Liang, Shun-QingLiu, PengpengSmith, Jordan LMintzer, EstherMaitland, StacyDong, XiaolongYang, QiyuanLee, JonathanHaynes, Cole MZhu, Lihua JulieWatts, Jonathan KSontheimer, Erik JWolfe, Scot AXue, Wen2023-06-272023-06-272022-01-21Liang SQ, Liu P, Smith JL, Mintzer E, Maitland S, Dong X, Yang Q, Lee J, Haynes CM, Zhu LJ, Watts JK, Sontheimer EJ, Wolfe SA, Xue W. Genome-wide detection of CRISPR editing in vivo using GUIDE-tag. Nat Commun. 2022 Jan 21;13(1):437. doi: 10.1038/s41467-022-28135-9. PMID: 35064134; PMCID: PMC8782884.2041-172310.1038/s41467-022-28135-935064134https://hdl.handle.net/20.500.14038/52198Analysis 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.enOpen Access: This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. © The Author(s) 2022Attribution 4.0 Internationalhttp://creativecommons.org/licenses/by/4.0/BiologicsCRISPR-Cas9 genome editingGenetic engineeringJournal ArticleNature communications