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A Variant CRISPR-Cas9 System Adds Versatility to Genome Engineering

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  COMMENTARY  A variant CRISPR-Cas9 system adds versatility to genome engineering  Ryan M. Walsh a,b and Konrad Hochedlinger a,b,1 a Howard Hughes Medical Institute, Massachusetts General Hospital Center for RegenerativeMedicine/Cancer Center, Boston, MA 02114; and   b Harvard Stem Cell Institute and Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138 Targeted genome engineering has been instru-mental for the study of biological processes,and it holds great promise for the treatment of disease. Historically, gene targeting by homol-ogous recombination has been the preferredmethod to modify speci 󿬁 c genes in mouseand human cells (Fig. 1). However, this ap-proach is hampered by low ef  󿬁 ciency, the re-quirement for drug selection to detect targetedcells, and the limited number of cell types andorganisms amenable to the method. To over-come these limitations, technologies based onsequence-speci 󿬁 c zinc  󿬁 nger (ZF) and tran-scription activator-like effector (TALE) pro-teins have been developed. These proteinscan be engineered to theoretically recognizeany DNA sequence in the genome. Whenfused to a nuclease domain and assembled inpairs  󿬂 anking a target site of interest, ZF andTALE nucleases (ZFNs and TALENs) will in-troduce double-strand breaks (DSBs) on DNA.DSBs serve as substrates for nonhomologousend joining (NHEJ) or homology-directed re-pair (HDR), which in turn facilitate the engineer-ing of targeted mutations, repair of endogenousmutations, or introduction of transgenic DNAelements. The clustered, regularly interspaced,short palindromic repeat (CRISPR)-CRISPR-associated (Cas) system represents the latest ad-dition to this arsenal of tools for site-speci 󿬁 cgenome engineering (see below). Although eachof these three gene modi 󿬁 cation approaches hasadvantages and disadvantages (Fig. 1), the paceand ease with which the CRISPR-Cas systemshave been adapted to modify genes in differentcelltypesandorganismssuggeststhatitmayvery well become the new method of choice for ge-nome engineering. In PNAS, Hou et al. intro-duce a unique variant of the Cas9 enzyme (1),which provides additional  󿬂 exibility and speci- 󿬁 city to the CRISPR system and to genome-modifying tools in general.CRISPR and Cas9 were initially identi 󿬁 ed ascomponents of the bacterial immune responseto bacteriophages (2). In this system, a shortCRISPR RNA component (crRNA) recognizesa complementary stretch of nucleotides (theprotospacer) within foreign DNA, thus confer-ring sequence speci 󿬁 city (Fig. 1). In addition, atransactivating CRISPR RNA, dubbed tracrRNA,is required to form ribonucleoprotein com-plexes with the Cas9 nuclease, generating site-speci 󿬁 c DSBs (3). By combining these two RNAcomponents of CRISPR into a single chimericmolecule termed  “ guide RNA ”  (gRNA) andexpressing it alongside Cas9 protein, severalgroups have demonstrated the adaptability of this system to modify endogenous loci ineukaryotic cells. DSBs generated by CRISPR are preferentially repaired by NHEJ, which isan error-prone process and thus useful to in-troduce insertions and deletions into mamma-lian cells (4, 5). In addition, DSBs are resolvedat lower frequency by HDR and therefore canbe used to modify endogenous loci (e.g., re-placement of point mutations, insertion of knock-in alleles) (4, 5). Notably, this approachrequires much shorter homology arms com-pared with traditional gene targeting systems.Although ZFNs and TALENs enable similargenome modi 󿬁 cations, the CRISPR-Cas systemoffers a few advantages. First and foremost, theCRISPR-Cas system is based on simple gRNA/DNA hybrids to confer sequence speci 󿬁 city,which enables rapid design and delivery of targeting constructs. In contrast, ZFNs andTALENs require modular assembly of pairs of proteins that recognize areas  󿬂 anking a targetsite, a process that is more tedious and time-consuming. Another advantage of the CRISPR-Cas system is its amenability to multiplexing,allowing for the simultaneous generation of upto  󿬁  ve mutations from a single transfectionevent (6). This ability is expected to facilitateepistatic gene studies and should allow for dis-section of phenotypes that are normally maskedby compensatory mechanisms. Furthermore,multiplexing may enable the study of complex genetic diseases in a Petri dish using humanembryonic stem cells (hESCs) or patient-derivedinduced pluripotent stem cells (iPSCs), as hasrecently been accomplished for monogenic dis-eases with TALENs (7).Although the CRISPR/Cas system undoubt-edly holds great potential for genome editing,there are a number of important limitations toconsider. Target site cleavage by the CRISPR-Cas system requires a so-called protospaceradjacent motif (PAM) immediately down-stream of the protospacer element to whichthe gRNA binds, restricting targeting range (Fig.1). Interestingly, the PAM sequence varies insize and nucleotide composition in differentbacterial strains from which Cas9 proteins havebeen isolated. The two previously characterizedCas9 proteins from  Streptococcus pyogenes (SpCas) and  Streptococcus thermophiles  (StCas)require PAMs of 5 ′ -NGG-3 ′  and 5 ′ -NNA-GAAW-3 ′ , respectively (2). While these sitesare found quite frequently in the genome,as often as every 8 bp (4), speci 󿬁 c genomicregions may be dif  󿬁 cult to target with thesesequence constraints. Another major concernof CRISPR-Cas technology is the potential foroff-target effects. This issue is particularly rele- vant with respect to potential therapeutic appli-cations of this technology. Two recent in-depthstudies report that up to  󿬁  ve mismatches in theprotospacer and a 5 ′ -NAG-3 ′  variation withinthe PAM sequence are tolerated by   S. pyogenes CRISPR-Cas9 without impairing the ability tocleave DNA (8, 9). This implies that dozens of nontarget loci may be cut by CRISPR-Cas9 whenaiming to cleave individual genes in vivo. Indeed,a recent report documented extensive cleavagewithin the genome when examining a numberof predicted off-targets sites (8). Different ap-proaches have been proposed to mitigate off-target effects including titration of gRNA andCas9 levels, rational design of gRNAs based onbioinformatic predictions (9), as well as the useof pairs of modi 󿬁 ed Cas9 enzymes that intro-duce single strand nicks, as opposed to DSBs, onopposite strands of a target site (akin to ZNFand TALEN design) (10). A  󿬁 nal possibility toincrease speci 󿬁 city and decrease off-target cleav-age is the isolation of alternative CRISPR-Cassystems with more stringent interactions be-tween gRNA, target sequence, and PAM fromother strains of archaea or bacteria (2).In PNAS, Hou et al. present the identi 󿬁 cationand analysis of a unique Cas9 nuclease isolatedfrom  Neisseria meningitidis  (NmCas9). Unlike Author contributions: R.M.W. and K.H. wrote the paper.The authors declare no con 󿬂 ict of interest.See companion article on page 15644. 1 To whom correspondence should be addressed. E-mail:khochedlinger@helix.mgh.harvard.edu. 15514 – 15515  |  PNAS  |  September 24, 2013  |  vol. 110  |  no. 39 www.pnas.org/cgi/doi/10.1073/pnas.1314697110  the SpCas9 and StCas9 enzymes, NmCas9binds a 24nt protospacer sequence on its tar-get DNA, conferring additional speci 󿬁 city overthe previous 20nt protospacer (Fig. 1). More-over, NmCas9 differs in its PAM, requiring a5 ′ -NNNNGATT-3 ′  or 5-NNNNGCTT-3 ′  se-quence to achieve ef  󿬁 cient target DNA cleavage.This extended PAM sequence should enhancespeci 󿬁 city and will increase the number of lociwithin the genome that are amenable to target-ing by CRISPR-Cas. Critically, NmCas9, likeSpCas9, is capable of achieving ef  󿬁 cient cleavageof target DNA sequences both in vitro and in vivo. To document the utility of this uniquesystem for generating mutations in hESCs, theauthors generated a puromycin-selectable epi-somal vector containing crRNA, tracrRNA,and Cas9 on a single construct. This modi 󿬁 ca-tion will afford increased ef  󿬁 cacy over alterna-tive genome editing systems that require two ormore plasmids. Indeed, the authors generatedan Oct4-GFP knock-in reporter in hESCs andiPSCs at ef  󿬁 ciencies of around 60%, which ishigher than comparable efforts using TALENs.This report further demonstrates the feasibility of inserting large genomic fragments into cellsby HDR using CRISPR-Cas technology, indicat-ing that it should now be possible to generatesimilar reporter alleles for other genes of inter-est. Additional single gene and multigene re-porter cell lines (derived through multiplex-ing) will be invaluable tools for tracking cell fateand isolating rare cell populations in differentbiological contexts.Of interest, CRISPR-Cas technology is notlimited to introducing mutations or reportersinto cells, but can, in principle, be used to repairdisease-associated mutations in patients. Inaddition, recent reports showed that transcrip-tional activators or repressors can be directedto de 󿬁 ned genomic elements to reactivate orsilence gene expression using CRISPR-Castechnology (10, 11). Together, these examplesdocument the remarkable versatility and ame-nability of the CRISPR-Cas system and itspotential to revolutionize the  󿬁 elds of reversegenetics and epigenetics. The unique Cas9 pro-tein, described by Hou et al., will certainly addto this powerful set of tools and may in factsolve some of the current limitations of CRISPR technology in basic biology and cell therapy. 1  Hou Z, et al. (2013) Ef 󿬁 cient genome engineering in humanpluripotent stem cells using Cas9 from  Neisseria meningitidis .  ProcNatl Acad Sci USA  110:15644 – 15649. 2  Horvath P, Barrangou R (2010) CRISPR/Cas, the immune system ofbacteria and archaea.  Science  327(5962):167 – 170. 3  Jinek M, et al. (2012) A programmable dual-RNA-guided DNAendonuclease in adaptive bacterial immunity.  Science  337(6096):816 – 821. 4  Cong L, et al. (2013) Multiplex genome engineering using CRISPR/ Cas systems.  Science  339(6121):819 – 823. 5  Mali P, et al. (2013) RNA-guided human genome engineering viaCas9.  Science  339(6121):823 – 826. 6  Wang H, et al. (2013) One-step generation of mice carryingmutations in multiple genes by CRISPR/Cas-mediated genomeengineering.  Cell   153(4):910 – 918. 7  Ding Q, et al. (2013a) A TALEN genome-editing system forgenerating human stem cell-based disease models.  Cell Stem Cell  12(2):238 – 251. 8  Fu Y, et al. (2013) High-frequency off-target mutagenesis inducedby CRISPR-Cas nucleases in human cells [published online ahead ofprint June 23, 2013].  Nat Biotechnol  , 10.1038/nbt.2623. 9  Hsu PD, et al. (2013) DNA targeting speci 󿬁 city of RNA-guided Cas9nucleases [published online ahead of print July 21, 2013].  NatBiotechnol  , 10.1038/nbt.2647. 10  Mali P, et al. (2013b) CAS9 transcriptional activators for targetspeci 󿬁 city screening and paired nickases for cooperative genomeengineering [published online ahead of print August 1, 2013].  NatBiotechnol  , 10.1038/nbt.2675. 11  Gilbert LA, et al. (2013) CRISPR-mediated modular RNA-guidedregulation of transcription in eukaryotes.  Cell   154(2):442 – 451. Fig. 1.  Different approaches to manipulate the mammalian genome. Shown are currently available methods to modify the genome in a site-speci 󿬁 c manner with a list of keyfeatures, advantages, and disadvantages of each approach. Illustration of homology-directed repair (HDR) to generate a knock-in allele. See text for details and abbreviations. Walsh and Hochedlinger PNAS  |  September 24, 2013  |  vol. 110  |  no. 39  |  15515      C     O     M     M     E     N     T     A     R     Y
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