
than its Streptococcus pyogenes ortholog, has a much smaller
REC lobe with substantially different orientation (Jinek et al.,
2014).
Protospacer Adjacent Motif: Cas9 Target Range and
Search Mechanism
A critical feature of the Cas9 system is the protospacer-adjacent
motif (PAM), which flanks the 30 end of the DNA target site
(Figure 2C) and dictates the DNA target search mechanism of
Cas9. In addition to facilitating self versus non-self discrimination
by Cas9 (Shah et al., 2013), because direct repeats do not
contain PAM sites, biochemical and structural characterization
of SpCas9 suggested that PAM recognition is involved in triggering the transition between Cas9 target binding and cleavage
conformations (Sternberg et al., 2014; Jinek et al., 2014; Nishimasu et al., 2014).
Single-molecule imaging indicated that Cas9-crRNAtracrRNA complexes first associate with PAM sequences
throughout the genome, allowing Cas9 to initiate DNA strand
separation via unknown mechanisms (Sternberg et al., 2014).
DNA competitor cleavage assays additionally suggested that
formation of the RNA-DNA heteroduplex is initiated at the PAM
site before proceeding PAM distally by interrogating the target
site upstream of the PAM for guide sequence complementarity
(Sternberg et al., 2014). Binding of the PAM and a matching
target then triggers Cas9 nuclease activity by activating the
HNH and RuvC domains, supported by the observation of
HNH domain flexibility within the Cas9-sgRNA-DNA ternary
complex (Nishimasu et al., 2014).
The complexity of the PAM sequences also determines the
overall DNA targeting space of Cas9. For example, the 50 -NGG
of SpCas9 allows it to target, on average, every 8 bp within the
human genome (Cong et al., 2013; Hsu et al., 2013). Additionally,
SpCas9 can target sites flanked by 50 -NAG PAMs (Jiang et al.,
2013; Hsu et al., 2013), albeit at a lower efficiency, further
expanding its editing versatility. The PAM is specific to each
Cas9 ortholog, even within the same species, such as 50 -NNA
GAAW for Streptococcus thermophilus CRISPR1 (Deveau
et al., 2008) and 50 -NGGNG for Streptococcus thermophilus
CRISPR3 (Horvath et al., 2008). Another Cas9 from Neisseria
meningitidis with a 50 -NNNNGATT PAM requirement (Zhang
et al., 2013) was recently applied in human pluripotent stem cells
(Hou et al., 2013).
Computational (Chylinski et al., 2013, 2014; Fonfara et al.,
2014) or metagenomic analysis of bacteria and archaea containing CRISPR loci could lead to the discovery of Cas9 nucleases
with additional PAMs to expand the targeting range of the

Cas9 toolkit. Delivery of multiple Cas9 proteins with different
PAM requirements facilitates orthogonal genome engineering,
in which independent but simultaneous functions are applied
at different loci within the same cell or cell population. NmCas9
and SpCas9, for example, can be employed for independent
transcriptional repression and nuclease activity (Esvelt et al.,
2013).
PAM specificity can also be modified. For instance, orthologous replacement of the PAM-interacting (PI) domain from
the Streptococcus thermophilus CRISPR3 Cas9 with the corresponding domain from Streptococcus pyogenes Cas9 successfully altered PAM recognition from 50 -NGGNG to 50 -NGG
(Nishimasu et al., 2014). PAM engineering strategies could also
be exploited to generate short Cas9 orthologs with flexible 50 NGG or 50 -NG PAM domains.
Genome Editing Using CRISPR-Cas9 in Eukaryotic Cells
To date, the Streptococcus pyogenes Cas9 (SpCas9) has been
used broadly to achieve efficient genome editing in a variety of
species and cell types, including human cell lines, bacteria, zebrafish, yeast, mouse, fruit fly, roundworm, rat, common crops,
pig, and monkey (see Sander and Joung [2014] for a detailed
list). SpCas9 is also dramatically expanding the catalog of genetically tractable model organisms, for example, by enabling the
introduction of multiplex mutations in cynomolgus monkeys
(Niu et al., 2014).
SpCas9 can be targeted either with a pair of crRNA and
tracrRNA (Cong et al., 2013) or with a chimeric sgRNA (Cong
et al., 2013; Mali et al., 2013a; Cho et al., 2013; Jinek et al.,
2013), as the crRNA or sgRNA contains a 20 nt guide sequence
that directly matches the target site. The only requirement for the
selection of Cas9 target sites is the presence of a protospaceradjacent motif (PAM) immediately downstream of the target site.
An early discrepancy in the use of SpCas9 editing of the
human genome was the drastically higher levels of NHEJinduced indels given the same target site, when using the engineered dual guide RNA system (Cong et al., 2013) compared
to the engineered sgRNA(+48) scaffold, which only contained
up to the 48th base of tracrRNA. Although sgRNA(+48) is sufficient for cleaving DNA in vitro (Jinek et al., 2012), extension of
the 30 tracrRNA sequence preserved several hairpin structures
(sgRNA(+72) and sgRNA(+84)) that were critical for effective
sgRNA-mediated genome editing in vivo (Mali et al., 2013a;
Hsu et al., 2013). The additional stem loops enhance the stability
of the sgRNA (Hsu et al., 2013) and are important for proper
Cas9-sgRNA-DNA ternary complex formation (Nishimasu
et al., 2014). These analyses of the sgRNA structure and function
indicate that careful sgRNA design is critical for optimal Cas9
activity, especially for testing novel Cas9 candidates derived
from metagenomic analysis.
One hallmark of the natural CRISPR-Cas9 system is its
inherent ability to efficiently cleave multiple distinct target
sequences in parallel (Barrangou et al., 2007; Garneau et al.,
2010) by converting a pre-crRNA transcript containing many
spacers into individual guide RNAs duplexes (mature crRNA
and tracrRNA) through hybridization with tracrRNA (Deltcheva
et al., 2011). Harnessing this unique aspect of CRISPR
interference would enable highly scalable multiplex perturbations. Indeed, coexpression of a CRISPR array containing
spacers targeting different genes (Cong et al., 2013) or a battery
of several sgRNAs (Mali et al., 2013a; Wang et al., 2013) together
with SpCas9 has led to efficient multiplex editing in mammalian
cells. Surprisingly, CRISPR arrays containing direct repeats
interspaced by designer spacers were processed into mature
guide RNAs without the introduction of bacterial RNase III.
Because RNase III is required for crRNA maturation in prokaryotic cells (Deltcheva et al., 2011), it is likely that endogenous
mammalian RNases play compensatory roles (Cong et al., 2013).
Specificity of Cas9 Nucleases
Because genome editing leads to permanent modifications
within the genome, the targeting specificity of Cas9 nucleases
is of particular concern, especially for clinical applications and
gene therapy. A combination of in vitro and in vivo assays has
been typically used to characterize the specificity of ZFNs and
TALENs (Gabriel et al., 2011), but systematic analysis has
remained challenging due to difficulties in synthesizing large
libraries of proteins with varying sequence specificity. However,
Cas9 target recognition is dictated by the Watson-Crick basepairing interactions of an RNA guide with its DNA target, enabling
experimentally tractable and systematic evaluation of the effect
of guide RNA-target DNA mismatches on Cas9 activity.
Streptococcus pyogenes Cas9 specificity has been extensively characterized by multiple groups using mismatched guide
RNA libraries, in vitro selection, and reporter assays (Fu et al.,
2013; Hsu et al., 2013; Mali et al., 2013b; Pattanayak et al.,
2013). In contrast to previous studies that suggested a seed
sequence model for Cas9 specificity, wherein the first 812
PAM-proximal guide sequence bases determine specificity
(Jinek et al., 2012; Cong et al., 2013), these studies collectively
demonstrate that Cas9 tolerates mismatches throughout the
guide sequence in a manner that is sensitive to the number,
position, and distribution of the mismatches (Fu et al., 2013;
Hsu et al., 2013; Mali et al., 2013b; Pattanayak et al., 2013).
Although the PAM-distal bases of the guide sequence are less
important for specificity, meaning that mismatches at those
positions often do not abolish Cas9 activity, all positions within
the guide contribute to overall specificity. Importantly, off-target
sites followed by the 50 -NAG PAM can also lead to off-target
cleavage, demonstrating the importance of considering both
NGG and NAG PAMs in off-target analysis.
Interestingly, Cas9 requires extensive homology between the
guide RNA and target DNA in order to cleave but can remain
semi-transiently bound with only a short stretch of complementary sequence between the guide RNA and target DNA. These
observations suggest that Cas9 has many off-target binding
sites but cleaves only a small fraction of them (Wu et al., 2014).
Thus, concerns about off-target activity could vary widely given
a desired application that exploits Cas9 for its DNA binding or
cleavage capabilities.
Enzymatic concentration is also an important factor in determining Cas9 off-target mutagenesis. This is particularly important because Cas9 can tolerate even five mismatches within
the target site (Fu et al., 2013). Mismatches appear to be better
tolerated when Cas9 is present at high concentrations (Hsu
et al., 2013; Pattanayak et al., 2013), leading to higher off-target

activity; decreasing Cas9 concentration significantly improves
the on- to off-target ratio at the expense of the efficiency of
on-target cleavage (Hsu et al., 2013). The duration of Cas9
expression is likely an additional factor that tunes off-target
activity, though its contributions remain to be carefully investigated.
While potential off-target sites have typically been computationally determined by searching for genomic sequences with
high sequence similarity to the desired target locus, wholegenome sequencing or other unbiased ways of labeling DNA
DSBs genome-wide may illuminate off-target sites that are not
predictable by first-order sequence comparison. Unbiased
genome-wide characterizations have been previously used to
characterize ZFN off-target mutagenesis (Gabriel et al., 2011)
and could easily be adapted for Cas9 nuclease activity. Such
data, perhaps in combination with thermodynamic characterization of guide RNA and target DNA hybridization, will likely provide a quantitative framework for assessing and predicting the
off-target activity of Cas9. Multiple groups now provide Cas9
target selection tools (e.g., http://tools.genome-engineering.
org, http://zifit.partners.org, and http://www.e-crisp.org).
Improving Cas9 Target Recognition Fidelity
Cas9 nucleases cleave DNA through the activity of their RuvC
and HNH nuclease domains, each of which nicks a strand of
DNA to generate a blunt-ended DSB (Figure 2C). SpCas9 can
be converted into a DNA nickase that creates a singlestranded break (SSB) by catalytically inactivating the RuvC or
HNH nuclease domains (Gasiunas et al., 2012; Jinek et al.,
2012; Sapranauskas et al., 2011) via point mutations
(Figure 6A). Because DNA single-strand breaks are repaired
via the high-fidelity base excision repair (BER) pathway (Dianov
and Hubscher, 2013), Cas9 nickases can be exploited for more
specific NHEJ as well as HR.
To improve on-target DSB specificity, a double-nicking
approach analogous to dimeric ZFNs or TALENs can be used
to increase the overall number of bases that are specifically
recognized in the target DNA. Using pairs of guide RNAs (Mali
et al., 2013b; Ran et al., 2013) and an SpCas9 HNH+/RuvC nickase mutant (D10A), properly spaced cooperative nicks can
mimic DSBs and mediate efficient indel formation (Figure 6B).
Because off-target nick sites are precisely repaired, this
multiplexed nicking strategy can improve specificity by up to
1,5003 relative to the wild-type Cas9 (Ran et al., 2013).
Because Cas9 nuclease or multiplex nicking activity both stimulate NHEJ, a population of cells cotargeted with a homology
donor would eventually possess a mix of indel mutants and
donor integrants. Single DNA nicks, however, are also able to
mediate donor recombination, albeit at a lower level than with
DSBs (Hsu et al., 2013). Cas9 nickases with single sgRNAs
can thus be used to mediate HR rather than NHEJ. Furthermore,
off-target integration is highly unlikely due to long homology
arms flanking the donor cassette.
In addition to the double-nicking strategy, sgRNAs truncated
by 2 or 3 nt have been reported to significantly increase targeting
specificity of SpCas9, potentially due to greater mismatch sensitivity (Fu et al., 2014). These truncated sgRNAs can be combined
Figure 6. Applications of Cas9 as a Genome

Engineering Platform
(A) The Cas9 nuclease cleaves DNA via its RuvC
and HNH nuclease domains, each of which nicks a
DNA strand to generate blunt-end DSBs. Either
catalytic domain can be inactivated to generate
nickase mutants that cause single-strand DNA
breaks.
(B) Two Cas9 nickase complexes with appropriately spaced target sites can mimic targeted DSBs
via cooperative nicks, doubling the length of target
recognition without sacrificing cleavage efficiency.
(C) Expression plasmids encoding the Cas9 gene
and a short sgRNA cassette driven by the U6 RNA
polymerase III promoter can be directly transfected into cell lines of interest.
(D) Purified Cas9 protein and in vitro transcribed
sgRNA can be microinjected into fertilized zygotes
for rapid generation of transgenic animal models.
(E) For somatic genetic modification, high-titer
viral vectors encoding CRISPR reagents can be
transduced into tissues or cells of interest.
(F) Genome-scale functional screening can be
facilitated by mass synthesis and delivery of guide
RNA libraries.
(G) Catalytically dead Cas9 (dCas9) can be converted into a general DNA-binding domain and
fused to functional effectors such as transcriptional activators or epigenetic enzymes. The
modularity of targeting and flexible choice of
functional domains enable rapid expansion of the
Cas9 toolbox.
(H) Cas9 coupled to fluorescent reporters facilitates live imaging of DNA loci for illuminating the
dynamics of genome architecture.
(I) Reconstituting split fragments of Cas9 via
chemical or optical induction of heterodimer
domains, such as the cib1/cry2 system from Arabidopsis, confers temporal control of dynamic
cellular processes.
with multiplex nicking to further reduce off-target mutagenesis

(Fu et al., 2014). Future structure-function analyses and Cas9
and protein engineering via rational design or directed evolution
may lead to further improvements in Cas9 specificity.
Applications of Cas9 in Research, Medicine, and
Biotechnology
Cas9 can be used to facilitate a wide variety of targeted genome
engineering applications. The wild-type Cas9 nuclease has
enabled efficient and targeted genome modification in many
species that have been intractable using traditional genetic
manipulation techniques. The ease of retargeting Cas9 by simply
designing a short RNA sequence also enables large-scale unbiased genome perturbation experiments to probe gene function
or elucidate causal genetic variants. In addition to facilitating covalent genome modifications, the wild-type Cas9 nuclease can
also be converted into a generic RNA-guided homing device
(dCas9) by inactivating the catalytic domains. The use of effector
fusions can greatly expand the repertoire of genome engineering
modalities achievable using Cas9. For example, a variety of proteins or RNAs can be tethered to Cas9 or sgRNA to alter transcription states of specific genomic loci, monitor chromatin
states, or even rearrange the three-dimensional organization of

the genome.
Rapid Generation of Cellular and Animal Models
Cas9-mediated genome editing has enabled accelerated generation of transgenic models and expands biological research
beyond traditional, genetically tractable animal model organisms
(Sander and Joung, 2014). By recapitulating genetic mutations
found in patient populations, CRISPR-based editing could be
used to rapidly model the causal roles of specific genetic variations instead of relying on disease models that only phenocopy a
particular disorder. This could be applied to develop novel transgenic animal models (Wang et al., 2013; Niu et al., 2014), to
engineer isogenic ES and iPS cell disease models with specific
mutations introduced or corrected, respectively, or in vivo and
ex vivo gene correction (Schwank et al., 2013; Wu et al., 2013).
For generation of cellular models, Cas9 can be easily introduced into the target cells using transient transfection of plasmids carrying Cas9 and the appropriately designed sgRNA
(Figure 6C). Additionally, the multiplexing capabilities of Cas9
offer a promising approach for studying common human
diseasessuch as diabetes, heart disease, schizophrenia, and
autismthat are typically polygenic. Large-scale genome-wide
association studies (GWAS), for example, have identified haplotypes that show strong association with disease risk. However, it
is often difficult to determine which of several genetic variants in
tight linkage disequilibrium with the haplotype or which of several
genes in the region are responsible for the phenotype. Using
Cas9, one could study the effect of each individual variant or
test the effect of manipulating each individual gene on an
isogenic background by editing stem cells and differentiating
them into cell types of interest.
For generation of transgenic animal models, Cas9 protein and
transcribed sgRNA can be directly injected into fertilized zygotes
to achieve heritable gene modification at one or multiple alleles
in models such as rodents and monkeys (Wang et al., 2013; Li
et al., 2013; Yang et al., 2013; Niu et al., 2014) (Figure 6D). By
bypassing the typical ES cell targeting stage in generating transgenic lines, the generation time for mutant mice and rats can be
reduced from more than a year to only several weeks. Such
advances will facilitate cost-effective and large-scale in vivo
mutagenesis studies in rodent models and can be combined
with highly specific editing (Fu et al., 2014; Ran et al., 2013) to
avoid confounding off-target mutagenesis. Successful multiplex
targeting in cynomolgus monkey models was also recently reported (Niu et al., 2014), suggesting the potential for establishing
more accurate modeling of complex human diseases such as
neuropsychiatric disorders using primate models. Additionally,
Cas9 could be harnessed for direct modification of somatic tissue, obviating the need for embryonic manipulation (Figure 6E)
as well as enabling therapeutic use for gene therapy.
One outstanding challenge with transgenic animal models
generated via zygotic injection of CRISPR reagents is genetic
mosaicism, partly due to a slow rate of nuclease-induced
mutagenesis. Studies to date have typically relied on the injection of Cas9 mRNA into zygotes (fertilized embryos at the
single-cell stage). However, because transcription and translation activity is suppressed in the mouse zygote, Cas9 mRNA
translation into active enzymatic form is likely delayed until
after the first cell division (Oh et al., 2000). Because NHEJmediated repair is thought to introduce indels of random
length, this translation delay likely plays a major role in contributing to genetic mosaicism in CRISPR-modified mice. To overcome this limitation, Cas9 protein and sgRNA could be directly
injected into single-cell fertilized embryos. The high rate of
nonmutagenic repair by the NHEJ process may additionally
contribute to undesired mosaicism because introducing indels
that mutate the Cas9 recognition site would then have to
compete with zygotic division rates. To increase the mutagenic
activity of NHEJ, a pair of sgRNAs flanking a small fragment of
the target gene may be used to increase the probability of gene
disruption.
Functional Genomic Screens
The efficiency of genome editing with Cas9 makes it possible
to alter many targets in parallel, thereby enabling unbiased
genome-wide functional screens to identify genes that play an
important role in a phenotype of interest. Lentiviral delivery of
sgRNAs directed against all genes (either together with Cas9
or to cells already expressing Cas9) can be used to perturb thousands of genomic elements in parallel. Recent papers have
demonstrated the ability to perform robust negative and positive
selection screens in human cells (Wang et al., 2014; Shalem

et al., 2014) by introducing loss-of-function mutations into early,
constitutive coding exons of a different gene in each cell
(Figure 6F). Genome-wide loss-of-function screens have previously used RNAi, but this approach leads to only partial
knockdown, has extensive off-target effects, and is limited to
transcribed (and usually protein-coding) genes. By contrast,
Cas9-mediated pooled sgRNA screens have been shown to provide increased screening sensitivity as well as consistency and
can be designed to target nearly any DNA sequence (Shalem
et al., 2014).
Future applications of single sgRNA libraries may also enable
the perturbation of noncoding genetic elements, while multiplex
sgRNA delivery may be used to dissect the function of large
genomic regions through tiled microdeletions. For example, systematic targeting of gene regulatory regions could facilitate the
discovery of distant enhancers, general promoter architectures,
and any additional regulatory elements that have an effect on
protein levels. An additional application could be to dissect large,
uncharacterized genomic regions that are implicated in
sequencing studies or GWAS.
Tethering dCas9 to different effector domains may also facilitate genomic screens beyond loss-of-function phenotypes.
dCas9 fused to epigenetic modifiers, for instance, could be
used to study the effects of methylation or certain chromatin
states on cellular differentiation or disease pathologies, whereas
transcriptional activators allow screening for gain-of-function
phenotypes. Using truncated sgRNAs or building redundancy
with several sgRNAs targeting each locus would be important
design principles for filtering out false positive signals and
improving the interpretability of screening data.
Transcriptional Modulation
dCas9 binding alone to DNA elements may repress transcription
by sterically hindering RNA polymerase machinery (Qi et al.,
2013), likely by stalling transcriptional elongation. This
CRISPR-based interference, or CRISPRi, works efficiently in
prokaryotic genomes but is less effective in eukaryotic cells
(Gilbert et al., 2013). The repressive function of CRISPRi can
be enhanced by tethering dCas9 to transcriptional repressor
domains such as KRAB or SID effectors, which promote epigenetic silencing (Gilbert et al., 2013; Konermann et al., 2013).
However, dCas9-mediated transcriptional repression needs to
be further improvedin the current generation of dCas9-based
eukaryotic transcription repressors, even the addition of helper
functional domains results in only partial transcriptional knockdown (Gilbert et al., 2013; Konermann et al., 2013).
Cas9 can also be converted into a synthetic transcriptional
activator by fusing it to VP16/VP64 or p65 activation domains
(Figure 6G). In general, targeting Cas9 activators with a single
sgRNA to a particular endogenous gene promoter leads to
only modest transcriptional upregulation (Konermann et al.,
2013; Maeder et al., 2013b; Perez-Pinera et al., 2013; Mali
et al., 2013b). By tiling a promoter with multiple sgRNAs, several
groups have reported strong synergistic effects with nonlinear
increases in activation (Perez-Pinera et al., 2013; Maeder et al.,
2013b; Mali et al., 2013b). Although the requirement for multiple
sgRNAs to achieve efficient transcription activation is potentially
advantageous for increased specificity, screening applications
employing libraries of sgRNAs will require highly efficient and

specific transcriptional control using individual guide RNAs.
Epigenetic Control
Complex genome functions are defined by the highly dynamic
landscape of epigenetic states. Epigenetic modifications that
tune histones are thus crucial for transcriptional regulation and
play important roles in a variety of biological functions. These
marks, such as DNA methylation or histone acetylation, are
established and maintained in mammalian cells by a variety of
enzymes that are recruited to specific genomic loci either directly
or indirectly through scaffolding proteins.
Previously, zinc finger proteins and TAL effectors have been
used in a small number of proof-of-concept studies to achieve
locus-specific targeting of epigenetic modifying enzymes (Beerli
et al., 2000a; Konermann et al., 2013; Maeder et al., 2013a; Mendenhall et al., 2013). Cas9 epigenetic effectors (epiCas9s) that
can artificially install or remove specific epigenetic marks at specific loci would serve as a more flexible platform to probe the
causal effects of epigenetic modifications in shaping the regulatory networks of the genome (Figure 6G). Of course, the potential
for off-target activity or crosstalk between effector domains and
endogenous epigenetic complexes would need to be carefully
characterized. One solution could be to harness prokaryotic
epigenetic enzymes to develop orthogonal epigenetic regulatory
systems that minimize crosstalk with endogenous proteins.
Live Imaging of the Cellular Genome
The spatial organization of functional and structural elements
within the cell contribute to the functional output of genomes,
which can be amplified or suppressed dynamically. However,
the way that genomes are modified and how their structural
organization in vivo modulates functional output remain unclear.
Genomic loci located megabases apart or on entirely different
chromosomes could be brought into close proximity given
appropriate chromosomal organization, thus mediating longrange trans interactions.
Studying the interactions of specific genes given changing
chromatin states would require a robust method to visualize
DNA in living cells. Traditional techniques for labeling DNA,
such as fluorescence in situ hybridization (FISH), require sample
fixation and are thus unable to capture live processes. Fluorescently tagged Cas9 labeling of specific DNA loci was recently
developed as a powerful live-cell-imaging alternative to DNAFISH (Chen et al., 2013) (Figure 6H). Advances in orthogonal
Cas9 proteins or modified sgRNAs will build out multi-color
and multi-locus capabilities to enhance the utility of CRISPRbased imaging for studying complex chromosomal architecture
and nuclear organization.
Inducible Regulation of Cas9 Activity
By exploiting the bilobed structure of Cas9, it may be possible to
split the protein into two units and control their reassembly
via small-molecule or light-inducible heterodimeric domains
(Figure 6I). Small-molecule induction would facilitate systemic
control of Cas9 in patients or animal models, whereas optical
regulation enables more spatially precise perturbation. For
example, the light-inducible dimerization domains CIB1 and
CRY2 or chemically inducible analogs ABI and PYL, which have
been successfully used to construct inducible TALEs (Konermann
et al., 2013), may be adapted to engineer inducible Cas9 systems.
Future Development of Cas9-Based Genome

Engineering Technologies
Unbiased Analysis of Cas9 Binding and Cleavage
Despite the rapid adoption of Cas9 as a platform technology for
genetic and epigenetic perturbation and significant progress in
understanding and improving Cas9 specificity, its on- and offtarget DNA binding and cleavage profiles still need to be thoroughly evaluated. Studies to date characterizing Cas9 off-target
activity have relied on in silico computational prediction or in vitro
selection. As a result, they have been unable to account for the
likelihood of Cas9 activity that is unpredictable by sequence homology to the sgRNA guide sequence.
Because the off-target activity of dCas9 binding for effector
domain localization may be much more extensive than Cas9mediated genome editing, unbiased profiling methods are
needed to refine our understanding of true positive Cas9
off-target activity that actually leads to undesired functional
outcomes. Cas9-based chromatin immunoprecipitation
sequencing (ChIP-seq) analysis at multiple target sites could
be a high-throughput solution for understanding binding degeneracy (Wu et al., 2014), whereas techniques for detecting and
labeling double-strand breaks (Crosetto et al., 2013) will help
to achieve a comprehensive map of Cas9-induced off-target
indels.
These data together will help to generate predictive models for
minimizing off-target activity in gene therapeutics or other applications requiring high levels of precision. Understanding Cas9
binding and cleavage in the context of chromatin accessibility
and epigenetic states will also inform better computational evaluation of guide RNA specificity. For example, particular sgRNAs
could be evaluated based on the genomic nature of its off-target
sites, which would vary by guide sequence. Degenerate targeting of transcriptionally silent genes for a cell type or tissue of interest would likely be preferred to off-target sites in the coding
region of essential housekeeping genes.
Although it is still unclear how Cas9 is affected by chromatin
accessibility and heterochromatin versus euchromatin, dCas9
transcriptional activators can upregulate transcription at sites
lacking DNase I hypersensitivity sites, indicating successful
binding to inaccessible chromatin (Perez-Pinera et al., 2013)
(Figure 6G). CpG methylation does not appear to affect DNA
cleavage in vitro, and Cas9 could introduce indels at a highly
methylated promoter in vivo (Hsu et al., 2013). It will be important
to evaluate Cas9 binding and cleavage of genomic loci in relevant primary cells with different chromatin states, ideally in postmitotic cells in which genomic architecture is stably defined.
Overall, these efforts aimed at improving our understanding of
Cas9 binding and cleavage specificity will complement existing
methods (Mali et al., 2013b; Ran et al., 2013; Fu et al., 2014) as
well as future protein engineering and metagenomic mining
efforts to improve Cas9 specificity and the selection of guide
RNA target sites.
Development of Versatile Delivery and Expression
Systems for Applications of Cas9
Viral vectors such as adeno-associated virus (AAV) or lentivirus
are commonly used for delivering genes of interest in vivo or
into cell types resistant to common transfection methods, such
as immune cells. AAV vectors have been commonly used for
attractive candidates for efficient gene delivery in vivo because

of their low immunogenic potential, reduced oncogenic risk
from host-genome integration, and well-characterized serotype
specificity (Figure 6E). However, the most commonly used
Cas9 nuclease-encoding gene from Streptococcus pyogenes
is >4 kb in length, which is difficult to transduce using AAV due
to its 4.7 kb packaging capacity. Non-viral approaches for introducing CRISPR reagents in vivo present a fertile ground for
developing novel delivery strategies, from liposomes and aptamers to cell-penetrating peptides and the molecular Trojan
horse (Niewoehner et al., 2014).
However, viral approaches are still highly desirable due to their
low immunogenicity and wide array of characterized tropisms.
The size constraints of viral vectors can be sidestepped by using
significantly smaller Cas9 orthologs derived from metagenomic
discovery, several of which have already been characterized
and validated in human cells (Cong et al., 2013; Hou et al.,
2013; Esvelt et al., 2013). Interestingly, short Cas9 variants
reported to date recognize much longer PAM sequences than
SpCas9 (50 -NNAGAAW from Streptococcus thermophilus
CRISPR1 or 50 -NNNNGATT from Neisseria meningitidis) (Zhang
et al., 2013; Garneau et al., 2010), whereas some longer orthologs have more relaxed PAMs (50 -NG from Francisella novicida)
(Fonfara et al., 2014). Although the effect of PAM restriction on
DNA targeting specificity remains to be investigated, the more
limited overall targeting range of short Cas9 variants may be
partially compensated for by decreasing the number of potential
off-target substrates genome-wide.
Moving beyond Endogenous Cellular Repair
The current generation of genome editing technologies depends
on the endogenous DNA repair machinery to introduce loss-offunction mutations or precise modifications (Figure 2A). Although
Cas9 can be used to generate indel mutations via NHEJ with high
efficiency, the absolute rate of HDR remains relatively low.
Although it is sufficient for the generation of cell lines, especially
when paired with drug selection or FACS enrichment, poor rates
of recombination greatly limit the practical utility of Cas9mediated targeted gene insertion in fertilized zygotes or somatic
tissue. Homologous recombination proteins are also mainly expressed in the G2 phase of the cell cycle, making HDR-based
gene editing difficult in postmitotic cells such as neurons or
cardiac myocytes. As a result, methods for stimulating HDRbased repair or alternative strategies for efficient gene insertion
are urgently needed. For instance, the highly efficient DNA damage repair system in Deinococcus radiodurans (Zahradka et al.,
2006) may be exploited to enable efficient genome editing in
mitotic as well as postmitotic cells.
Furthermore, the majority of CRISPR-based technology development has focused on the signature Cas9 nuclease from type II
CRISPR systems. However, there remains a wide diversity of
CRISPR types and functions. Cas RAMP module (Cmr) proteins
identified in Pyrococcus furiosus and Sulfolobus solfataricus
(Hale et al., 2012) constitute an RNA-targeting CRISPR immune
system, forming a complex guided by small CRISPR RNAs that
target and cleave complementary RNA instead of DNA. Cmr protein homologs can be found throughout bacteria and archaea,
typically relying on a 50 site tag sequence on the target-matching
crRNA for Cmr-directed cleavage.
Unlike RNAi, which is targeted largely by a 6 nt seed region and

to a lesser extent 13 other bases, Cmr crRNAs contain 3040 nt
of target complementarity. Cmr-CRISPR technologies for RNA
targeting are thus a promising target for orthogonal engineering
and minimal off-target modification. Although the modularity of
Cmr systems for RNA-targeting in mammalian cells remains to
be investigated, Cmr complexes native to P. furiosus have
already been engineered to target novel RNA substrates (Hale
et al., 2009, 2012).
Cas9 as a Therapeutic Molecule for Treating Genetic
Disorders
Although Cas9 has already been widely used as a research tool,
a particularly exciting future direction is the development of Cas9
as a therapeutic technology for treating genetic disorders. For a
monogenic recessive disorder due to loss-of-function mutations
(such as cystic fibrosis, sickle-cell anemia, or Duchenne
muscular dystrophy), Cas9 may be used to correct the causative
mutation. This has many advantages over traditional methods of
gene augmentation that deliver functional genetic copies via viral
vector-mediated overexpressionparticularly that the newly
functional gene is expressed in its natural context. For dominant-negative disorders in which the affected gene is haplosufficient (such as transthyretin-related hereditary amyloidosis or
dominant forms of retinitis pigmentosum), it may also be
possible to use NHEJ to inactivate the mutated allele to achieve
therapeutic benefit. For allele-specific targeting, one could
design guide RNAs capable of distinguishing between singlenucleotide polymorphism (SNP) variations in the target gene,
such as when the SNP falls within the PAM sequence.
Some monogenic diseases also result from duplication of
genomic sequences. For these diseases, the multiplexing capability of Cas9 may be exploited for deletion of the duplicated
elements. For example, trinucleotide repeat disorders could be
treated using two simultaneous DSBs to excise the repeat
region. The success of this strategy will likely be higher for
diseases such as Friedreichs ataxia, in which duplications occur
in noncoding regions of the target gene, because NHEJ-mediated repair may lead to imperfect or frameshifted repair junctions.
In addition to repairing mutations underlying inherited disorders, Cas9-mediated genome editing might be used to introduce
protective mutations in somatic tissues to combat nongenetic or
complex diseases. For example, NHEJ-mediated inactivation of
the CCR5 receptor in lymphocytes (Lombardo et al., 2007) may
be a viable strategy for circumventing HIV infection, whereas
deletion of PCSK9 (Cohen et al., 2005) or angiopoietin (Musunuru
et al., 2010) may provide therapeutic effects against statin-resistant hypercholesterolemia or hyperlipidemia. Although these
targets may be also addressed using siRNA-mediated protein
knockdown, a unique advantage of NHEJ-mediated gene inactivation is the ability to achieve permanent therapeutic benefit
without the need for continuing treatment. As with all gene therapies, it will of course be important to establish that each proposed therapeutic use has a favorable benefit-risk ratio.
Cas9 could be used beyond the direct genome modification of
somatic tissue, such as for engineering therapeutic cells.
Chimeric antigen receptor (CAR) T cells can be modified
ex vivo and reinfused into a patient to specifically target certain

cancers (Couzin-Frankel, 2013). The ease of design and testing
of Cas9 may also facilitate the treatment of highly rare genetic
variants through personalized medicine. Supporting these
tremendous possibilities are a number of animal model studies
as well as clinical trials using programmable nucleases that
already provide important insights into the future development
of Cas9-based therapeutics.
Recently, hydrodynamic delivery of plasmid DNA encoding
Cas9 and sgRNA along with a repair template into the liver of
an adult mouse model of tyrosinemia was shown to be able to
correct the mutant Fah gene and rescue expression of the
wild-type Fah protein in 1 out of 250 cells (Yin et al., 2014). In
addition, clinical trials successfully used ZF nucleases to combat
HIV infection by ex vivo knockout of the CCR5 receptor. In all
patients, HIV DNA levels decreased, and in one out of four
patients, HIV RNA became undetectable (Tebas et al., 2014).
Both of these results demonstrate the promise of programmable
nucleases as a new therapeutic platform.
However, numerous challenges still lie ahead. Most importantly, successful clinical translation will depend on appropriate
and efficacious delivery systems to target specific disease
tissues. To achieve high levels of therapeutic efficacy and simultaneously address a broad spectrum of genetic disorders,
homologous recombination efficiency will need to be significantly improved. Although permanent genome modification
has advantages over monoclonal antibody or siRNA treatments,
which require repeated administration of the therapeutic molecule, the long-term implications remain unclear. As researchers
further develop and test Cas9 toward clinical translation, it will
be paramount to thoroughly characterize the safety as well as
physiological effects of Cas9 using a variety of preclinical
models.
Conclusions
The story of how a mysterious prokaryotic viral defense system
became one of the most powerful and versatile platforms for
engineering biology highlights the importance of basic science
research. Just as recombinant DNA technology benefited from
basic investigation of the restriction enzymes that are central
to warfare between phage and bacteria, the latest generation
of Cas9-based genome engineering tools are also based on
components from the microbial antiphage defense system. It is
highly likely that the future solutions for efficient and precise
gene modification will be found in as of yet unexplored corners
of the rich biological diversity of nature.
SUPPLEMENTAL INFORMATION
CRISPR community for this beautiful story. P.D.H. is a James Mills Pierce
Fellow. This work is supported by the NIMH through a NIH Directors Pioneer
Award (DP1-MH100706), the NINDS through a NIH Transformative R01 grant
(R01-NS 07312401), NSF, the Keck, McKnight, Damon Runyon, Searle
Scholars, Klingenstein, Vallee, Merkin, and Simons Foundations, and Bob
Metcalfe. CRISPR reagents are available to the academic community through
Addgene, and associated protocols, support forum, and computational tools
are available via the Zhang lab website (http://www.genome-engineering.org).
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CRISPR-Cas9 For Genome Engineering

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Leading Edge

Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA 02141, USA

of the genome and its functions. In biotechnology, precise

Figure 1. Applications of Genome Engineering

geted DNA double-strand breaks (DSBs) could greatly stimulate

Figure 2. Genome Editing Technologies

RNAs rather than a library of large, bulky proteins. The ease of

scribed as a single RNA before subsequent processing into

Figure 3. Key Studies Characterizing and Engineering CRISPR Systems

degrade viral transcripts in a RNAi-like mechanism (Makarova

Figure 4. Natural Mechanisms of Microbial

2008), although later investigation of a different type III-B system

Around this time, two studies characterizing the functional

In 2011, Siksnys and colleagues first demonstrated that the

conformation in which the HNH domain active site is blocked

Figure 5. Structural and Metagenomic Diversity of Cas9 Orthologs

1268 Cell 157, June 5, 2014 2014 Elsevier Inc.

Chylinski et al., 2013, 2014; Fonfara et al., 2014), consisting of

with additional PAMs to expand the targeting range of the

et al., 2013; Pattanayak et al., 2013), leading to higher off-target

Figure 6. Applications of Cas9 as a Genome

with multiplex nicking to further reduce off-target mutagenesis

states, or even rearrange the three-dimensional organization of

selection screens in human cells (Wang et al., 2014; Shalem

employing libraries of sgRNAs will require highly efficient and

Future Development of Cas9-Based Genome

attractive candidates for efficient gene delivery in vivo because

Unlike RNAi, which is targeted largely by a 6 nt seed region and

ex vivo and reinfused into a patient to specifically target certain

Cell 157, June 5, 2014 2014 Elsevier Inc. 1275

resulting from frequent nonsense mutations in PCSK9. Nat. Genet. 37,

1276 Cell 157, June 5, 2014 2014 Elsevier Inc.

Pourcel, C., Salvignol, G., and Vergnaud, G. (2005). CRISPR elements in

Mojica, F.J., Dez-Villasenor, C., Garca-Martnez, J., and Soria, E. (2005).

Cell 157, June 5, 2014 2014 Elsevier Inc. 1277

1278 Cell 157, June 5, 2014 2014 Elsevier Inc.

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