Double Nicking by RNA-guided CRISPR Cas9 for Enhanced Genome Editing Specificity

Cell. 2013 Sep 12;154(6):1380-9. doi: 10.1016/j.cell.2013.08.021. Epub 2013 Aug 29.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Base Sequence
  • DNA Breaks, Double-Stranded*
  • Gene Targeting / methods*
  • Genome*
  • Mice
  • Molecular Sequence Data
  • RNA, Guide / genetics
  • Streptococcus pyogenes / enzymology
  • Streptococcus pyogenes / genetics
  • Zygote / metabolism

Substances

  • RNA, Guide