Massively targeted evaluation of therapeutic CRISPR off-targets in cells
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Methods for sensitive and high-throughput evaluation of CRISPR RNA-guided nucleases (RGNs) off-targets (OTs) are essential for advancing RGN-based gene therapies. Here we report SURRO-seq for simultaneously evaluating thousands of therapeutic RGN OTs in cells. SURRO-seq captures RGN-induced indels in cells by pooled lentiviral OTs libraries and deep sequencing, an approach comparable and complementary to OTs detection by T7 endonuclease 1, GUIDE-seq, and CIRCLE-seq. Application of SURRO-seq to 8150 OTs from 110 therapeutic RGNs identifies significantly detectable indels in 783 OTs, of which 37 OTs are found in cancer genes and 23 OTs are further validated in five human cell lines by targeted amplicon sequencing. Finally, SURRO-seq reveals that thermodynamically stable wobble base pair (rG•dT) and free binding energy strongly affect RGN specificity. Our study emphasizes the necessity of thoroughly evaluating therapeutic RGN OTs to minimize inevitable off-target effects.
| Originalsprog | Engelsk |
|---|---|
| Artikelnummer | 4049 |
| Tidsskrift | Nature Communications |
| Vol/bind | 13 |
| Antal sider | 14 |
| ISSN | 2041-1723 |
| DOI | |
| Status | Udgivet - 2022 |
Bibliografisk note
Funding Information:
This project was partially supported by the Sanming Project of Medicine in Shenzhen (SZSM201612074, to L.B. and Y.L.), Qingdao-Europe Advanced Institute for Life Sciences Grant, Guangdong Science and Technology Department (No. 2019B1515120034), the Science, Technology, and Innovation Commission of Shenzhen Municipality (grant no. JCYJ20200109150410232), Guangdong Provincial Key Laboratory of Genome Read and Write (No. 2017B030301011 to X.Xu.), Guangdong Provincial Academician Workstation of BGI Synthetic Genomics (No. 2017B090904014 to X.Xu.), Danish Research Council (9041-00317B to J.G. and Y.L.), European Union’s Horizon 2020 research and innovation program under grant agreement No 899417 (Y.L.), the Novo Nordisk Foundation (NNF21OC0068988 to J.G. and Y.L.; NNF21OC0071031 to Y.L.), the DFF Sapere Aude Starting grant (8048-00072 A to L.L.) and the National Human Genome Research Institute of the National Institutes of Health (RM1HG008525 to G.C.). We thank the China National GeneBank for the support of executing the project under the framework of Genome Read and Write.
Publisher Copyright:
© 2022, The Author(s).
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