Lars Bolund Institute of Regenerative Medicine, Qingdao-Europe Advanced Institute for Life Sciences, BGI-Qingdao, BGI-Shenzhen, Qingdao, China.
Department of Biology, Copenhagen University, Copenhagen, Denmark.
Nat Commun. 2022 Jul 13;13(1):4049. doi: 10.1038/s41467-022-31543-6.
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.
用于敏感和高通量评估 CRISPR RNA 引导的核酸酶（RGNs）脱靶（OTs）的方法对于推进基于 RGN 的基因治疗至关重要。在这里，我们报告了 SURRO-seq，用于在细胞中同时评估数千种治疗性 RGN OTs。SURRO-seq 通过汇集慢病毒 OTs 文库和深度测序来捕获 RGN 诱导的细胞内缺失，这是一种与 T7 内切酶 1、GUIDE-seq 和 CIRCLE-seq 相当且互补的方法。应用 SURRO-seq 对 110 种治疗性 RGN 中的 8150 个 OTs 进行分析，在 783 个 OTs 中鉴定出明显可检测的缺失，其中 37 个 OTs 位于癌症基因中，23 个 OTs 在五个人类细胞系中通过靶向扩增子测序进一步验证。最后，SURRO-seq 表明，热力学稳定的摆动碱基对（rG•dT）和自由结合能强烈影响 RGN 的特异性。我们的研究强调了彻底评估治疗性 RGN OTs 的必要性，以最小化不可避免的脱靶效应。
