A seed motif for target RNA capture enables efficient immune defence by a type III-B CRISPR-Cas system
A seed motif for target RNA capture enables efficient immune defence by a type III-B CRISPR-Cas system
复制标题
用于捕获目标 RNA 的种子基序可通过 III-B 型 CRISPR-Cas 系统实现有效的免疫防御
DOI：
10.1080/15476286.2019.1618693
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发表时间：
2019-09-02
期刊：
影响因子：
4.1
通讯作者：
Li, Yingjun
中科院分区：
文献类型：
--
作者：
Pan, Saifu;Li, Qi;Li, Yingjun
摘要CRISPR-Cas系统在古细菌和细菌中提供了由小RNA（crRNA）引导的针对外源核酸的适应性防御。据报道，III型CRISPR系统携带RNA酶、RNA活化的DNA酶和环寡腺苷酸（cOA）合成酶活性，并且与其他CRISPR系统显著不同。然而，对于增强靶特异性至关重要的靶识别的详细特征在III型CRISPR系统中仍然未知。本文证明了S. islandicus产生两个恒定长度的crRNA，与间隔区的长度无关。crRNA的3 ′端突变或靶标截短极大地影响Cmr-α效应复合物对靶标的捕获和切割。此外，我们发现Cmr-α RNP复合物在靶RNA上的标签近端位点的切割相对于其他位点延迟，这可能为Cas 10提供了更多的时间来作为抵御入侵者的屏障。使用体内诱变试验，我们发现位于crRNA标签远端区域的种子基序是Cmr-α系统捕获靶RNA所需的，从而增强靶特异性和效率。这些发现进一步完善了III-B型CRISPR-Cas系统的免疫防御模型，从捕获，切割和调节开始。
ABSTRACT CRISPR-Cas systems provide an adaptive defence against foreign nucleic acids guided by small RNAs (crRNAs) in archaea and bacteria. The Type III CRISPR systems are reported to carry RNase, RNA-activated DNase and cyclic oligoadenylate (cOA) synthetase activity, and are significantly different from other CRISPR systems. However, detailed features of target recognition, which are essential for enhancing target specificity remain unknown in Type III CRISPR systems. Here, we show that the Type III-B Cmr-α system in S. islandicus generates two constant lengths of crRNA independent of the length of the spacer. Either mutation at the 3ʹ-end of crRNA or target truncation greatly influences the target capture and cleavage by the Cmr-α effector complex. Furthermore, we found that cleavage at the tag-proximal site on the target RNA by the Cmr-α RNP complex is delayed relative to the other sites, which probably provides Cas10 more time to function as a guard against invaders. Using a mutagenesis assay in vivo, we discovered that a seed motif located at the tag-distal region of the crRNA is required by Cmr1α for target RNA capture by the Cmr-α system thereby enhancing target specificity and efficiency. These findings further refine the model for immune defence of Type III-B CRISPR-Cas system, commencing on capture, cleavage and regulation.