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precision and efficiency. To investigate the specificity of RNA-guided genome modification, we analyzed single-nucleotide mismatches between the spacer and its mammalian protospacer target (Fig. 3A). We observed that single-base mismatch up to 11 bp 5′ of the PAM completely abolished genomic cleavage by SpCas9, whereas spacers with mutations farther upstream retained activity against the protospacer target (Fig. 3B). A B human EMX1 locus pre-crRNA tracrRNA protospacer (1) pre-crRNA + tracrRNA processing spacer (30 bp) chimeric RNA guide sequence (20 bp) Fig. 2. SpCas9 can be reprogrammed to target multiple genomic loci in mammalian cells. (A) Schematic of the human EMX1 locus showing the location of five protospacers indicated by blue lines with corresponding PAM in magenta. (B) Schematic of the pre-crRNA:tracrRNA complex (top) showing hybridization between the direct repeat (gray) region of the precrRNA and tracrRNA. Schematic of a chimeric RNA design (12) (bottom). A human EMX1 locus C wt crRNA mismatchcontaining guide sequences human EMX1 locus left TALEN binding site protospacer (1) protospacer (1) This is consistent with previous bacterial and in vitro studies of Cas9 specificity (12, 20). Furthermore, SpCas9 is able to mediate genomic cleavage as efficiently as a pair of TALE nucleases (TALENs) targeting the same EMX1 protospacer (Fig.3,CandD). Targeted modification of genomes ideally avoids mutations arising from the error-prone NHEJ mechanism. The wild-type SpCas9 is able PAM PAM right TALEN binding site Fig. 3. Evaluation of the SpCas9 specificity and comparison of efficiency with TALENs. (A) EMX1-targeting chimeric crRNAs with single point mutations were generated to evaluate the effects of spacer-protospacer mismatches. (B) protospacer (5) C EMX1 protospacer target to mediate site-specific DSBs, which can be repaired through either NHEJ or homology-directed repair (HDR). We engineered an aspartate-toalanine substitution (D10A) in the RuvC I domain of SpCas9 to convert the nuclease into a DNA nickase (SpCas9n, Fig. 4A) (12, 13, 20), because nicked genomic DNA is typically repaired either seamlessly or through high-fidelity HDR. SURVEYOR (Fig. 4B) and sequencing of protospacer (4) protospacer (2) protospacer (3) tracrRNA sequence is shown in red and the 20-bp spacer sequence in blue. (C) SURVEYOR assay comparing the efficacy of Cas9-mediated cleavage at five protospacers in the human EMX1 locus. Each protospacer was targeted by using either processed pre-crRNA:tracrRNA complex (crRNA) or chimeric RNA (chiRNA). Arrowheads indicate cleavage products for each protospacer target. B 684bp 367bp 317bp indel (%) indel (%) chimeric RNA with mismatched guide sequence 2kb m17 m15 m13 m11 m9 m7 m5 m4 m3 m2 m1 wt 5.6 7.5 8.8 9.7 D TALEN chimeric RNA crRNA control 684bp 367bp 317bp 3.6 3.8 4.5 11 7.4 8.6 22 29 25 REPORTS SURVEYOR assay comparing the cleavage efficiency of different mutant chimeric RNAs. (C) Schematic showing the design of TALENs that target EMX1. (D) SURVEYOR gel comparing the efficiency of TALEN and SpCas9 (N =3). www.sciencemag.org SCIENCE VOL 339 15 FEBRUARY 2013 821 on February 14, 2013 www.sciencemag.org Downloaded from
REPORTS 822 A B chimeric U6 RNA EF1α F G D10A crRNA array design crRNA array design human EMX1 locus deletion result NLS hSpCas9n NLS RuvC I RuvC II RuvC III Surveyor target 684bp 577bp 381bp 367bp 317bp 196bp indel (%) DR-EMX1(1)- DR-PVALB(9)- DR HNH 327 amplicons did not detect any indels induced by SpCas9n. However, nicked DNA can in rare cases be processed via a DSB intermediate and result in a NHEJ event (27). We then tested Cas9mediated HDR at the same EMX1 locus with a homology repair template to introduce a pair of restriction sites near the protospacer (Fig. 4C). SpCas9 and SpCas9n catalyzed integration of the repair template into EMX1 locus at similar levels (Fig. 4D), which we further verified via Sanger sequencing (Fig. 4E). These results demonstrate the utility of CRISPR for facilitating targeted genomic insertions. Given the 14-bp (12 bp from the seed sequence and 2 bp from PAM) target specificity (Fig. 3B) of the wild-type SpCas9, the use of a nickase may reduce off-target mutations. Lastly, the natural architecture of CRISPR loci with arrayed spacers (fig. S1) suggests the possibility of multiplexed genome engineering. By using a single CRISPR array encoding a pair of EMX1- and PVALB-targeting spacers, we de- DR- EMX1(1)- DR SpCas9 SpCas9n chimeric RNA DR- PVALB(9)- DR 684bp 367bp 317bp indel (%) EMX1(1) PVALB(9) DR DR DR EMX1 PVALB untransfected EMX1 PVALB EMX1 PVALB EMX1 PVALB 27 7.3 24 15 EMX1(1) EMX1(8) 118bp deletion junction 5.3 4.6 3.5 tected efficient cleavage at both loci (Fig. 4F). We further tested targeted deletion of larger genomic regions through concurrent DSBs by using spacers against two targets within EMX1 spaced by 119 bp and observed a 1.6% deletion efficacy (3 out of 182 amplicons, Fig. 4G), thus demonstrating the CRISPR/Cas system can mediate multiplexed editing within a single genome. The ability to use RNA to program sequencespecific DNA cleavage defines a new class of genome engineering tools. Here, we have shown that the S. pyogenes CRISPR system can be heterologously reconstituted in mammalian cells to facilitate efficient genome editing; an accompanying study has independently confirmed high-efficiency RNA-guided genome targeting in several human cell lines (28). However, several aspects of the CRISPR/Cas system can be further improved to increase its efficiency and versatility. The requirement for an NGG PAM restricts the target space of SpCas9 to every 8 bp on average in the human 15 FEBRUARY 2013 VOL 339 SCIENCE www.sciencemag.org C human EMX1 locus D E HR Template SpCas9 SpCas9n HR template 2281bp 1189bp 1092bp HR (%) HindIII, NheI 0.70 0.46 HindIII NheI 200bp Fig. 4. Applications of Cas9 for homologous recombination and multiplex genome engineering. (A) Mutation of the RuvC I domain converts Cas9 into a nicking enzyme (SpCas9n). HNH, histidine-asparaginehistidine endonuclease domain. (B) Coexpressionof EMX1-targeting chimeric RNA with SpCas9 leads to indels, whereas SpCas9n does not (N =3).(C) Schematic representation of the recombination strategy. A homology repair (HR) template is designed to insert restriction sites into EMX1 locus. Primers used to amplify the modified region are shown as red arrows. (D) Restriction fragment length polymorphism gel analysis. Arrows indicate fragments generated by HindIII digestion. (E) Example chromatogram showing successful recombination. (F) SpCas9 can facilitate multiplex genome modification by using a crRNA array that contains two spacers targeting EMX1 and PVALB.Schematic showing the design of the crRNA array (top). Both spacers mediate efficient protospacer cleavage (bottom). (G) SpCas9 can be used to achieve precise genomic deletion. Two spacers targeting EMX1 (top) mediated a 118-bp genomic deletion (bottom). genome (fig. S7), not accounting for potential constraints posed by crRNA secondary structure or genomic accessibility resulting from chromatin and DNA methylation states. Some of these restrictions may be overcome by exploiting the family of Cas9 enzymes and its differing PAM requirements (22, 23) across the microbial diversity (17). Indeed, other CRISPR loci are likely to be transplantable into mammalian cells; for example, the Streptococcus thermophilus LMD-9 CRISPR1 system can also mediate mammalian genome cleavage (fig. S8). Lastly, the ability to carry out multiplex genome editing in mammalian cells enables powerful applications across basic science, biotechnology, and medicine (29). References and Notes 1. M. H. Porteus, D. Baltimore, Science 300, 763 (2003). 2. J. C. Miller et al., Nat. Biotechnol. 25, 778 (2007). 3. J. D. Sander et al., Nat. Methods 8, 67 (2011). on February 14, 2013 www.sciencemag.org Downloaded from
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