ssODN-mediated knock-in with CRISPR-Cas for large genomic regions in zygotes

Nat Commun. 2016 Jan 20;7:10431. doi: 10.1038/ncomms10431.

Abstract

The CRISPR-Cas system is a powerful tool for generating genetically modified animals; however, targeted knock-in (KI) via homologous recombination remains difficult in zygotes. Here we show efficient gene KI in rats by combining CRISPR-Cas with single-stranded oligodeoxynucleotides (ssODNs). First, a 1-kb ssODN co-injected with guide RNA (gRNA) and Cas9 messenger RNA produce GFP-KI at the rat Thy1 locus. Then, two gRNAs with two 80-bp ssODNs direct efficient integration of a 5.5-kb CAG-GFP vector into the Rosa26 locus via ssODN-mediated end joining. This protocol also achieves KI of a 200-kb BAC containing the human SIRPA locus, concomitantly knocking out the rat Sirpa gene. Finally, three gRNAs and two ssODNs replace 58-kb of the rat Cyp2d cluster with a 6.2-kb human CYP2D6 gene. These ssODN-mediated KI protocols can be applied to any target site with any donor vector without the need to construct homology arms, thus simplifying genome engineering in living organisms.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antigens, Differentiation / genetics
  • CRISPR-Cas Systems / genetics*
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics
  • Female
  • Gene Knock-In Techniques
  • Genetic Engineering / methods*
  • Homologous Recombination / genetics
  • Humans
  • Male
  • Mice
  • Oligodeoxyribonucleotides / genetics*
  • Rats
  • Receptors, Immunologic / genetics
  • Zygote / metabolism*

Substances

  • Antigens, Differentiation
  • Oligodeoxyribonucleotides
  • Receptors, Immunologic
  • SIRPA protein, human