Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases.
Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases.
复制标题
DOI：
10.1101/gr.162339.113
复制
发表时间：
2014-01
期刊：
影响因子：
7
通讯作者：
Kim JS
中科院分区：
文献类型：
--
作者：
Cho SW;Kim S;Kim Y;Kweon J;Kim HS;Bae S;Kim JS
RNA引导的内核酸酶（RGENS），源自原核适应性免疫系统CRISPR/CAS，可以在细胞和生物中实现靶向基因组工程。 rgens是核糖核蛋白，由引导RNA和CAS9组成，RNA和Cas9是蛋白质成分的蛋白质成分。这些酶裂解染色体DNA的序列是互补的，以靶向方式引导RNA，产生特定位点的DNA双链断裂（DSB），其修复会引起靶向基因组的修饰。尽管对RGEN介导的基因组编辑的兴趣广泛，但这些核酸酶仍受到脱靶突变和与脱靶DNA裂解相关的有害染色体易位的限制。在这里，我们表明，通过在基因组中选择独特的目标序列并修改指南RNA和Cas9，可以在深度测序的检测限制下降低rgens的脱靶效应。我们发现，指导RNA的组成和结构都会影响细胞中的RGEN活性，以减少脱靶效应。 rgens从靶向位点有效区分了靶向位点，而靶向位置则有两个基础。此外，外显子组测序分析表明，在四个克隆人群的突变细胞中，没有两个rgens诱导脱靶突变。此外，由D10A Cas9和Guide RNA组成的配对Cas9 nickase在人体细胞中产生两个单链断裂（SSB）或在不同的DNA链上产生两个单链断裂（SSB）或划痕，在人类细胞中非常具体，避免了脱靶突变而不牺牲基因组编辑的效率。有趣的是，配对的nickase以靶向方式诱导染色体缺失，而不会引起不必要的易位。我们的结果强调了选择独特的目标序列并优化引导RNA和CAS9以避免或减少RGEN诱导的脱靶突变的重要性。
RNA-guided endonucleases (RGENs), derived from the prokaryotic adaptive immune system known as CRISPR/Cas, enable targeted genome engineering in cells and organisms. RGENs are ribonucleoproteins that consist of guide RNA and Cas9, a protein component originated from Streptococcus pyogenes. These enzymes cleave chromosomal DNA, whose sequence is complementary, to guide RNA in a targeted manner, producing site-specific DNA double-strand breaks (DSBs), the repair of which gives rise to targeted genome modifications. Despite broad interest in RGEN-mediated genome editing, these nucleases are limited by off-target mutations and unwanted chromosomal translocations associated with off-target DNA cleavages. Here, we show that off-target effects of RGENs can be reduced below the detection limits of deep sequencing by choosing unique target sequences in the genome and modifying both guide RNA and Cas9. We found that both the composition and structure of guide RNA can affect RGEN activities in cells to reduce off-target effects. RGENs efficiently discriminated on-target sites from off-target sites that differ by two bases. Furthermore, exome sequencing analysis showed that no off-target mutations were induced by two RGENs in four clonal populations of mutant cells. In addition, paired Cas9 nickases, composed of D10A Cas9 and guide RNA, which generate two single-strand breaks (SSBs) or nicks on different DNA strands, were highly specific in human cells, avoiding off-target mutations without sacrificing genome-editing efficiency. Interestingly, paired nickases induced chromosomal deletions in a targeted manner without causing unwanted translocations. Our results highlight the importance of choosing unique target sequences and optimizing guide RNA and Cas9 to avoid or reduce RGEN-induced off-target mutations.
登录
查看更多内容
影响因子：
46.9
作者：
通讯作者：
--
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle
影响因子：
56.9
作者：
Boch, Jens;Scholze, Heidi;Bonas, Ulla
通讯作者：
Bonas, Ulla
影响因子：
46.9
作者：
Cho, Seung Woo;Kim, Sojung;Kim, Jin-Soo
通讯作者：
Kim, Jin-Soo
影响因子：
7
作者：
Kim, Hye Joo;Lee, Hyung Joo;Kim, Jin-Soo
通讯作者：
Kim, Jin-Soo