Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ

BMC Genomics. 2016 Nov 28;17(1):979. doi: 10.1186/s12864-016-3331-9.


Background: Although CRISPR/Cas enables one-step gene cassette knock-in, assembling targeting vectors containing long homology arms is a laborious process for high-throughput knock-in. We recently developed the CRISPR/Cas-based precise integration into the target chromosome (PITCh) system for a gene cassette knock-in without long homology arms mediated by microhomology-mediated end-joining.

Results: Here, we identified exonuclease 1 (Exo1) as an enhancer for PITCh in human cells. By combining the Exo1 and PITCh-directed donor vectors, we achieved convenient one-step knock-in of gene cassettes and floxed allele both in human cells and mouse zygotes.

Conclusions: Our results provide a technical platform for high-throughput knock-in.

Keywords: CRISPR/Cas; Cloning-free; Exo1; Flox; Gene cassette; High throughput; Knock-in; MMEJ; Mouse; Reporter.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • CRISPR-Cas Systems
  • Cell Line
  • Chromosomes
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • Exodeoxyribonucleases / metabolism
  • Gene Knock-In Techniques*
  • Gene Targeting
  • Genetic Loci
  • Homologous Recombination*
  • Humans
  • Mice
  • Transcription Activator-Like Effector Nucleases
  • Zygote*


  • Exodeoxyribonucleases
  • Transcription Activator-Like Effector Nucleases
  • exodeoxyribonuclease I