Targeted and genome-wide sequencing reveal single nucleotide variations impacting specificity of Cas9 in human stem cells.
Targeted and genome-wide sequencing reveal single nucleotide variations impacting specificity of Cas9 in human stem cells.
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DOI：
10.1038/ncomms6507
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发表时间：
2014-11-26
影响因子：
16.6
通讯作者：
Church, George
中科院分区：
文献类型：
--
作者：
Yang, Luhan;Grishin, Dennis;Wang, Gang;Aach, John;Zhang, Cheng-Zhong;Chari, Raj;Homsy, Jason;Cai, Xuyu;Zhao, Yue;Fan, Jian-Bing;Seidman, Christine;Seidman, Jonathan;Pu, William;Church, George
CRISPR/Cas9在位点特异性基因靶向方面表现出高效率。然而，Cas9核酸酶的潜在脱靶效应是任何治疗应用的主要安全问题。在这里，我们通过CRISPR/Cas9在人诱导的多能干细胞中敲除Tafazzin基因，效率为54%。我们结合全基因组测序和深度靶向测序来表征Cas9编辑的脱靶效应。cas9修饰的hiPSC克隆的全基因组测序既没有检测到总体基因组改变，也没有检测到突变率升高。在Cas9处理的细胞群体中，对计算机预测的脱靶位点进行深度测序进一步证实了Cas9的高特异性。然而，在我们的实验中，我们发现了一个由常见的种系单核苷酸变异（SNV）产生的单一高效脱靶位点。基于计算机分析，我们估计snv在人类基因组中产生脱靶位点的可能性约为1.5-8.5%，这取决于基因组和位点选择方法，但也注意到这些位点的突变率可能很低，可能不会产生功能后果。我们的研究证明了使用CRISPR/Cas9进行高特异性克隆离体基因编辑的可行性，并强调了在个性化CRISPR设计之前进行全基因组测序的价值。微生物rna引导的CRISPR/Cas9系统具有强大的基因组编辑活性，但Cas9核酸酶的脱靶效应直到最近才开始被分析。在这里，作者提供了证据，证明Cas9核酸酶在人类诱导的多能干细胞中靶向Tafazzin基因具有高特异性，并证明了人类基因组的单核苷酸变异对Cas9特异性的影响。
CRISPR/Cas9 has demonstrated a high-efficiency in site-specific gene targeting. However, potential off-target effects of the Cas9 nuclease represent a major safety concern for any therapeutic application. Here, we knock out the Tafazzin gene by CRISPR/Cas9 in human-induced pluripotent stem cells with 54% efficiency. We combine whole-genome sequencing and deep-targeted sequencing to characterise the off-target effects of Cas9 editing. Whole-genome sequencing of Cas9-modified hiPSC clones detects neither gross genomic alterations nor elevated mutation rates. Deep sequencing of in silico predicted off-target sites in a population of Cas9-treated cells further confirms high specificity of Cas9. However, we identify a single high-efficiency off-target site that is generated by a common germline single-nucleotide variant (SNV) in our experiment. Based on in silico analysis, we estimate a likelihood of SNVs creating off-target sites in a human genome to be ~1.5–8.5%, depending on the genome and site-selection method, but also note that mutations might be generated at these sites only at low rates and may not have functional consequences. Our study demonstrates the feasibility of highly specific clonal ex vivo gene editing using CRISPR/Cas9 and highlights the value of whole-genome sequencing before personalised CRISPR design. The microbial RNA-guided CRISPR/Cas9 system has robust genome-editing activities, but the off-target effects of the Cas9 nuclease have only recently begun to be analysed. Here the authors provide evidence for high specificity of the Cas9 nuclease on targeting of the Tafazzin gene in human-induced pluripotent stem cells and demonstrate the impact of single-nucleotide variations of the human genome on Cas9 specificity.
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来自1,092个人基因组的遗传变异的综合图。
影响因子：
64.8
作者：
通讯作者：
--
影响因子：
82.9
作者：
通讯作者：
--
影响因子：
30.8
作者：
Bione, S;DAdamo, P;Toniolo, D
通讯作者：
Toniolo, D
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle
影响因子：
64.5
作者：
Niu, Yuyu;Shen, Bin;Sha, Jiahao
通讯作者：
Sha, Jiahao