HeritablegenetargetinginthemouseandratusingaCRISPR-Cassystem.doc

Heritable gene targeting in the mouse and rat using a CRISPR-Cas system To the Editor: CRISPR-Cas systems have been developed as an efficient gene editing technology in cells and model organisms. Here we use a CRISPR-Cas system to induce genomic DNA fragment deletion in mice by co-injecting two single-guide RNAs (sgRNAs) targeting the Uhrf2 locus with Cas9 mRNA.Furthermore, we report the generation of a Mc3R and Mc4R double-gene knockout rat by means of a single microinjection. High germline-transmission efficiency was observed in both mice and rats. The clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated protein (Cas) system has evolved in bacteria and archaea as an RNA-based adaptive immune system against viral and plasmid invasion1. Based on gene conservation and locus organization, three major types of CRISPR systems have been identified2,3. In the type II systems,the complex of a CRISPR RNA (crRNA) annealed to a trans-activating crRNA (tracrRNA) is sufficient to guide the Cas9 endonuclease to a specific genomic sequence to generate double-strand breaks in target DNA4. Previous studies established a strategy for multiplex genome engineering with the Cas9 RNA-guided endonuclease in mammalian cells5,6. Recently, efficient genome editing by the CRISPR-Cas system has been shown in multiple organisms, including zebrafish, mice and bacteria7–9. Several groups have demonstrated that compared with zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs),CRISPR-Cas–mediated gene targeting has similar or greater efficiency in cells and zebrafish5–7,10. Although it has been demonstrated that multiple genes can be disrupted in individual mouse embryos using CRISPR-Cas–mediated systems9, germline transmission of Cas9-mediated mutations in animals has not yet been reported. In addition, whether long, specific, genomic DNA target fragments can be deleted by the CRISPR-Cas system is still unknown. Moreover, the utili