In vivo blunt-end cloning through CRISPR/Cas9-facilitated non-homologous end-joining

Nucleic Acids Res. 2016 May 5;44(8):e76. doi: 10.1093/nar/gkv1542. Epub 2016 Jan 13.

Abstract

The CRISPR/Cas9 system facilitates precise DNA modifications by generating RNA-guided blunt-ended double-strand breaks. We demonstrate that guide RNA pairs generate deletions that are repaired with a high level of precision by non-homologous end-joining in mammalian cells. We present a method called knock-in blunt ligation for exploiting these breaks to insert exogenous PCR-generated sequences in a homology-independent manner without loss of additional nucleotides. This method is useful for making precise additions to the genome such as insertions of marker gene cassettes or functional elements, without the need for homology arms. We successfully utilized this method in human and mouse cells to insert fluorescent protein cassettes into various loci, with efficiencies up to 36% in HEK293 cells without selection. We also created versions of Cas9 fused to the FKBP12-L106P destabilization domain in an effort to improve Cas9 performance. Our in vivo blunt-end cloning method and destabilization-domain-fused Cas9 variant increase the repertoire of precision genome engineering approaches.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CRISPR-Associated Proteins / genetics*
  • CRISPR-Cas Systems / genetics*
  • Cell Line
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics*
  • DNA Breaks, Double-Stranded
  • DNA End-Joining Repair / genetics*
  • Genetic Engineering / methods*
  • Genome / genetics
  • HEK293 Cells
  • Humans
  • Mice
  • RNA Editing / genetics
  • RNA, Guide / genetics

Substances

  • CRISPR-Associated Proteins
  • RNA, Guide