4.8 Article

Massively targeted evaluation of therapeutic CRISPR off-targets in cells

Journal

NATURE COMMUNICATIONS
Volume 13, Issue 1, Pages -

Publisher

NATURE PORTFOLIO
DOI: 10.1038/s41467-022-31543-6

Keywords

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Funding

  1. Sanming Project of Medicine in Shenzhen [SZSM201612074]
  2. Qingdao-Europe Advanced Institute for Life Sciences Grant
  3. Guangdong Science and Technology Department [2019B1515120034]
  4. Science, Technology, and Innovation Commission of Shenzhen Municipality [JCYJ20200109150410232]
  5. Guangdong Provincial Key Laboratory of Genome Read and Write [2017B030301011]
  6. Guangdong Provincial Academician Workstation of BGI Synthetic Genomics [2017B090904014]
  7. Danish Research Council [9041-00317B]
  8. European Union [899417]
  9. Novo Nordisk Foundation [NNF21OC0068988, NNF21OC0071031]
  10. DFF Sapere Aude Starting grant [8048-00072A]
  11. National Human Genome Research Institute of the National Institutes of Health [RM1HG008525]
  12. China National GeneBank

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Evaluation methods for CRISPR RNA-guided nucleases off-targets, like SURRO-seq, are crucial for minimizing off-target effects and advancing gene therapies. The study highlights the impact of thermodynamically stable base pairs and free binding energy on RGN specificity.
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 center dot 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. Thorough evaluation of CRISPR RNA-guided nucleases off-targets in cells is required for advancing gene therapies. Here the authors report SURRO-seq for the simultaneous investigation of thousands of off-target sites for therapeutic RNA-guided nucleases in cells.

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