不同的CRISPR-Cas9酶对单链DNA的切割
Single-Stranded DNA Cleavage by Divergent CRISPR-Cas9 Enzymes.
作者信息
Ma Enbo, Harrington Lucas B, O'Connell Mitchell R, Zhou Kaihong, Doudna Jennifer A
机构信息
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Center for RNA Systems Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
出版信息
Mol Cell. 2015 Nov 5;60(3):398-407. doi: 10.1016/j.molcel.2015.10.030.
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.
Cas9对双链DNA（dsDNA）的切割是II型CRISPR-Cas免疫系统的一个标志。Cas9-导向RNA复合物识别DNA中的20个碱基对序列，并产生一个位点特异性双链断裂，这是一种用于基因组编辑的强大活性。所有已研究的Cas9酶对DNA的识别都需要在靶位点旁边有一个原间隔相邻基序（PAM）。我们发现，来自进化上不同细菌的Cas9酶可以通过一种RNA导向、不依赖PAM的识别机制识别并切割单链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不同，较小的II-C型Cas9蛋白具有有限的dsDNA结合和解旋活性以及混杂的导向RNA特异性。这些结果表明，II-C型Cas9酶在基因组编辑中的低效是由于其切割dsDNA的能力有限，并表明ssDNA切割是Cas9酶家族的一种原始功能。
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