1. Genome-wide detection of CRISPR editing in vivo using GUIDE-tag.
- Author
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Liang 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, and Xue W
- Subjects
- Animals, Base Sequence, Biotin metabolism, Biotinylation, CRISPR-Associated Protein 9 metabolism, Cell Line, Tumor, DNA metabolism, Genes, Reporter, Genome, Liver metabolism, Lung metabolism, Mice, Ribonucleoproteins metabolism, CRISPR-Cas Systems genetics, Gene Editing, RNA, Guide, CRISPR-Cas Systems genetics
- 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., (© 2022. The Author(s).)
- Published
- 2022
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