a State Key Laboratory of Agricultural Microbiology and College of Life Science and Technology, Huazhong Agricultural University , Wuhan , China.
b Archaea Centre, Department of Biology, University of Copenhagen , Copenhagen , Denmark.
RNA Biol. 2019 Sep;16(9):1166-1178. doi: 10.1080/15476286.2019.1618693. Epub 2019 May 26.
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 generates two constant lengths of crRNA independent of the length of the spacer. Either mutation at the 3'-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 , 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.
CRISPR-Cas 系统在古菌和细菌中由小 RNA（crRNA）指导，提供对外来核酸的适应性防御。已报道的 III 型 CRISPR 系统具有核糖核酸酶、RNA 激活的 DNA 酶和环寡腺苷酸（cOA）合成酶活性，与其他 CRISPR 系统有显著不同。然而，在 III 型 CRISPR 系统中，对于增强靶特异性至关重要的靶标识别的详细特征仍然未知。在这里，我们展示了 中的 III-B 型 Cmr-α 系统独立于间隔子的长度产生两种恒定长度的 crRNA。crRNA 的 3'端突变或靶标截断都会极大地影响 Cmr-α 效应复合物的靶标捕获和切割。此外，我们发现 Cmr-α RNP 复合物在靶 RNA 上标签近端位点的切割相对于其他位点延迟，这可能为 Cas10 提供更多的时间作为抵御入侵者的保护。通过突变分析，我们发现位于 crRNA 标签远端区域的种子基序对于 Cmr1α 捕获靶 RNA 是必需的，从而提高了靶标特异性和效率。这些发现进一步完善了 III-B 型 CRISPR-Cas 系统免疫防御的模型，从捕获、切割和调控开始。
