Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9.
Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9.
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DOI：
10.1038/nbt.3437
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发表时间：
2016-02
影响因子：
46.9
通讯作者：
Root DE
中科院分区：
文献类型：
--
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
基于CRISPR-Cas9的基因筛选是生物学中一种强大的新工具。通过简单地改变单向导RNA（sgRNA）的序列，Cas9可以相对容易地被重编程为靶向基因组中的不同位点，但是单个sgRNA的中靶活性和脱靶效应可以变化很大。在这里，我们使用最近设计的sgRNA设计规则来创建人类和小鼠全基因组文库，进行阳性和阴性选择筛选，并观察到使用这些规则产生了改进的结果。此外，我们分析了数千种sgRNA的脱靶活性，并开发了一种预测脱靶位点的指标。我们将这些来自大规模经验数据的发现结合起来，以改进我们的计算设计规则，并创建优化的sgRNA文库，使靶向活性最大化，脱靶效应最小化，从而实现更有效和高效的遗传筛选和基因组工程。
CRISPR-Cas9-based genetic screens are a powerful new tool in biology. By simply altering the sequence of the single-guide RNA (sgRNA), Cas9 can be reprogrammed to target different sites in the genome with relative ease, but the on-target activity and off-target effects of individual sgRNAs can vary widely. Here, we use recently-devised sgRNA design rules to create human and mouse genome-wide libraries, perform positive and negative selection screens and observe that the use of these rules produced improved results. Additionally, we profile the off-target activity of thousands of sgRNAs and develop a metric to predict off-target sites. We incorporate these findings from large-scale, empirical data to improve our computational design rules and create optimized sgRNA libraries that maximize on-target activity and minimize off-target effects to enable more effective and efficient genetic screens and genome engineering.