A role for alternative end-joining factors in homologous recombination and genome editing in Chinese hamster ovary cells

DNA Repair (Amst). 2019 Oct;82:102691. doi: 10.1016/j.dnarep.2019.102691. Epub 2019 Aug 15.


CRISPR technologies greatly foster genome editing in mammalian cells through site-directed DNA double strand breaks (DSBs). However, precise editing outcomes, as mediated by homologous recombination (HR) repair, are typically infrequent and outnumbered by undesired genome alterations. By using knockdown and overexpression studies in Chinese hamster ovary (CHO) cells as well as characterizing repaired DNA junctions, we found that efficient HR-mediated genome editing depends on alternative end-joining (alt-EJ) DNA repair activities, a family of incompletely characterized DNA repair pathways traditionally considered to oppose HR. This dependency was influenced by the CRISPR nuclease type and the DSB-to-mutation distance, but not by the DNA sequence surrounding the DSBs or reporter cell line. We also identified elevated Mre11 and Pari, and low Rad51 expression levels as the most rate-limiting factors for HR in CHO cells. Counteracting these three bottlenecks improved precise genome editing by up to 75%. Altogether, our study provides novel insights into the complex interplay of alt-EJ and HR repair pathways, highlighting their relevance for developing improved genome editing strategies.

Keywords: Alternative end-joining pathways; Gene correction; Genome editing; Homologous recombination.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CHO Cells
  • CRISPR-Associated Protein 9 / metabolism
  • Cricetinae
  • Cricetulus
  • DNA End-Joining Repair / genetics*
  • Deoxyribonucleases, Type II Site-Specific / metabolism
  • Gene Editing*
  • Homologous Recombination / genetics*


  • CRISPR-Associated Protein 9
  • endodeoxyribonuclease FokI
  • Deoxyribonucleases, Type II Site-Specific