1. Genome-wide detection of CRISPR editing in vivo using GUIDE-tag.
- Author
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Liang, Shun-Qing, Liu, Pengpeng, Smith, Jordan L., Mintzer, Esther, Maitland, Stacy, Dong, Xiaolong, Yang, Qiyuan, Lee, Jonathan, Haynes, Cole M., Zhu, Lihua Julie, Watts, Jonathan K., Sontheimer, Erik J., Wolfe, Scot A., and Xue, Wen
- Subjects
DNA repair ,GENOME editing ,DOUBLE-strand DNA breaks ,CRISPRS ,LIVER analysis ,GENOMICS - 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. In vivo assessment of nuclease off-target activity has primarily been indirect or through ChIP-based detection of double-strand break DNA repair factors, which can be cumbersome. Here, the authors show that GUIDE-tag, enables one-step off-target genome editing analysis in mouse liver and lung. [ABSTRACT FROM AUTHOR]
- Published
- 2022
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