CRISPR -cas9 genome editng

JACRISPRCas911.pdf

Home >Biology homework help >CRISPR -cas9 genome editng

Development and Applications of CRISPR-Cas9 for Genome Engineering

Patrick D. Hsu1,2,3, Eric S. Lander1, and Feng Zhang1,2,*

1Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA 02141, USA

2McGovern 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

Abstract

Recent advances in genome engineering technologies based on the CRISPR-associated RNA-

guided 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.

Supplemental Information Supplemental Information includes one movie and can be found with this article at http://dx.doi.org/ 10.1016/j.cell.2014.05.010.

HHS Public Access Author manuscript Cell. Author manuscript; available in PMC 2015 February 27.

Published in final edited form as: Cell. 2014 June 5; 157(6): 1262–1278. doi:10.1016/j.cell.2014.05.010.

A u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

http://dx.doi.org/10.1016/j.cell.2014.05.010

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 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 106–109 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 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 CRISPR-associated endonuclease Cas9 as a platform

technology for achieving targeted perturbation of endogenous genomic elements and also

discuss challenges and future avenues for innovation.

Hsu et al. Page 2

Cell. Author manuscript; available in PMC 2015 February 27.

A u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

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 targeted 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 DSB-induced HR and NHEJ as powerful pathways for the

versatile and precise modification of eukaryotic genomes.

To achieve effective genome editing via introduction of site-specific DNA DSBs, four major

classes of customizable DNA-binding 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 dueto 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 DNA-binding 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

Hsu et al. Page 3

Cell. Author manuscript; available in PMC 2015 February 27.

A u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

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 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 array—a 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 transcribed 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

Hsu et al. Page 4

Cell. Author manuscript; available in PMC 2015 February 27.

A u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

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 I–III) (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 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 inadaptive 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, 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 protospacer

Hsu et al. Page 5

Cell. Author manuscript; available in PMC 2015 February 27.

A u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

adjacent 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 5′ 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 systems—a

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).

Hsu et al. Page 6

Cell. Author manuscript; available in PMC 2015 February 27.

A u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

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 α-helical recognition (REC) lobe and a nuclease

(NUC) lobe consisting of the HNH domain, assembled RuvC subdomains, and a PAM-

interacting (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. Asaresult, 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 incomplex 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 3′ 8–12 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).

Hsu et al. Page 7

Cell. Author manuscript; available in PMC 2015 February 27.

A u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

http://www.genome-engineering.org

http://www.egenome.org

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 IICRISPR loci are further

subdivided into three subtypes (IIA–IIC) (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 israther 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; Chylinski et al., 2013, 2014; Fonfara et al., 2014),

consisting of the RuvC and HNH nuclease domains and the REC domain, an α-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).

Hsu et al. Page 8

Cell. Author manuscript; available in PMC 2015 February 27.

A u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

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 3′ 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-crRNA-tracrRNA 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 5′-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 5′-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 5′-NNA GAAW for Streptococcus

thermophilus CRISPR1 (Deveau et al., 2008) and 5′-NGGNG for Streptococcus

thermophilus CRISPR3 (Horvath et al., 2008). Another Cas9 from Neisseria meningitidis

with a 5′-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 5′-NGGNG to 5′-NGG (Nishimasu et al., 2014). PAM engineering

strategies could also be exploited to generate short Cas9 orthologs with flexible 5′-NGG or

5′-NG PAM domains.

Hsu et al. Page 9

Cell. Author manuscript; available in PMC 2015 February 27.

A u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

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

protospacer-adjacent 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 NHEJ-induced 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 3′ 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

Hsu et al. Page 10

Cell. Author manuscript; available in PMC 2015 February 27.

A u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

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 base-pairing 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

8–12 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 5′-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

Hsu et al. Page 11

Cell. Author manuscript; available in PMC 2015 February 27.

A u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

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 single-stranded 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 Hübscher, 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 1,500× 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 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 co-valent 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

Hsu et al. Page 12

Cell. Author manuscript; available in PMC 2015 February 27.

A u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

http://tools.genome-engineering

http://zifit.partners.org

http://www.e-crisp.org

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 diseases—such as diabetes, heart disease, schizophrenia, and

autism—that 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

Hsu et al. Page 13

Cell. Author manuscript; available in PMC 2015 February 27.

A u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

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

NHEJ-mediated 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.

Hsu et al. Page 14

Cell. Author manuscript; available in PMC 2015 February 27.

A u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

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 improved—

in 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

Hsu et al. Page 15

Cell. Author manuscript; available in PMC 2015 February 27.

A u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

entirely different chromosomes could be brought into close proximity given appropriate

chromosomal organization, thus mediating long-range 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 DNA-FISH (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 CRISPR-based

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 off-target DNA binding and cleavage profiles still need to be thor oughly 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 ho-mology to the sgRNA guide

sequence.

Because the off-target activity of dCas9 binding for effector domain localization may be

much more extensive than Cas9-mediated 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 degen eracy (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.

Hsu et al. Page 16

Cell. Author manuscript; available in PMC 2015 February 27.

A u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

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 post-mitotic 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 (5′-NNAGAAW from Streptococcus

thermophilus CRISPR1 or 5′-NNNNGATT from Neisseria meningitidis) (Zhang et al.,

2013; Garneau et al., 2010), whereas some longer orthologs have more relaxed PAMs (5′-

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.

Hsu et al. Page 17

Cell. Author manuscript; available in PMC 2015 February 27.

A u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

Moving beyond Endogenous Cellular Repair

The current generation of genome editing technologies depends on the endogenous DNA

repair machinery to introduce loss-of-function 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 Cas9-mediated 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

HDR-based 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 5′ site tag sequence on the target-

matching crRNA for Cmr-directed cleavage.

Unlike RNAi, whichis targeted largely by a 6 nt seed region and to a lesser extent 13 other

bases, Cmr crRNAs contain 30–40 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 overexpression

—particularly 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 single-nucleotide polymorphism (SNP) variations in the target gene, such as when

the SNP falls within the PAM sequence.

Hsu et al. Page 18

Cell. Author manuscript; available in PMC 2015 February 27.

A u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

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 Friedreich's 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 (Cohenet al., 2005) orangiopoietin (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

Hsu et al. Page 19

Cell. Author manuscript; available in PMC 2015 February 27.

A u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

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.

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

Acknowledgments

We gratefully acknowledge Sigrid Knemeyer for help with illustration; Ian Slay-maker for structural guidance; Chengwei Luo for expertise in phylogenetic analysis; Emmanuelle Charpentier, Philippe Horvath, Charles Jennings, Ellen Law, Luciano Marraffini, Francisco Mojica, Hiroshi Nishimasu, Virginijus Siksnys, and Alexandro Trevino for discussion and comments; and the 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 Director's 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).

References

Barrangou, R.; van der Oost, J. CRISPR-Cas Systems: RNA-Mediated Adaptive Immunity in Bacteria and Archaea. Heidelberg, Germany: Springer; 2013.

Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero DA, Horvath P. CRISPR provides acquired resistance against viruses in prokaryotes. Science. 2007; 315:1709– 1712. [PubMed: 17379808]

Beerli RR, Dreier B, Barbas CF 3rd. Positive and negative regulation of endogenous genes by designed transcription factors. Proc Natl Acad Sci USA. 2000a; 97:1495–1500. [PubMed: 10660690]

Beerli RR, Schopfer U, Dreier B, Barbas CF 3rd. Chemically regulated zinc finger transcription factors. J Biol Chem. 2000b; 275:32617–32627. [PubMed: 10924515]

Bibikova M, Carroll D, Segal DJ, Trautman JK, Smith J, Kim YG, Chandrasegaran S. Stimulation of homologous recombination through targeted cleavage by chimeric nucleases. Mol Cell Biol. 2001; 21:289–297. [PubMed: 11113203]

Bibikova M, Golic M, Golic KG, Carroll M. Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases. Genetics. 2002; 161:1169–1175. [PubMed: 12136019]

Bibikova M, Beumer K, Trautman JK, Carroll D. Enhancing gene targeting with designed zinc finger nucleases. Science. 2003; 300:764. [PubMed: 12730594]

Boch J, Scholze H, Schornack S, Landgraf A, Hahn S, Kay S, Lahaye T, Nickstadt A, Bonas U. Breaking the code of DNA binding specificity of TAL-type III effectors. Science. 2009; 326:1509– 1512. [PubMed: 19933107]

Hsu et al. Page 20

Cell. Author manuscript; available in PMC 2015 February 27.

A u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

http://www.genome-engineering.org

Bolotin A, Quinquis B, Sorokin A, Ehrlich SD. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extra-chromosomal origin. Microbiology. 2005; 151:2551–2561. [PubMed: 16079334]

Brouns SJ, Jore MM, Lundgren M, Westra ER, Slijkhuis RJ, Snijders AP, Dickman MJ, Makarova KS, Koonin EV, van der Oost J. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science. 2008; 321:960–964. [PubMed: 18703739]

Capecchi MR. Altering the genome by homologous recombination. Science. 1989; 244:1288–1292. [PubMed: 2660260]

Chen B, Gilbert LA, Cimini BA, Schnitzbauer J, Zhang W, Li GW, Park J, Blackburn EH, Weissman JS, Qi LS, Huang B. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell. 2013; 155:1479–1491. [PubMed: 24360272]

Cho SW, Kim S, Kim JM, Kim JS. Targeted genome engineering in human cells with the Cas9 RNA- guided endonuclease. Nat Biotechnol. 2013; 31:230–232. [PubMed: 23360966]

Choulika A, Perrin A, Dujon B, Nicolas JF. Induction of homologous recombination inmammalian chromosomesby using the I-SceI system of Saccharomyces cerevisiae. Mol Cell Biol. 1995; 15:1968–1973. [PubMed: 7891691]

Christian M, Cermak T, Doyle EL, Schmidt C, Zhang F, Hummel A, Bogdanove AJ, Voytas DF. Targeting DNA double-strand breaks with TAL effector nucleases. Genetics. 2010; 186:757–761. [PubMed: 20660643]

Chylinski K, Le Rhun A, Charpentier E. The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems. RNA Biol. 2013; 10:726–737. [PubMed: 23563642]

Chylinski, K.; Makarova, KS.; Charpentier, E.; Koonin, EV. Classification and evolution of type II CRISPR-Cas systems. Nucleic Acids Res. 2014. Published online Apr 11, 2014. http://dx.doi.org/ 10.1093/nar/gku241

Cohen J, Pertsemlidis A, Kotowski IK, Graham R, Garcia CK, Hobbs HH. Low LDL cholesterol in individuals of African descent resulting from frequent nonsense mutations in PCSK9. Nat Genet. 2005; 37:161–165. [PubMed: 15654334]

Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013; 339:819–823. [PubMed: 23287718]

Couzin-Frankel J. Breakthrough of the year 2013. Cancer immunotherapy. Science. 2013; 342:1432– 1433. [PubMed: 24357284]

Crosetto N, Mitra A, Silva MJ, Bienko M, Dojer N, Wang Q, Karaca E, Chiarle R, Skrzypczak M, Ginalski K, et al. Nucleotide-resolution DNA double-strand break mapping by next-generation sequencing. Nat Methods. 2013; 10:361–365. [PubMed: 23503052]

Deltcheva E, Chylinski K, Sharma CM, Gonzales K, Chao Y, Pirzada ZA, Eckert MR, Vogel J, Charpentier E. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature. 2011; 471:602–607. [PubMed: 21455174]

Deveau H, Barrangou R, Garneau JE, Labonté J, Fremaux C, Boyaval P, Romero DA, Horvath P, Moineau S. Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. J Bacteriol. 2008; 190:1390–1400. [PubMed: 18065545]

Dianov GL, Hübscher U. Mammalian base excision repair: the forgotten archangel. Nucleic Acids Res. 2013; 41:3483–3490. [PubMed: 23408852]

Esvelt KM, Mali P, Braff JL, Moosburner M, Yaung SJ, Church GM. Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat Methods. 2013; 10:1116–1121. [PubMed: 24076762]

Fonfara I, Le Rhun A, Chylinski K, Makarova KS, Lécrivain AL, Bzdrenga J, Koonin EV, Charpentier E. Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems. Nucleic Acids Res. 2014; 42:2577–2590. [PubMed: 24270795]

Fu Y, Foden JA, Khayter C, Maeder ML, Reyon D, Joung JK, Sander JD. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol. 2013; 31:822– 826. [PubMed: 23792628]

Hsu et al. Page 21

Cell. Author manuscript; available in PMC 2015 February 27.

A u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

http://dx.doi.org/10.1093/nar/gku241

Fu Y, Sander JD, Reyon D, Cascio VM, Joung JK. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat Biotechnol. 2014; 32:279–284. [PubMed: 24463574]

Gabriel R, Lombardo A, Arens A, Miller JC, Genovese P, Kaeppel C, Nowrouzi A, Bartholomae CC, Wang J, Friedman G, et al. An unbiased genome-wide analysis of zinc-finger nuclease specificity. Nat Biotechnol. 2011; 29:816–823. [PubMed: 21822255]

Garneau JE, Dupuis ME, Villion M, Romero DA, Barrangou R, Boyaval P, Fremaux C, Horvath P, Magadán AH, Moineau S. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature. 2010; 468:67–71. [PubMed: 21048762]

Gasiunas G, Barrangou R, Horvath P, Siksnys V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci USA. 2012; 109:E2579–E2586. [PubMed: 22949671]

Gilbert LA, Larson MH, Morsut L, Liu Z, Brar GA, Torres SE, Stern-Ginossar N, Brandman O, Whitehead EH, Doudna JA, et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell. 2013; 154:442–451. [PubMed: 23849981]

Gonzaelz B, Schwimmer LJ, Fuller RP, Ye Y, Asawapornmongkol L, Barbas CF. Modular system for the construction of zinc-finger libraries and proteins. Nat Protoc. 2010; 5:791–810. [PubMed: 20360772]

Haft DH, Selengut J, Mongodin EF, Nelson KE. A guild of 45 CRISPR-associated (Cas) protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes. PLoS Comput Biol. 2005; 1:e60. [PubMed: 16292354]

Hale CR, Zhao P, Olson S, Duff MO, Graveley BR, Wells L, Terns RM, Terns MP. RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex. Cell. 2009; 139:945–956. [PubMed: 19945378]

Hale CR, Majumdar S, Elmore J, Pfister N, Compton M, Olson S, Resch AM, Glover CV 3rd, Graveley BR, Terns RM, Terns MP. Essential features and rational design of CRISPR RNAs that function with the Cas RAMP module complex to cleave RNAs. Mol Cell. 2012; 45:292–302. [PubMed: 22227116]

Horvath P, Romero DA, Coûté -Monvoisin AC, Richards M, Deveau H, Moineau S, Boyaval P, Fremaux C, Barrangou R. Diversity, activity, and evolution of CRISPR loci in Streptococcus thermophilus. J Bacteriol. 2008; 190:1401–1412. [PubMed: 18065539]

Horvath P, Coûté -Monvoisin AC, Romero DA, Boyaval P, Fremaux C, Barrangou R. Comparative analysis of CRISPR loci in lactic acid bacteria genomes. Int J Food Microbiol. 2009; 131:62–70. [PubMed: 18635282]

Hou Z, Zhang Y, Propson NE, Howden SE, Chu LF, Sontheimer EJ, Thomson JA. Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis. Proc Natl Acad Sci USA. 2013; 110:15644–15649. [PubMed: 23940360]

Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu X, Shalem O, et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol. 2013; 31:827– 832. [PubMed: 23873081]

Ishino Y, Shinagawa H, Makino K, Amemura M, Nakata A. Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. J Bacteriol. 1987; 169:5429–5433. [PubMed: 3316184]

Jansen R, Embden JD, Gaastra W, Schouls LM. Identification of genes that are associated with DNA repeats in prokaryotes. Mol Microbiol. 2002; 43:1565–1575. [PubMed: 11952905]

Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol. 2013; 31:233–239. [PubMed: 23360965]

Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA- guided DNA endonuclease in adaptive bacterial immunity. Science. 2012; 337:816–821. [PubMed: 22745249]

Jinek M, East A, Cheng A, Lin S, Ma E, Doudna J. RNA-programmed genome editing in human cells. eLife. 2013; 2:e00471. [PubMed: 23386978]

Jinek M, Jiang F, Taylor DW, Sternberg SH, Kaya E, Ma E, Anders C, Hauer M, Zhou K, Lin S, et al. Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. Science. 2014; 343:1247997. [PubMed: 24505130]

Hsu et al. Page 22

Cell. Author manuscript; available in PMC 2015 February 27.

A u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

Juillerat A, Dubois G, Valton J, Thomas S, Stella S, Maréchal A, Langevin S, Benomari N, Bertonati C, Silva GH, et al. Comprehensive analysis of the specificity of transcription activator-like effector nucleases. Nucleic Acids Res. 2014; 42:5390–5402. [PubMed: 24569350]

Konermann S, Brigham MD, Trevino AE, Hsu PD, Heidenreich M, Cong L, Platt RJ, Scott DA, Church GM, Zhang F. Optical control of mammalian endogenous transcription and epigenetic states. Nature. 2013; 500:472–476. [PubMed: 23877069]

Li W, Teng F, Li T, Zhou Q. Simultaneous generation and germline transmission of multiple gene mutations in rat using CRISPR-Cas systems. Nat Biotechnol. 2013; 31:684–686. [PubMed: 23929337]

Lombardo A, Genovese P, Beausejour CM, Colleoni S, Lee YL, Kim KA, Ando D, Urnov FD, Galli C, Gregory PD, et al. Gene editing in human stem cells using zinc finger nucleases and integrase- defective lentiviral vector delivery. Nat Biotechnol. 2007; 25:1298–1306. [PubMed: 17965707]

Maeder ML, Thibodeau-Beganny S, Osiak A, Wright DA, Anthony RM, Eichtinger M, Jiang T, Foley JE, Winfrey RJ, Townsend JA, et al. Rapid “open-source” engineering of customized zinc-finger nucleases for highly efficient gene modification. Mol Cell. 2008; 31:294–301. [PubMed: 18657511]

Maeder ML, Angstman JF, Richardson ME, Linder SJ, Cascio VM, Tsai SQ, Ho QH, Sander JD, Reyon D, Bernstein BE, et al. Targeted DNA demethylation and activation of endogenous genes using programmable TALE-TET1 fusion proteins. Nat Biotechnol. 2013a; 31:1137–1142. [PubMed: 24108092]

Maeder ML, Linder SJ, Cascio VM, Fu Y, Ho QH, Joung JK. CRISPR RNA-guided activation of endogenous human genes. Nat Methods. 2013b; 10:977–979. [PubMed: 23892898]

Makarova KS, Grishin NV, Shabalina SA, Wolf YI, Koonin EV. A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. Biol Direct. 2006; 1:7. [PubMed: 16545108]

Makarova KS, Aravind L, Wolf YI, Koonin EV. Unification of Cas protein families and a simple scenario for the origin and evolution of CRISPR-Cas systems. Biol Direct. 2011a; 6:38. [PubMed: 21756346]

Makarova KS, Haft DH, Barrangou R, Brouns SJ, Charpentier E, Horvath P, Moineau S, Mojica FJ, Wolf YI, Yakunin AF, et al. Evolution and classification of the CRISPR-Cas systems. Nat Rev Microbiol. 2011b; 9:467–477. [PubMed: 21552286]

Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM. RNA-guided human genome engineering via Cas9. Science. 2013a; 339:823–826. [PubMed: 23287722]

Mali P, Aach J, Stranges PB, Esvelt KM, Moosburner M, Kosuri S, Yang L, Church GM. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol. 2013b; 31:833–838. [PubMed: 23907171]

Marraffini LA, Sontheimer EJ. CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science. 2008; 322:1843–1845. [PubMed: 19095942]

Marraffini LA, Sontheimer EJ. Self versus non-self discrimination during CRISPR RNA-directed immunity. Nature. 2010; 463:568–571. [PubMed: 20072129]

Mendenhall EM, Williamson KE, Reyon D, Zou JY, Ram O, Joung JK, Bernstein BE. Locus-specific editing of histone modifications at endogenous enhancers. Nat Biotechnol. 2013; 31:1133–1136. [PubMed: 24013198]

Miller JC, Holmes MC, Wang J, Guschin DY, Lee YL, Rupniewski I, Beausejour CM, Waite AJ, Wang NS, Kim KA, et al. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol. 2007; 25:778–785. [PubMed: 17603475]

Miller JC, Tan S, Qiao G, Barlow KA, Wang J, Xia DF, Meng X, Paschon DE, Leung E, Hinkley SJ, et al. A TALE nuclease architecture for efficient genome editing. Nat Biotechnol. 2011; 29:143– 148. [PubMed: 21179091]

Mojica FJ, Díez-Villaseñor C, Soria E, Juez G. Biological significance of a family of regularly spaced repeats in the genomes of Archaea, Bacteria and mitochondria. Mol Microbiol. 2000; 36:244–246. [PubMed: 10760181]

Hsu et al. Page 23

Cell. Author manuscript; available in PMC 2015 February 27.

A u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

Mojica FJ, Díez-Villaseñor C, García-Martínez J, Soria E. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J Mol Evol. 2005; 60:174–182. [PubMed: 15791728]

Moscou MJ, Bogdanove AJ. A simple cipher governs DNA recognition by TAL effectors. Science. 2009; 326:1501. [PubMed: 19933106]

Musunuru K, Pirruccello JP, Do R, Peloso GM, Guiducci C, Sougnez C, Garimella KV, Fisher S, Abreu J, Barry AJ, et al. Exome sequencing, ANGPTL3 mutations, and familial combined hypolipidemia. N Engl J Med. 2010; 363:2220–2227. [PubMed: 20942659]

Niewoehner J, Bohrmann B, Collin L, Urich E, Sade H, Maier P, Rueger P, Stracke JO, Lau W, Tissot AC, et al. Increased brain penetration and potency of a therapeutic antibody using amonovalent molecular shuttle. Neuron. 2014; 81:49–60. [PubMed: 24411731]

Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata SI, Dohmae N, Ishitani R, Zhang F, Nureki O. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell. 2014; 156:935–949. [PubMed: 24529477]

Niu Y, Shen B, Cui Y, Chen Y, Wang J, Wang L, Kang Y, Zhao X, Si W, Li W, et al. Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell. 2014; 156:836–843. [PubMed: 24486104]

Oh B, Hwang S, McLaughlin J, Solter D, Knowles BB. Timely translation during the mouse oocyte-to- embryo transition. Development. 2000; 127:3795–3803. [PubMed: 10934024]

Pattanayak V, Lin S, Guilinger JP, Ma E, Doudna JA, Liu DR. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat Biotechnol. 2013; 31:839–843. [PubMed: 23934178]

Perez-Pinera P, Kocak DD, Vockley CM, Adler AF, Kabadi AM, Polstein LR, Thakore PI, Glass KA, Ousterout DG, Leong KW, et al. RNA-guided gene activation by CRISPR-Cas9-based transcription factors. Nat Methods. 2013; 10:973–976. [PubMed: 23892895]

Plessis A, Perrin A, Haber JE, Dujon B. Site-specific recombination determined by I-SceI, a mitochondrial group I intron-encoded endonuclease expressed in the yeast nucleus. Genetics. 1992; 130:451–460. [PubMed: 1551570]

Pourcel C, Salvignol G, Vergnaud G. CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology. 2005; 151:653–663. [PubMed: 15758212]

Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, Lim WA. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell. 2013; 152:1173–1183. [PubMed: 23452860]

Quiberoni A, Moineau S, Rousseau GM, Reinheimer J, Ackermann HW. Streptococcus thermophilus bacteriophages. Int Dairy J. 2010; 20:657–664.

Ran FA, Hsu PD, Lin CY, Gootenberg JS, Konermann S, Trevino AE, Scott DA, Inoue A, Matoba S, Zhang Y, Zhang F. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell. 2013; 154:1380–1389. [PubMed: 23992846]

Rouet P, Smih F, Jasin M. Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease. Mol Cell Biol. 1994; 14:8096–8106. [PubMed: 7969147]

Rudin N, Sugarman E, Haber JE. Genetic and physical analysis of double-strand break repair and recombination in Saccharomyces cerevisiae. Genetics. 1989; 122:519–534. [PubMed: 2668114]

Sander JD, Joung JK. CRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotechnol. 2014; 32:347–355. [PubMed: 24584096]

Sander JD, Dahlborg EJ, Goodwin MJ, Cade L, Zhang F, Cifuentes D, Curtin SJ, Blackburn JS, Thibodeau-Beganny S, Qi Y, et al. Selection-free zinc-finger-nuclease engineering by context- dependent assembly (CoDA). Nat Methods. 2011; 8:67–69. [PubMed: 21151135]

Sapranauskas R, Gasiunas G, Fremaux C, Barrangou R, Horvath P, Siksnys V. The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli. Nucleic Acids Res. 2011; 39:9275–9282. [PubMed: 21813460]

Hsu et al. Page 24

Cell. Author manuscript; available in PMC 2015 February 27.

A u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

Schwank G, Koo BK, Sasselli V, Dekkers JF, Heo I, Demircan T, Sasaki N, Boymans S, Cuppen E, van der Ent CK, et al. Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell Stem Cell. 2013; 13:653–658. [PubMed: 24315439]

Shah SA, Erdmann S, Mojica FJ, Garrett RA. Protospacer recognition motifs: mixed identities and functional diversity. RNA Biol. 2013; 10:891–899. [PubMed: 23403393]

Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelsen TS, Heckl D, Ebert BL, Root DE, Doench JG, Zhang F. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science. 2014; 343:84–87. [PubMed: 24336571]

Smith J, Grizot S, Arnould S, Duclert A, Epinat JC, Chames P, Prieto J, Redondo P, Blanco FJ, Bravo J, et al. A combinatorial approach to create artificial homing endonucleases cleaving chosen sequences. Nucleic Acids Res. 2006; 34:e149. [PubMed: 17130168]

Sternberg SH, Redding S, Jinek M, Greene EC, Doudna JA. DNA interrogation by the CRISPR RNA- guided endonuclease Cas9. Nature. 2014; 507:62–67. [PubMed: 24476820]

Tang TH, Bachellerie JP, Rozhdestvensky T, Bortolin ML, Huber H, Drungowski M, Elge T, Brosius J, Hüttenhofer A. Identification of 86 candidates for small non-messenger RNAs from the archaeon Archaeoglobus fulgidus. Proc Natl Acad Sci USA. 2002; 99:7536–7541. [PubMed: 12032318]

Tebas P, Stein D, Tang WW, Frank I, Wang SQ, Lee G, Spratt SK, Surosky RT, Giedlin MA, Nichol G, et al. Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV. N Engl J Med. 2014; 370:901–910. [PubMed: 24597865]

Urnov FD, Miller JC, Lee YL, Beausejour CM, Rock JM, Augustus S, Jamieson AC, Porteus MH, Gregory PD, Holmes MC. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature. 2005; 435:646–651. [PubMed: 15806097]

Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell. 2013; 153:910–918. [PubMed: 23643243]

Wang T, Wei JJ, Sabatini DM, Lander ES. Genetic screens in human cells using the CRISPR-Cas9 system. Science. 2014; 343:80–84. [PubMed: 24336569]

Wu Y, Liang D, Wang Y, Bai M, Tang W, Bao S, Yan Z, Li D, Li J. Correction of a genetic disease in mouse via use of CRISPR-Cas9. Cell Stem Cell. 2013; 13:659–662. [PubMed: 24315440]

Wu, X.; Scott, DA.; Kriz, AJ.; Chiu, AC.; Hsu, PD.; Dadon, DB.; Cheng, AW.; Trevino, AE.; Konermann, S.; Chen, S., et al. Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells. Nature Biotechnol. 2014. Published online Apr 20, 2014. http://dx.doi.org/ 10.1038/nbt.2889

Xu GL, Bestor TH. Cytosine methylation targetted to pre-determined sequences. Nat Genet. 1997; 17:376–378. [PubMed: 9398832]

Yang H, Wang H, Shivalila CS, Cheng AW, Shi L, Jaenisch R. One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell. 2013; 154:1370–1379. [PubMed: 23992847]

Yin, H.; Xue, W.; Chen, S.; Bogorad, RL.; Benedetti, E.; Grompe, M.; Koteliansky, V.; Sharp, PA.; Jacks, T.; Anderson, DG. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nature Biotechnol. 2014. Published online Mar 30, 2014. http://dx.doi.org/10.1038/ nbt.2884

Zahradka K, Slade D, Bailone A, Sommer S, Averbeck D, Petranovic M, Lindner AB, Radman M. Reassembly of shattered chromosomes in Deinococcus radiodurans. Nature. 2006; 443:569–573. [PubMed: 17006450]

Zhang Y, Heidrich N, Ampattu BJ, Gunderson CW, Seifert HS, Schoen C, Vogel J, Sontheimer EJ. Processing-independent CRISPR RNAs limit natural transformation in Neisseria meningitidis. Mol Cell. 2013; 50:488–503. [PubMed: 23706818]

Hsu et al. Page 25

Cell. Author manuscript; available in PMC 2015 February 27.

A u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

http://dx.doi.org/10.1038/nbt.2889

http://dx.doi.org/10.1038/nbt.2884

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).

Hsu et al. Page 26

Cell. Author manuscript; available in PMC 2015 February 27.

A u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

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 error-prone 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 micro-homology, 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.

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.

Hsu et al. Page 27

Cell. Author manuscript; available in PMC 2015 February 27.

A u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

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.

Hsu et al. Page 28

Cell. Author manuscript; available in PMC 2015 February 27.

A u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

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 3′ 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.

Hsu et al. Page 29

Cell. Author manuscript; available in PMC 2015 February 27.

A u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

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.

Hsu et al. Page 30

Cell. Author manuscript; available in PMC 2015 February 27.

A u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

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.

Hsu et al. Page 31

Cell. Author manuscript; available in PMC 2015 February 27.

A u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

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.

Hsu et al. Page 32

Cell. Author manuscript; available in PMC 2015 February 27.

A u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

Figure 6. Applications of Cas9 as a Genome Engineering Platform (A) The Cas9 nuclease cleaves DNA via its RuvC and HNH nuclease domains, each

ofwhich 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.

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.

Hsu et al. Page 33

Cell. Author manuscript; available in PMC 2015 February 27.

A u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

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.

Hsu et al. Page 34

Cell. Author manuscript; available in PMC 2015 February 27.

A u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip