Mapping the genetic landscape of DNA double-strand break repair

Jeffrey A Hussmann; Jia Ling; Purnima Ravisankar; Jun Yan; Ann Cirincione; Albert Xu; Danny Simpson; Dian Yang; Anne Bothmer; Cecilia Cotta-Ramusino; Jonathan S Weissman; Britt Adamson

doi:10.1016/j.cell.2021.10.002

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.

Authors

Affiliations

¹ Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA; Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge, MA 02142, USA.
² Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ 08544, USA.
³ Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA.
⁴ Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA.
⁵ Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA; Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge, MA 02142, USA.
⁶ Editas Medicine, Cambridge, MA 02141, USA.
⁷ Editas Medicine, Cambridge, MA 02141, USA. Electronic address: cecilia.cotta@gmail.com.
⁸ Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA; Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge, MA 02142, USA; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Electronic address: weissman@wi.mit.edu.
⁹ Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ 08544, USA; Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA. Electronic address: badamson@princeton.edu.

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, CRISPR-Cas Systems / metabolism
Reproducibility of Results

Substances

RNA, Guide, CRISPR-Cas Systems
CRISPR-Associated Protein 9

Abstract

Publication types

MeSH terms

Substances

Grants and funding