CRISPR-Cas9 Genome Editing in Human Cells Occurs via the Fanconi Anemia Pathway

Nat Genet. 2018 Aug;50(8):1132-1139. doi: 10.1038/s41588-018-0174-0. Epub 2018 Jul 27.

Abstract

CRISPR-Cas genome editing creates targeted DNA double-strand breaks (DSBs) that are processed by cellular repair pathways, including the incorporation of exogenous DNA via single-strand template repair (SSTR). To determine the genetic basis of SSTR in human cells, we developed a coupled inhibition-cutting system capable of interrogating multiple editing outcomes in the context of thousands of individual gene knockdowns. We found that human Cas9-induced SSTR requires the Fanconi anemia (FA) pathway, which is normally implicated in interstrand cross-link repair. The FA pathway does not directly impact error-prone, non-homologous end joining, but instead diverts repair toward SSTR. Furthermore, FANCD2 protein localizes to Cas9-induced DSBs, indicating a direct role in regulating genome editing. Since FA is itself a genetic disease, these data imply that patient genotype and/or transcriptome may impact the effectiveness of gene editing treatments and that treatments biased toward FA repair pathways could have therapeutic value.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems / genetics*
  • Cell Line
  • Cell Line, Tumor
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics*
  • DNA Breaks, Double-Stranded
  • DNA End-Joining Repair / genetics
  • Fanconi Anemia / genetics*
  • Gene Editing / methods
  • Genotype
  • HCT116 Cells
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Jurkat Cells
  • K562 Cells
  • MCF-7 Cells
  • Signal Transduction / genetics*