Massively targeted evaluation of therapeutic CRISPR off-targets in cells.
Massively targeted evaluation of therapeutic CRISPR off-targets in cells.
复制标题
细胞中治疗性CRISPR脱靶的大规模靶向评估。
DOI：
10.1038/s41467-022-31543-6
复制
发表时间：
2022-07-13
影响因子：
16.6
通讯作者：
中科院分区：
文献类型：
--
作者：
用于CRISPR RNA引导的核酸酶（RGN）脱靶（OT）的敏感和高通量评估的方法对于推进基于RGN的基因疗法至关重要。在这里，我们报告了SURRO-seq，用于同时评估细胞中数千种治疗性RGN OT。SURRO-seq通过合并的慢病毒OT文库和深度测序捕获细胞中RGN诱导的插入缺失，这是一种与通过T7内切核酸酶1、GUIDE-seq和CIRCLE-seq进行的OT检测相当且互补的方法。将SURRO-seq应用于来自110个治疗性RGN的8150个OT，在783个OT中鉴定出显著可检测的indel，其中37个OT在癌症基因中发现，23个OT通过靶向扩增子测序在5个人类细胞系中进一步验证。最后，SURRO-seq揭示了化学稳定的摆动碱基对（rG·dT）和自由结合能强烈影响RGN特异性。我们的研究强调了彻底评估治疗性RGN OT的必要性，以尽量减少不可避免的脱靶效应。对细胞中CRISPR RNA引导的核酸酶脱靶的彻底评估是推进基因疗法所必需的。在这里，作者报告了SURRO-seq，用于同时研究细胞中治疗性RNA引导核酸酶的数千个脱靶位点。
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. 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.
登录
查看更多内容
DeepCRISPR：通过深度学习优化 CRISPR 引导 RNA 设计
影响因子：
12.3
作者：
Chuai G;Ma H;Yan J;Chen M;Hong N;Xue D;Zhou C;Zhu C;Chen K;Duan B;Gu F;Qu S;Huang D;Wei J;Liu Q
通讯作者：
Liu Q
影响因子：
7
作者：
Cho SW;Kim S;Kim Y;Kweon J;Kim HS;Bae S;Kim JS
通讯作者：
Kim JS
影响因子：
46.9
作者：
Doench JG;Fusi N;Sullender M;Hegde M;Vaimberg EW;Donovan KF;Smith I;Tothova Z;Wilen C;Orchard R;Virgin HW;Listgarten J;Root DE
通讯作者：
Root DE
影响因子：
12.4
作者：
Chung, Cheng-Han;Allen, Alexander G.;Dampier, Will
通讯作者：
Dampier, Will
影响因子：
46.9
作者：
Hendel A;Bak RO;Clark JT;Kennedy AB;Ryan DE;Roy S;Steinfeld I;Lunstad BD;Kaiser RJ;Wilkens AB;Bacchetta R;Tsalenko A;Dellinger D;Bruhn L;Porteus MH
通讯作者：
Porteus MH