CRISPR/Cas9 systems targeting β-globin and CCR5 genes have substantial off-target activity.
CRISPR/Cas9 systems targeting β-globin and CCR5 genes have substantial off-target activity.
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DOI：
10.1093/nar/gkt714
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发表时间：
2013-11
影响因子：
14.9
通讯作者：
Bao G
中科院分区：
文献类型：
--
作者：
Cradick TJ;Fine EJ;Antico CJ;Bao G
精确修饰内源基因的能力可以显著促进生物学研究和疾病治疗，并且成簇的规则间隔短回文重复序列（CRISPR）系统具有成为基因组工程的强大工具的潜力。然而，CRISPR系统的靶特异性在很大程度上是未知的。在这里，我们证明了靶向人血红蛋白β和C-C趋化因子受体5型基因的CRISPR/Cas9系统具有显著的脱靶切割，特别是分别在血红蛋白δ和C-C趋化因子受体2型基因内，导致总染色体缺失。CRISPR/Cas9系统的引导链被设计为与潜在脱靶位点的序列具有一系列错配。使用T7核酸内切酶I突变检测试验和桑格测序进行脱靶分析。我们发现，对靶和脱靶切割的修复导致了各种各样的插入、缺失和点突变。因此，CRISPR/Cas9系统需要仔细设计以避免潜在的脱靶切割位点，包括与引导链前间隔区邻近基序近端的12个碱基错配的那些。
The ability to precisely modify endogenous genes can significantly facilitate biological studies and disease treatment, and the clustered regularly interspaced short palindromic repeats (CRISPR) systems have the potential to be powerful tools for genome engineering. However, the target specificity of CRISPR systems is largely unknown. Here we demonstrate that CRISPR/Cas9 systems targeting the human hemoglobin β and C-C chemokine receptor type 5 genes have substantial off-target cleavage, especially within the hemoglobin δ and C-C chemokine receptor type 2 genes, respectively, causing gross chromosomal deletions. The guide strands of the CRISPR/Cas9 systems were designed to have a range of mismatches with the sequences of potential off-target sites. Off-target analysis was performed using the T7 endonuclease I mutation detection assay and Sanger sequencing. We found that the repair of the on-and off-target cleavage resulted in a wide variety of insertions, deletions and point mutations. Therefore, CRISPR/Cas9 systems need to be carefully designed to avoid potential off-target cleavage sites, including those with mismatches to the 12-bases proximal to the guide strand protospacer-adjacent motif.
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DOI：
10.1038/nrg2749
发表时间：
2010-03
期刊：
Nature reviews. Genetics
影响因子：
--
作者：
通讯作者：
--
影响因子：
14.9
作者：
Yu AM;McVey M
通讯作者：
McVey M
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle
DOI：
10.1007/978-1-60761-753-2_14
发表时间：
2010-01-01
期刊：
ENGINEERED ZINC FINGER PROTEINS: METHODS AND PROTOCOLS
影响因子：
--
作者：
Cornu, Tatjana I.;Cathomen, Toni
通讯作者：
Cathomen, Toni
影响因子：
7
作者：
Lee, Hyung Joo;Kim, Eunji;Kim, Jin-Soo
通讯作者：
Kim, Jin-Soo