PEAC-seq adopts Prime Editor to detect CRISPR off-target and DNA translocation

Zhenxing Yu, Zhike Lu, Jingjing Li, Yingying Wang, Panfeng Wu, Yini Li, Yangfan Zhou, Bailun Li, Heng Zhang, Yingzheng Liu and Lijia Ma ()
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Zhenxing Yu: Fudan University
Zhike Lu: Westlake Laboratory of Life Sciences and Biomedicine
Jingjing Li: Westlake University
Yingying Wang: Fudan University
Panfeng Wu: Westlake University
Yini Li: Fudan University
Yangfan Zhou: Westlake Laboratory of Life Sciences and Biomedicine
Bailun Li: Westlake Laboratory of Life Sciences and Biomedicine
Heng Zhang: Westlake University
Yingzheng Liu: Westlake Laboratory of Life Sciences and Biomedicine
Lijia Ma: Westlake Laboratory of Life Sciences and Biomedicine

Nature Communications, 2022, vol. 13, issue 1, 1-13

Abstract: Abstract CRISPR technology holds significant promise for biological studies and gene therapies because of its high flexibility and efficiency when applied in mammalian cells. But endonuclease (e.g., Cas9) potentially generates undesired edits; thus, there is an urgent need to comprehensively identify off-target sites so that the genotoxicities can be accurately assessed. To date, it is still challenging to streamline the entire process to specifically label and efficiently enrich the cleavage sites from unknown genomic locations. Here we develop PEAC-seq, in which we adopt the Prime Editor to insert a sequence-optimized tag to the editing sites and enrich the tagged regions with site-specific primers for high throughput sequencing. Moreover, we demonstrate that PEAC-seq could identify DNA translocations, which are more genotoxic but usually overlooked by other off-target detection methods. As PEAC-seq does not rely on exogenous oligodeoxynucleotides to label the editing site, we also conduct in vivo off-target identification as proof of concept. In summary, PEAC-seq provides a comprehensive and streamlined strategy to identify CRISPR off-targeting sites in vitro and in vivo, as well as DNA translocation events. This technique further diversified the toolkit to evaluate the genotoxicity of CRISPR applications in research and clinics.

Date: 2022
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DOI: 10.1038/s41467-022-35086-8

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