Single-Stranded DNA Cleavage by Divergent CRISPR-Cas9 Enzymes.
Single-Stranded DNA Cleavage by Divergent CRISPR-Cas9 Enzymes.
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DOI：
10.1016/j.molcel.2015.10.030
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发表时间：
2015-11-05
期刊：
影响因子：
16
通讯作者：
Doudna JA
中科院分区：
文献类型：
--
作者：
Ma E;Harrington LB;O'Connell MR;Zhou K;Doudna JA
双链DNA(DsDNA)被Cas9切割是II型CRISPR-Cas免疫系统的标志。Cas9-引导RNA复合体识别DNA中的20个碱基对的序列，并产生位点特异性的双链断裂，这是一种用于基因组编辑的强大活动。所有研究的Cas9酶对DNA的识别都需要在靶点附近有一个邻近基序(PAM)的保护基序。我们证明了来自进化分化细菌的Cas9酶可以通过RNA引导的、PAM不依赖的识别机制识别和切割单链DNA(SsDNA)。比较分析表明，与广泛用于基因组工程的II-A型化脓性链球菌Cas9相比，较小的II-C型Cas9蛋白具有有限的dsDNA结合和解离活性以及混杂的Guide-RNA特异性。这些结果表明，II型-C型Cas9酶对基因组编辑的效率低下是由于切割dsDNA的能力有限，并提示单链DNA切割是Cas9酶家族的祖先功能。
Double-stranded DNA (dsDNA) cleavage by Cas9 is a hallmark of type II CRISPR-Cas immune systems. Cas9–guide RNA complexes recognize 20-base-pair sequences in DNA and generate a site-specific double-strand break, a robust activity harnessed for genome editing. DNA recognition by all studied Cas9 enzymes requires a protospacer adjacent motif (PAM) next to the target site. We show that Cas9 enzymes from evolutionarily divergent bacteria can recognize and cleave single-stranded DNA (ssDNA) by an RNA-guided, PAM-independent recognition mechanism. Comparative analysis shows that in contrast to the type II-A S. pyogenes Cas9 that is widely used for genome engineering, the smaller type II-C Cas9 proteins have limited dsDNA binding and unwinding activity and promiscuous guide-RNA specificity. These results indicate that inefficiency of type II-C Cas9 enzymes for genome editing results from a limited ability to cleave dsDNA, and suggest that ssDNA cleavage was an ancestral function of the Cas9 enzyme family.