Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells
Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells
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DOI：
10.1038/nmeth.3284
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发表时间：
2015-03-01
期刊：
影响因子：
48
通讯作者：
Kim, Jin-Soo
中科院分区：
文献类型：
--
作者：
Kim, Daesik;Bae, Sangsu;Kim, Jin-Soo
尽管通过 CRISPR-Cas9 系统进行的 RNA 引导基因组编辑现已广泛应用于生物医学研究，但 Cas9 核酸酶的全基因组靶标特异性仍然存在争议。在这里，我们推出 Digenome-seq，即体外 Cas9 消化的全基因组测序，以分析人类细胞中全基因组 Cas9 脱靶效应。这种体外消化产生的序列读数在切割位点具有相同的 5' 末端，可以通过计算来识别。我们验证了诱导插入或缺失的脱靶位点，频率低于 0.1%，接近靶向深度测序的检测限。我们还表明，Cas9 核酸酶具有高度特异性，仅在整个基因组中的几个而非数千个位点诱导脱靶突变，并且可以通过用修饰的 sgRNA 替换“混杂”的单引导 RNA (sgRNA) 来避免 Cas9 脱靶效应。 Digenome-seq 是一种稳健、灵敏、公正且经济高效的方法，用于分析包括 Cas9 在内的可编程核酸酶的全基因组脱靶效应。
Although RNA-guided genome editing via the CRISPR-Cas9 system is now widely used in biomedical research, genome-wide target specificities of Cas9 nucleases remain controversial. Here we present Digenome-seq, in vitro Cas9-digested whole-genome sequencing, to profile genome-wide Cas9 off-target effects in human cells. This in vitro digest yields sequence reads with the same 5' ends at cleavage sites that can be computationally identified. We validated off-target sites at which insertions or deletions were induced with frequencies below 0.1%, near the detection limit of targeted deep sequencing. We also showed that Cas9 nucleases can be highly specific, inducing off-target mutations at merely several, rather than thousands of, sites in the entire genome and that Cas9 off-target effects can be avoided by replacing 'promiscuous' single guide RNAs (sgRNAs) with modified sgRNAs. Digenome-seq is a robust, sensitive, unbiased and cost-effective method for profiling genome-wide off-target effects of programmable nucleases including Cas9.