生物
基因组编辑
清脆的
Cas9
引导RNA
基因组
基因组工程
遗传学
计算生物学
突变
基因
DNA
核酸酶
突变体
作者
F. Ann Ran,Patrick Hsu,Chie Yu Lin,Jonathan S. Gootenberg,Silvana Konermann,Alexandro E. Trevino,David Scott,Atsuyuki Inoue,Shogo Matoba,Yi Zhang,Feng Zhang
出处
期刊:Cell
[Elsevier]
日期:2013-09-01
卷期号:154 (6): 1380-1389
被引量:2902
标识
DOI:10.1016/j.cell.2013.08.021
摘要
Summary
Targeted genome editing technologies have enabled a broad range of research and medical applications. The Cas9 nuclease from the microbial CRISPR-Cas system is targeted to specific genomic loci by a 20 nt guide sequence, which can tolerate certain mismatches to the DNA target and thereby promote undesired off-target mutagenesis. Here, we describe an approach that combines a Cas9 nickase mutant with paired guide RNAs to introduce targeted double-strand breaks. Because individual nicks in the genome are repaired with high fidelity, simultaneous nicking via appropriately offset guide RNAs is required for double-stranded breaks and extends the number of specifically recognized bases for target cleavage. We demonstrate that using paired nicking can reduce off-target activity by 50- to 1,500-fold in cell lines and to facilitate gene knockout in mouse zygotes without sacrificing on-target cleavage efficiency. This versatile strategy enables a wide variety of genome editing applications that require high specificity.
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