Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq
Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq
Abstract Genome editing using nucleases such as CRISPR-Cas induces programmable DNA damage at a target genomic site but can also affect off-target sites. Here, we develop a powerful, sensitive assay for the unbiased identification of off-target sites that we term DISCOVER-Seq. This approach takes advantage of the recruitment of endogenous DNA repair factors for genome-wide identification of Cas-induced double-strand breaks. One such factor, MRE11, is recruited precisely to double-strand breaks, enabling molecular characterization of nuclease cut sites with single-base resolution. DISCOVER-Seq detects off-targets in cellular models and in vivo upon adenoviral gene editing of mouse livers, paving the way for real-time off-target discovery during therapeutic gene editing. DISCOVER-Seq is furthermore applicable to multiple types of Cas nucleases and provides an unprecedented view of events that precede repair of the affected sites.
Wienert Beeke、Richardson Christopher D、Yeh Charles D、Vu Jonathan T、Judge Luke M、Morlock Michaela、Watry Hannah L、Porritt Michelle J、Maresca Marcello、Kazane Katelynn R、Corn Jacob E、Conklin Bruce R、Wyman Stacia K、Akcakaya Pinar
Innovative Genomics Institute, University of California||Department of Molecular and Cell Biology, University of California||Gladstone InstitutesInnovative Genomics Institute, University of California||Department of Molecular and Cell Biology, University of CaliforniaInnovative Genomics Institute, University of California||Department of Molecular and Cell Biology, University of California||Department of BiologyInnovative Genomics Institute, University of CaliforniaGladstone Institutes||Department of Pediatrics, UCSFDiscovery Biology, Discovery Sciences, IMED Biotech UnitInnovative Genomics Institute, University of California||Gladstone InstitutesDiscovery Biology, Discovery Sciences, IMED Biotech UnitDiscovery Biology, Discovery Sciences, IMED Biotech UnitInnovative Genomics Institute, University of California||Department of Molecular and Cell Biology, University of CaliforniaInnovative Genomics Institute, University of California||Department of Molecular and Cell Biology, University of California||Department of BiologyGladstone Institutes||Departments of Medicine, Ophthalmology & Pharmacology, UCSFInnovative Genomics Institute, University of CaliforniaDiscovery Biology, Discovery Sciences, IMED Biotech Unit
生物科学研究方法、生物科学研究技术分子生物学基础医学
Wienert Beeke,Richardson Christopher D,Yeh Charles D,Vu Jonathan T,Judge Luke M,Morlock Michaela,Watry Hannah L,Porritt Michelle J,Maresca Marcello,Kazane Katelynn R,Corn Jacob E,Conklin Bruce R,Wyman Stacia K,Akcakaya Pinar.Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq[EB/OL].(2025-03-28)[2025-06-23].https://www.biorxiv.org/content/10.1101/469635.点此复制
评论