Journal
SCIENCE
Volume 364, Issue 6437, Pages 286-+Publisher
AMER ASSOC ADVANCEMENT SCIENCE
DOI: 10.1126/science.aav9023
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Funding
- Li Ka Shing Foundation
- Heritage Medical Research Institute
- National Heart, Lung, and Blood Institute of the NIH [DP2-HL-141006]
- Fanconi Anemia Research Foundation
- CIRM [TRAN1-09292]
- Sir Keith Murdoch Fellowship from the American Australian Association
- NHMRC Early Career Fellowship
- NIH Instrumentation grant [S10 OD018174]
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CRISPR-Cas genome editing induces targeted DNA damage but can also affect off-target sites. Current off-target discovery methods work using purified DNA or specific cellular models but are incapable of direct detection in vivo. We developed DISCOVER-Seq (discovery of in situ Cas off-targets and verification by sequencing), a universally applicable approach for unbiased off-target identification that leverages the recruitment of DNA repair factors in cells and organisms. Tracking the precise recruitment of MRE11 uncovers the molecular nature of Cas activity in cells with single-base resolution. DISCOVER-Seq works with multiple guide RNA formats and types of Cas enzymes, allowing characterization of new editing tools. Off-targets can be identified in cell lines and patient-derived induced pluripotent stem cells and during adenoviral editing of mice, paving the way for in situ off-target discovery within individual patient genotypes during therapeutic genome editing.
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