| First Author | Ran FA | Year | 2013 |
| Journal | Cell | Volume | 154 |
| Issue | 6 | Pages | 1380-9 |
| PubMed ID | 23992846 | Mgi Jnum | J:204542 |
| Mgi Id | MGI:5532785 | Doi | 10.1016/j.cell.2013.08.021 |
| Citation | Ran FA, et al. (2013) Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154(6):1380-9 |
| abstractText | Targeted genome editing technologies have enabled a broad range of research and medical applications. The Cas9 nuclease from the microbial CRISPR-Cas system is targeted to specific genomic loci by a 20 nt guide sequence, which can tolerate certain mismatches to the DNA target and thereby promote undesired off-target mutagenesis. Here, we describe an approach that combines a Cas9 nickase mutant with paired guide RNAs to introduce targeted double-strand breaks. Because individual nicks in the genome are repaired with high fidelity, simultaneous nicking via appropriately offset guide RNAs is required for double-stranded breaks and extends the number of specifically recognized bases for target cleavage. We demonstrate that using paired nicking can reduce off-target activity by 50- to 1,500-fold in cell lines and to facilitate gene knockout in mouse zygotes without sacrificing on-target cleavage efficiency. This versatile strategy enables a wide variety of genome editing applications that require high specificity. |
