RS-1 enhances CRISPR/Cas9- and TALEN-mediated knock-in efficiency

Nat Commun. 2016 Jan 28;7:10548. doi: 10.1038/ncomms10548.


Zinc-finger nuclease, transcription activator-like effector nuclease and CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) are becoming major tools for genome editing. Importantly, knock-in in several non-rodent species has been finally achieved thanks to these customizable nucleases; yet the rates remain to be further improved. We hypothesize that inhibiting non-homologous end joining (NHEJ) or enhancing homology-directed repair (HDR) will improve the nuclease-mediated knock-in efficiency. Here we show that the in vitro application of an HDR enhancer, RS-1, increases the knock-in efficiency by two- to five-fold at different loci, whereas NHEJ inhibitor SCR7 has minimal effects. We then apply RS-1 for animal production and have achieved multifold improvement on the knock-in rates as well. Our work presents tools to nuclease-mediated knock-in animal production, and sheds light on improving gene-targeting efficiencies on pluripotent stem cells.

Publication types

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

MeSH terms

  • Animals
  • Benzamides / pharmacology*
  • CRISPR-Cas Systems
  • DNA End-Joining Repair / drug effects*
  • Female
  • Gene Knock-In Techniques*
  • Gene Targeting
  • Pyrimidines / pharmacology*
  • Rabbits
  • Recombinational DNA Repair / drug effects*
  • Schiff Bases / pharmacology*
  • Sulfonamides / pharmacology*


  • 3-((benzylamino)sulfonyl)-4-bromo-N-(4-bromophenyl)benzamide
  • 5,6-bis(benzylideneamino)-2-mercaptopyrimidin-4-ol
  • Benzamides
  • Pyrimidines
  • Schiff Bases
  • Sulfonamides