Cut Site Selection by the Two Nuclease Domains of the Cas9 RNA-guided Endonuclease
Cut Site Selection by the Two Nuclease Domains of the Cas9 RNA-guided Endonuclease
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DOI：
10.1074/jbc.m113.539726
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发表时间：
2014-05-09
影响因子：
4.8
通讯作者：
Bailey, Scott
中科院分区：
文献类型：
--
作者：
Chen, Hongfan;Choi, Jihoon;Bailey, Scott
背景：Cas9 rna引导的核酸内切酶已被用于基因组操作和调控。结果：我们鉴定了嗜热链球菌LMG18311 Cas9对靶标的识别和裂解。结论：Cas9的两个核酸酶结构域通过不同的机制选择裂解位点。意义：这些发现有助于揭示cas9介导的DNA切割的分子基础。Cas9是一种来自CRISPR-Cas（聚集规律间隔短回文重复crispr相关）系统的rna引导DNA内切酶，已被用于多种模式生物的基因组编辑和基因调控。在这里，我们描述了嗜热链球菌LMG18311 （LMG18311 Cas9）的Cas9同源物。该系统的体外重构证实，LMG18311 Cas9与反式激活RNA （tracrRNA）和CRISPR RNA （crRNA）一起切割双链DNA，其特异性取决于crRNA的序列。切割不仅需要crRNA和靶标之间的互补，还需要一个称为PAM的短基序的存在。这里我们确定了LMG18311 Cas9的PAM序列要求。我们还表明，DNA目标切割的效率和切割位点的位置都取决于PAM序列的位置。
Background: The Cas9 RNA-guided endonuclease has been adapted for genome manipulation and regulation. Results: We have characterized target recognition and cleavage by Streptococcus thermophilus LMG18311 Cas9. Conclusion: The two nuclease domains of Cas9 select their cleavage sites by different mechanisms. Significance: These findings contribute to the molecular basis of Cas9-mediated DNA cleavage.Cas9, the RNA-guided DNA endonuclease from the CRISPR-Cas (clustered regularly interspaced short palindromic repeat-CRISPR-associated) system, has been adapted for genome editing and gene regulation in multiple model organisms. Here we characterize a Cas9 ortholog from Streptococcus thermophilus LMG18311 (LMG18311 Cas9). In vitro reconstitution of this system confirms that LMG18311 Cas9 together with a trans-activating RNA (tracrRNA) and a CRISPR RNA (crRNA) cleaves double-stranded DNA with a specificity dictated by the sequence of the crRNA. Cleavage requires not only complementarity between crRNA and target but also the presence of a short motif called the PAM. Here we determine the sequence requirements of the PAM for LMG18311 Cas9. We also show that both the efficiency of DNA target cleavage and the location of the cleavage sites vary based on the position of the PAM sequence.