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- W4206225117 abstract "Abstract Analysis of off-target editing is an important aspect of the development of safe nuclease-based genome editing therapeutics. in vivo assessment of nuclease off-target activity has primarily been indirect (based on discovery in vitro, in cells or via computational prediction) or through ChIP-based detection of double-strand break (DSB) DNA repair factors, which can be cumbersome. Herein we describe GUIDE-tag, which enables one-step, off-target genome editing analysis in mouse liver and lung. The GUIDE-tag system utilizes tethering between the Cas9 nuclease and the DNA donor to increase the capture rate of nuclease-mediated DSBs and UMI incorporation via Tn5 tagmentation to avoid PCR bias. These components can be delivered as SpyCas9-mSA ribonucleoprotein complexes and biotin-dsDNA donor for in vivo editing analysis. GUIDE-tag enables detection of off-target sites where editing rates are ≥ 0.2%. UDiTaS analysis utilizing the same tagmented genomic DNA detects low frequency translocation events with off-target sites and large deletions in vivo. The SpyCas9-mSA and biotin-dsDNA system provides a method to capture DSB loci in vivo in a variety of tissues with a workflow that is amenable to analysis of gross genomic alterations that are associated with genome editing." @default.
- W4206225117 created "2022-01-26" @default.
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- W4206225117 date "2022-01-21" @default.
- W4206225117 modified "2023-10-16" @default.
- W4206225117 title "Genome-wide detection of CRISPR editing in vivo using GUIDE-tag" @default.
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- W4206225117 doi "https://doi.org/10.1038/s41467-022-28135-9" @default.
- W4206225117 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/35064134" @default.
- W4206225117 hasPublicationYear "2022" @default.
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