Primed adaptation tolerates extensive structural and size variations of the CRISPR RNA guide in Haloarcula hispanica
Primed adaptation tolerates extensive structural and size variations of the CRISPR RNA guide in Haloarcula hispanica
复制标题
启动适应可耐受西班牙 Haloarcula 中 CRISPR RNA 向导的广泛结构和大小变化
DOI：
10.1093/nar/gkz244
复制
发表时间：
2019-06-20
影响因子：
14.9
通讯作者：
Xiang, Hua
中科院分区：
文献类型：
--
作者：
Gong, Luyao;Li, Ming;Xiang, Hua
摘要 最近关于 CRISPR 适应的研究表明，启动是间隔区获取的主要途径，至少对于最流行的 I 型系统而言是这样。启动是由完全/部分匹配入侵者 DNA 的 CRISPR RNA 引导的，但这种 RNA 引导的可塑性尚未得到表征。在这项研究中，我们广泛修改了 Haloarcula hispanica 中引发 crRNA 的两个保守手柄，并改变了其中央间隔部分的大小。有趣的是，引发不受 3' 柄完全删除的影响，这严重损害了 crRNA 的稳定性和干扰效应。删除 3' 手柄后，进一步截断 5' 手柄表明其间隔区近端 6 个核苷酸可以提供引发所需的最不保守的序列。随后的扫描突变进一步鉴定了 5' 手柄内的关键核苷酸。此外，与干扰相比，启动也被证明可以容忍间隔件部分更宽的尺寸变化。这些数据共同说明了启动对 crRNA 引导的广泛结构/大小变化的高度耐受性，这突出了 crRNA-效应核糖核蛋白复合物的结构灵活性。观察到的高引发有效性表明，引发适应通过快速且持续地多重干扰途径，促进快速复制和不断进化的病毒DNA的清除。
Abstract Recent studies on CRISPR adaptation revealed that priming is a major pathway of spacer acquisition, at least for the most prevalent type I systems. Priming is guided by a CRISPR RNA which fully/partially matches the invader DNA, but the plasticity of this RNA guide has not yet been characterized. In this study, we extensively modified the two conserved handles of a priming crRNA in Haloarcula hispanica, and altered the size of its central spacer part. Interestingly, priming is insusceptible to the full deletion of 3′ handle, which seriously impaired crRNA stability and interference effects. With 3′ handle deletion, further truncation of 5′ handle revealed that its spacer-proximal 6 nucleotides could provide the least conserved sequence required for priming. Subsequent scanning mutation further identified critical nucleotides within 5′ handle. Besides, priming was also shown to tolerate a wider size variation of the spacer part, compared to interference. These data collectively illustrate the high tolerance of priming to extensive structural/size variations of the crRNA guide, which highlights the structural flexibility of the crRNA-effector ribonucleoprotein complex. The observed high priming effectiveness suggests that primed adaptation promotes clearance of the fast-replicating and ever-evolving viral DNA, by rapidly and persistently multiplexing the interference pathway.