Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9

Nat Commun. 2014 Nov 20:5:5560. doi: 10.1038/ncomms6560.


Genome engineering using programmable nucleases enables homologous recombination (HR)-mediated gene knock-in. However, the labour used to construct targeting vectors containing homology arms and difficulties in inducing HR in some cell type and organisms represent technical hurdles for the application of HR-mediated knock-in technology. Here, we introduce an alternative strategy for gene knock-in using transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) mediated by microhomology-mediated end-joining, termed the PITCh (Precise Integration into Target Chromosome) system. TALEN-mediated PITCh, termed TAL-PITCh, enables efficient integration of exogenous donor DNA in human cells and animals, including silkworms and frogs. We further demonstrate that CRISPR/Cas9-mediated PITCh, termed CRIS-PITCh, can be applied in human cells without carrying the plasmid backbone sequence. Thus, our PITCh-ing strategies will be useful for a variety of applications, not only in cultured cells, but also in various organisms, including invertebrates and vertebrates.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Bombyx
  • CRISPR-Cas Systems*
  • DNA / metabolism*
  • Deoxyribonucleases*
  • Gene Knock-In Techniques / methods*
  • Genetic Engineering
  • Genetic Vectors
  • Homologous Recombination
  • Humans
  • Molecular Sequence Data
  • Plasmids
  • Saccharomyces cerevisiae
  • Xenopus


  • DNA
  • Deoxyribonucleases

Associated data

  • GENBANK/LC008486
  • GENBANK/LC008487
  • GENBANK/LC008488
  • GENBANK/LC008489
  • GENBANK/LC008490
  • GENBANK/LC008491
  • GENBANK/LC008492