脑膜炎奈瑟菌Cas9的脱氧核糖核酸酶H活性
DNase H Activity of Neisseria meningitidis Cas9.
作者信息
Zhang Yan, Rajan Rakhi, Seifert H Steven, Mondragón Alfonso, Sontheimer Erik J
机构信息
RNA Therapeutics Institute, Program in Molecular Medicine, University of Massachusetts Medical School, 368 Plantation Street, Worcester, MA 01605-2324, USA.
Department of Molecular Biosciences, Northwestern University, 2205 Tech Drive, Evanston, IL 60208-3500, USA.
出版信息
Mol Cell. 2015 Oct 15;60(2):242-55. doi: 10.1016/j.molcel.2015.09.020.
Type II CRISPR systems defend against invasive DNA by using Cas9 as an RNA-guided nuclease that creates double-stranded DNA breaks. Dual RNAs (CRISPR RNA [crRNA] and tracrRNA) are required for Cas9's targeting activities observed to date. Targeting requires a protospacer adjacent motif (PAM) and crRNA-DNA complementarity. Cas9 orthologs (including Neisseria meningitidis Cas9 [NmeCas9]) have also been adopted for genome engineering. Here we examine the DNA cleavage activities and substrate requirements of NmeCas9, including a set of unusually complex PAM recognition patterns. Unexpectedly, NmeCas9 cleaves single-stranded DNAs in a manner that is RNA guided but PAM and tracrRNA independent. Beyond the need for guide-target pairing, this "DNase H" activity has no apparent sequence requirements, and the cleavage sites are measured from the 5' end of the DNA substrate's RNA-paired region. These results indicate that tracrRNA is not strictly required for NmeCas9 enzymatic activation, and expand the list of targeting activities of Cas9 endonucleases.
II型CRISPR系统通过使用Cas9作为RNA引导的核酸酶来抵御入侵的DNA，该核酸酶可产生双链DNA断裂。迄今为止观察到的Cas9靶向活性需要双RNA（CRISPR RNA [crRNA]和反式激活crRNA [tracrRNA]）。靶向需要一个原间隔序列临近基序（PAM）和crRNA与DNA的互补性。Cas9直系同源物（包括脑膜炎奈瑟菌Cas9 [NmeCas9]）也已被用于基因组工程。在这里，我们研究了NmeCas9的DNA切割活性和底物要求，包括一组异常复杂的PAM识别模式。出乎意料的是，NmeCas9以一种由RNA引导但不依赖PAM和tracrRNA的方式切割单链DNA。除了需要引导序列与靶序列配对外，这种“核酸酶H”活性没有明显的序列要求，切割位点是从DNA底物RNA配对区域的5'端开始测量的。这些结果表明，tracrRNA对于NmeCas9的酶激活不是严格必需的，并扩展了Cas9内切核酸酶的靶向活性列表。
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