肠弯曲菌 Cas9 通过 CRISPR RNA 依赖性结合和切割内源性 RNA。
CRISPR RNA-Dependent Binding and Cleavage of Endogenous RNAs by the Campylobacter jejuni Cas9.
机构信息
Institute of Molecular Infection Biology (IMIB)/Research Center for Infectious Diseases (ZINF), University of Würzburg, Josef-Schneider-Str. 2 / D15, D-97080 Würzburg, Germany.
Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695, USA.
出版信息
Mol Cell. 2018 Mar 1;69(5):893-905.e7. doi: 10.1016/j.molcel.2018.01.032.
Cas9 nucleases naturally utilize CRISPR RNAs (crRNAs) to silence foreign double-stranded DNA. While recent work has shown that some Cas9 nucleases can also target RNA, RNA recognition has required nuclease modifications or accessory factors. Here, we show that the Campylobacter jejuni Cas9 (CjCas9) can bind and cleave complementary endogenous mRNAs in a crRNA-dependent manner. Approximately 100 transcripts co-immunoprecipitated with CjCas9 and generally can be subdivided through their base-pairing potential to the four crRNAs. A subset of these RNAs was cleaved around or within the predicted binding site. Mutational analyses revealed that RNA binding was crRNA and tracrRNA dependent and that target RNA cleavage required the CjCas9 HNH domain. We further observed that RNA cleavage was PAM independent, improved with greater complementarity between the crRNA and the RNA target, and was programmable in vitro. These findings suggest that C. jejuni Cas9 is a promiscuous nuclease that can coordinately target both DNA and RNA.
Cas9 核酸酶天然利用 CRISPR RNA（crRNA）来沉默外源双链 DNA。虽然最近的研究表明，一些 Cas9 核酸酶也可以靶向 RNA，但 RNA 的识别需要核酸酶的修饰或辅助因子。在这里，我们证明了空肠弯曲菌 Cas9（CjCas9）可以依赖于 crRNA 结合并切割互补的内源性 mRNA。大约 100 个转录本与 CjCas9 共免疫沉淀，并且通常可以根据其与四个 crRNA 的碱基配对潜力进行细分。这些 RNA 中的一部分在预测的结合位点周围或内部被切割。突变分析表明，RNA 结合依赖于 crRNA 和 tracrRNA，并且靶 RNA 的切割需要 CjCas9 的 HNH 结构域。我们进一步观察到，RNA 切割不依赖于 PAM，在 crRNA 和 RNA 靶之间具有更大的互补性时会得到改善，并且在体外具有可编程性。这些发现表明，空肠弯曲菌 Cas9 是一种混杂的核酸酶，可以协调靶向 DNA 和 RNA。
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