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Science. 2019 Apr 19;364(6437):286-289. doi: 10.1126/science.aav9023. Epub 2019 Apr 18.

Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq.

Author information

1
Innovative Genomics Institute, University of California Berkeley, Berkeley, CA 94704, USA.
2
Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA 94720, USA.
3
Gladstone Institutes, San Francisco, CA 94158, USA.
4
Discovery Biology, AstraZeneca, 43150 Gothenburg, Sweden.
5
Department of Pediatrics, University of California San Francisco, San Francisco, CA 94143, USA.
6
Departments of Medicine, Ophthalmology, and Pharmacology, University of California San Francisco, San Francisco, California 94143, USA.
7
Innovative Genomics Institute, University of California Berkeley, Berkeley, CA 94704, USA. jacob.corn@biol.ethz.ch.
#
Contributed equally

Abstract

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.

Comment in

PMID:
31000663
PMCID:
PMC6589096
DOI:
10.1126/science.aav9023
[Indexed for MEDLINE]
Free PMC Article

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