Mapping the genetic landscape of DNA double-strand break repair

Cell. 2021 Oct 28;184(22):5653-5669.e25. doi: 10.1016/j.cell.2021.10.002. Epub 2021 Oct 20.

Abstract

Cells repair DNA double-strand breaks (DSBs) through a complex set of pathways critical for maintaining genomic integrity. To systematically map these pathways, we developed a high-throughput screening approach called Repair-seq that measures the effects of thousands of genetic perturbations on mutations introduced at targeted DNA lesions. Using Repair-seq, we profiled DSB repair products induced by two programmable nucleases (Cas9 and Cas12a) in the presence or absence of oligonucleotides for homology-directed repair (HDR) after knockdown of 476 genes involved in DSB repair or associated processes. The resulting data enabled principled, data-driven inference of DSB end joining and HDR pathways. Systematic interrogation of this data uncovered unexpected relationships among DSB repair genes and demonstrated that repair outcomes with superficially similar sequence architectures can have markedly different genetic dependencies. This work provides a foundation for mapping DNA repair pathways and for optimizing genome editing across diverse modalities.

Keywords: CRISPR-Cas9; DNA repair; double-strand breaks; functional genomics; genome editing.

Publication types

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

MeSH terms

  • CRISPR-Associated Protein 9 / metabolism
  • Cell Line
  • Cluster Analysis
  • DNA Breaks, Double-Stranded*
  • DNA Repair / genetics
  • Gene Editing
  • Gene Expression Regulation
  • Genome, Human
  • Genomics*
  • Humans
  • Phenotype
  • RNA, Guide / metabolism
  • Reproducibility of Results

Substances

  • RNA, Guide
  • CRISPR-Associated Protein 9