CRISPR/Cas9 systems targeting β-globin and CCR5 genes have substantial off-target activity

被引:470
作者
Cradick, Thomas J.
Fine, Eli J.
Antico, Christopher J.
Bao, Gang [1 ]
机构
[1] Georgia Inst Technol, Dept Biomed Engn, Atlanta, GA 30332 USA
基金
美国国家卫生研究院;
关键词
ZINC-FINGER NUCLEASES; CHROMOSOMAL DELETIONS; EMBRYO MICROINJECTION; ADAPTIVE IMMUNITY; KNOCKOUT RATS; DNA; CLEAVAGE; BACTERIA; CELLS; CAS9;
D O I
10.1093/nar/gkt714
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 beta and C-C chemokine receptor type 5 genes have substantial off-target cleavage, especially within the hemoglobin delta 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.
引用
收藏
页码:9584 / 9592
页数:9
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