Massively targeted evaluation of therapeutic CRISPR off-targets in cells

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.

Automated summary

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.