High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity.
High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity.
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DOI：
10.1038/nbt.2673
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发表时间：
2013-09
影响因子：
46.9
通讯作者：
中科院分区：
文献类型：
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作者：
RNA可编程的Cas9内切核酸酶在与20个碱基对的指导RNA互补的位点处切割双链DNA。Cas9系统已用于修饰多种细胞和生物体的基因组，证明了其作为一种简便的基因组工程工具的潜力。我们使用体外选择和高通量测序来确定八种Cas9：向导RNA复合物切割10^12个潜在脱靶DNA序列中的每一个的倾向。选择结果预测了人类基因组中的五个脱靶位点，这些位点被证实在表达两种Cas9：向导RNA复合物之一时在HEK293T细胞中经历基因组切割。与以前的模型相比，我们的结果表明Cas9：向导RNA特异性延伸超过7至12个碱基对的种子序列。我们的研究结果还表明，在体外和细胞中的活性和特异性之间存在权衡，因为较短、活性较低的引导RNA比较长、活性更高的引导RNA更具特异性。高浓度的Cas9：向导RNA复合物可以切割含有PAM附近或PAM内的突变的脱靶位点，当酶浓度受限时，所述脱靶位点不被切割。
The RNA-programmable Cas9 endonuclease cleaves double-stranded DNA at sites complementary to a 20-base-pair guide RNA. The Cas9 system has been used to modify genomes in multiple cells and organisms, demonstrating its potential as a facile genome-engineering tool. We used in vitro selection and high-throughput sequencing to determine the propensity of eight Cas9:guide RNA complexes to cleave each of 10^12 potential off-target DNA sequences. The selection results predicted five off-target sites in the human genome that were confirmed to undergo genome cleavage in HEK293T cells upon expression of one of two Cas9:guide RNA complexes. In contrast to previous models, our results show that Cas9:guide RNA specificity extends past a 7- to 12-base pair seed sequence. Our results also suggest a tradeoff between activity and specificity both in vitro and in cells as a shorter, less-active guide RNA is more specific then a longer, more-active guide RNA. High concentrations of Cas9:guide RNA complexes can cleave off-target sites containing mutations near or within the PAM that are not cleaved when enzyme concentrations are limiting.
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影响因子：
46.9
作者：
Hockemeyer, Dirk;Soldner, Frank;Beard, Caroline;Gao, Qing;Mitalipova, Maisam;DeKelver, Russell C.;Katibah, George E.;Amora, Ranier;Boydston, Elizabeth A.;Zeitler, Bryan;Meng, Xiangdong;Miller, Jeffrey C.;Zhang, Lei;Rebar, Edward J.;Gregory, Philip D.;Urnov, Fyodor D.;Jaenisch, Rudolf
通讯作者：
Jaenisch, Rudolf
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle
影响因子：
46.9
作者：
通讯作者：
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DOI：
10.1126/science.1232033
发表时间：
2013-02-15
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Mali P;Yang L;Esvelt KM;Aach J;Guell M;DiCarlo JE;Norville JE;Church GM
通讯作者：
Church GM
影响因子：
14.9
作者：
Sapranauskas R;Gasiunas G;Fremaux C;Barrangou R;Horvath P;Siksnys V
通讯作者：
Siksnys V