Efficient Homologous Recombination in Mice Using Long Single Stranded DNA and CRISPR Cas9 Nickase

G3 (Bethesda). 2019 Jan 9;9(1):281-286. doi: 10.1534/g3.118.200758.

Abstract

The CRISPR/Cas9 nickase mutant is less prone to off-target double-strand (ds)DNA breaks than wild-type Cas9 because to produce dsDNA cleavage it requires two guide RNAs to target the nickase to nearby opposing strands. Like wild-type Cas9 lesions, these staggered lesions are repaired by either non-homologous end joining or, if a repair template is provided, by homologous recombination (HR). Here, we report very efficient (up to 100%) recovery of heterozygous insertions in Mus musculus produced by long (>300 nt), single-stranded DNA donor template-guided repair of paired-nickase lesions.

Keywords: Cas9 nickase; Long single stranded DNA; ssODN.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • CRISPR-Cas Systems / genetics*
  • DNA / genetics
  • DNA End-Joining Repair / genetics
  • DNA Repair / genetics
  • DNA, Single-Stranded / genetics
  • Deoxyribonuclease I / genetics*
  • Heterozygote
  • Homologous Recombination / genetics*
  • Mice
  • Mutation / genetics
  • RNA, Guide, CRISPR-Cas Systems / genetics*

Substances

  • DNA, Single-Stranded
  • RNA, Guide, CRISPR-Cas Systems
  • DNA
  • Deoxyribonuclease I

Associated data

  • figshare/10.25387/g3.7315478