Chat with us, powered by LiveChat JA summary in CSE formatting of the CRISPR genome editing technique. All information must be from the article and no other outside sources!!!? RUBRIC and article are attac | WriteDen

JA summary in CSE formatting of the CRISPR genome editing technique. All information must be from the article and no other outside sources!!!? RUBRIC and article are attac

Assignment: JA summary in CSE formatting of the CRISPR genome editing technique. All information must be from the article and no other outside sources!!! 

RUBRIC and article are attached in PDF form. 

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


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.


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.

©2014 Elsevier Inc. *Correspondence: [email protected]

Supplemental Information Supplemental Information includes one movie and can be found with this article at 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.

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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


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.

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Programmable Nucleases as Tools for Efficient and Precise Genome


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


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

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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.,


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

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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,


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

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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.,


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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:// and

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).

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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).

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