GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases.
GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases.
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DOI：
10.1038/nbt.3117
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发表时间：
2015-02
影响因子：
46.9
通讯作者：
Joung, J. Keith
中科院分区：
文献类型：
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作者：
Tsai, Shengdar Q.;Zheng, Zongli;Nguyen, Nhu T.;Liebers, Matthew;Topkar, Ved V.;Thapar, Vishal;Wyvekens, Nicolas;Khayter, Cyd;Iafrate, A. John;Le, Long P.;Aryee, Martin J.;Joung, J. Keith
CRISPR RNA引导的核酸酶（RGN）是广泛使用的基因组编辑试剂，但一直缺乏描述其全基因组脱靶切割活性的方法。在这里，我们描述了一种用于全局检测由RGN和潜在的其他核酸酶引入的DNA双链断裂（DSB）的方法。这种方法被称为测序实现的全基因组无偏见DSB鉴定（GUIDE-Seq），依赖于将双链寡脱氧核苷酸捕获到断裂中。将GUIDE-Seq应用于两种人类细胞系中的十三个RGN，揭示了RGN脱靶活性的广泛变异性以及脱靶序列的未被认识到的特征。大多数识别的位点未被现有的计算方法或ChIP-Seq检测到。GUIDE-Seq还鉴定了RGN非依赖性基因组断裂点“热点”。最后，GUIDE-Seq揭示截短的向导RNA表现出显著减少的RGN诱导的脱靶DSB。我们的实验定义了迄今为止全基因组识别RGN脱靶效应的最严格框架，并提供了一种在临床使用前评估这些核酸酶安全性的方法。
CRISPR RNA-guided nucleases (RGNs) are widely used genome-editing reagents, but methods to delineate their genome-wide off-target cleavage activities have been lacking. Here we describe an approach for global detection of DNA double-stranded breaks (DSBs) introduced by RGNs and potentially other nucleases. This method, called Genome-wide Unbiased Identification of DSBs Enabled by Sequencing (GUIDE-Seq), relies on capture of double-stranded oligodeoxynucleotides into breaks Application of GUIDE-Seq to thirteen RGNs in two human cell lines revealed wide variability in RGN off-target activities and unappreciated characteristics of off-target sequences. The majority of identified sites were not detected by existing computational methods or ChIP-Seq. GUIDE-Seq also identified RGN-independent genomic breakpoint ‘hotspots’. Finally, GUIDE-Seq revealed that truncated guide RNAs exhibit substantially reduced RGN-induced off-target DSBs. Our experiments define the most rigorous framework for genome-wide identification of RGN off-target effects to date and provide a method for evaluating the safety of these nucleases prior to clinical use.
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影响因子：
14.9
作者：
Sander JD;Ramirez CL;Linder SJ;Pattanayak V;Shoresh N;Ku M;Foden JA;Reyon D;Bernstein BE;Liu DR;Joung JK
通讯作者：
Joung JK
影响因子：
14.9
作者：
Orlando SJ;Santiago Y;DeKelver RC;Freyvert Y;Boydston EA;Moehle EA;Choi VM;Gopalan SM;Lou JF;Li J;Miller JC;Holmes MC;Gregory PD;Urnov FD;Cost GJ
通讯作者：
Cost GJ
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：
通讯作者：
--
影响因子：
64.5
作者：
Hsu PD;Lander ES;Zhang F
通讯作者：
Zhang F