Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) adaptive immune systems are found in bacteria and archaea to protect the hosts against the invasion of viruses and plasmids [1-3]. Three types (I-III) of CRISPR systems with different features have been identified. The CRISPR-associated protein Cas9 that belongs to the type II CRISPR/Cas system has attracted much attention due to its potential use in genomic engineering. Cas9 contains one HNH motif and three RuvC-like motifs, homologous to HNH and RuvC endonucleases, respectively (Supplementary information, Figure S1)[4-8]. Recent studies showed that Cas9 displayed strong DNA cleavage activity in bacteria and in test tubes. Its nuclease activity is guided by two non-coding RNA elements of the system; one is crRNA (CRISPR RNA) that contains about 20 base pairs (bp) of unique target sequence (called spacer sequence) and the other is tracrRNA (trans-activating crRNA). These two RNA elements form a crRNA: tracrRNA duplex that directs Cas9 to target DNA via complementary base pairing between the spacer on the crRNA and the complementary sequence (called protospacer) on the target DNA. The 3 nucleotides (nt) located immediately at the 3′ side next to the protospacer sequence constitute the protospacer adjacent motif (PAM) that is required to ensure the cleavage specificity in target sequences [9, 10]. Theoretically, CRISPR systems can be used in higher eukaryotes through ectopically expressing the enzyme (Cas9) and the RNAs, much like the zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs)(Figure 1A). To test this, we first asked whether Cas9 could perform DNA cleavage in zebrafish. We generated codon-optimized Cas9 (Supplementary information, Table S1 and Data S2) and cloned it into an expression vector. A chimeric RNA that is a single engineered RNA molecule combining features of both crRNA and tracrRNA [9] was designed to target one of the two sites (EGFP-A and-B) in the pEGFP-N1 plasmid (Figure 1B, Supplementary information, Figures S2 and S3). DNA fragments corresponding to Cas9 and the chimeric RNA were transcribed to RNAs in vitro by the T7 RNA polymerase and the Cas9 RNA was further modified by adding a translation-required cap at the 5′-end and a poly-A tail at the 3′-end to make it resemble an authentic mRNA. The Cas9 mRNA, chimeric RNA, and pEGFP-N1 plasmid were co-injected into one-cell zebrafish embryos. Similar strategies have previously been employed to test ZFN and TALEN activity [11, 12]. The embryos were cultured for 12 h before being harvested for total DNA extraction.

The targeted region on the EGFP plasmid was amplified from the extracted DNA; the purified PCR products were then denatured and reannealed to form hybridized DNA, followed by digestion with the T7 endonuclease 1 (T7EN1)[13] that can recognize and cleave mismatched DNA (Supplementary information, Figure S4). Gel electrophoresis of T7EN1-digestion products clearly showed two smaller fragments besides the amplicon of the targeted region, which were not observed in the control (Figure 1C and Supplementary information, Figure S3), suggesting that the target DNA was cleaved by Cas9. To precisely locate the site of cleavage, we cloned the PCR products and analyzed the clones by DNA sequencing. There were 2 mutant clones out of 48 sequenced. The cutting occurred about 4 nt away from the PAM (Figure 1D). These results demonstrate that Cas9/RNA can sitespecifically cut DNA in eukaryotic cells. To improve the cutting efficiency of the Cas9 and …