Efficient homologous recombination in mice using long single stranded DNA and CRISPR Cas9 nickase

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 nonhomologous 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.