biostudies-literatureLiang SQAmerican Cancer SocietyNCATS NIH HHSNIA NIH HHSNHLBI NIH HHSNHGRI NIH HHSNCI NIH HHSNIGMS NIH HHS437https://www.ebi.ac.uk/biostudies/studies/S-EPMC8782884biostudies-literatureUnknown13(1)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.Nature communicationsGenome-wide detection of CRISPR editing in vivo using GUIDE-tag.PMC8782884P01 HL131471R01 AG040061T32 GM107000129056-RSG-16-093R01 GM115911F30 CA239483UG3 HL147367DP2 HL137167T32 CA130807R01 HL150669UG3 TR002668U24 HG010423Dong XLiu PLee JLiang SQWolfe SAXue WHaynes CMMaitland SZhu LJWatts JKSontheimer EJSmith JLYang QMintzer EfalseGenome-wide detection of CRISPR editing in vivo using GUIDE-tag.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.2022-01-01T00:00:00Z2022 Jan2025-04-04T19:53:50.755Z2025-04-04T19:53:50.755ZS-EPMC87828843506413410.1038/s41467-022-28135-9