引导RNA
Cas9
基因组编辑
生物
清脆的
核酸酶
核糖核酸
亚基因组mRNA
遗传学
基因组
DNA
基因组工程
计算生物学
基因
作者
Patrick Hsu,David Scott,Joshua A. Weinstein,F. Ann Ran,Silvana Konermann,Vineeta Agarwala,Yinqing Li,Eli J. Fine,Xuebing Wu,Ophir Shalem,Thomas J. Cradick,Luciano A. Marraffini,Gang Bao,Feng Zhang
摘要
Analyses of the determinants of the specificity of Cas9 nuclease provide rules for selecting optimal target sites. The Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing1,2,3,4,5,6,7,8,9,10. Here, we characterize SpCas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target effects. Our study evaluates >700 guide RNA variants and SpCas9-induced indel mutation levels at >100 predicted genomic off-target loci in 293T and 293FT cells. We find that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. We also show that SpCas9-mediated cleavage is unaffected by DNA methylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modification. To facilitate mammalian genome engineering applications, we provide a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses.
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