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Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq
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Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq
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Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq
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Journal Article
Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq
2019
Overview
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.
Publisher
American Association for the Advancement of Science,The American Association for the Advancement of Science
Subject
/ Animals
/ Chromatin Immunoprecipitation
/ Clustered Regularly Interspaced Short Palindromic Repeats
/ CRISPR
/ CRISPR-Associated Protein 9 - chemistry
/ CRISPR-Associated Protein 9 - metabolism
/ DNA
/ DNA Repair Enzymes - metabolism
/ Editing
/ Embryos
/ Genomes
/ Humans
/ Induced Pluripotent Stem Cells
/ MRE11 Homologue Protein - genetics
/ MRE11 Homologue Protein - metabolism
/ Mutation
/ Proteins
/ Repair
/ RNA
/ RNA, Guide, CRISPR-Cas Systems
