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 plasmids1,2,3. 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)4,5,6,7,8. 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 sequences9,10.

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 site-specifically cut DNA in eukaryotic cells.

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