Spacer acquisition from RNA mediated by a natural reverse transcriptase-Cas1 fusion protein associated to a type III-D CRISPR-Cas System in Vibrio vulnificus

Association of reverse transcriptases (RTs) to CRISPR-Cas system has recently taken attention because the RT activity appears to facilitate the RT-dependent acquisition of spacers from RNA molecules. In spite of the great phylogenetically diversity of RTs associated to CRISPR-Cas systems, current knowledge on this type of spacer acquisition process offers limited insight. Here, we characterize the in vivo acquisition of spacers mediated by a RT-Cas1 fusion protein belonging to clade 6 linked to a type III-D system from Vibrio vulnificus strain YJ016 and show that the adaptive module consisting of the RT-Cas1 fusion, two distinct Cas2 proteins (A and B) and one of the two CRISPR arrays are completely functional in a heterologous host (E. coli). We found that mutation in the active site of the RT domain significantly decreased the acquisition of new spacers and show that this RT-Cas1 associated adaptation module is able to incorporate into the CRISPR array spacers from RNA molecules. We demonstrate that the two different Cas2 protein of the adaptation module, present in all members of this clade are required for spacer acquisition. Furthermore, we found that there are several sequence specific requirements for the correct acquisition and integration of spacers that can be acquired from any region of the genome with no bias along the 5´and 3´end of coding sequences, which could make this RT-Cas1 system a good alternative for biotechnological applications. Our study provides novel insights into the acquisition of spacer from RNA molecules mediated by RT-Cas1 fusion proteins.

逆转录酶（reverse transcriptases, RTs）与CRISPR-Cas系统的关联近来备受关注，因其逆转录酶活性可促进依赖RT的RNA来源间隔序列（spacers）的获取过程。尽管与CRISPR-Cas系统相关的RTs具有极高的系统发育多样性，但目前学界对这类间隔序列获取过程的认知仍较为有限。本研究对来自创伤弧菌（Vibrio vulnificus）YJ016菌株III-D型CRISPR-Cas系统第6进化枝的RT-Cas1融合蛋白介导的体内间隔序列获取过程进行了表征，并证实由该RT-Cas1融合蛋白、两种不同的Cas2蛋白（A与B）以及其中一个CRISPR阵列组成的适应性模块，可在异源宿主大肠杆菌（E. coli）中完全发挥功能。研究发现，RT结构域活性位点的突变会显著降低新间隔序列的获取效率；同时证实，该RT-Cas1关联的适应性模块能够将RNA分子来源的间隔序列整合至CRISPR阵列中。我们证明了该适应性模块中存在于此进化枝所有成员中的两种不同Cas2蛋白，是间隔序列获取所必需的。此外，我们发现间隔序列的正确获取与整合存在若干序列特异性要求，且可从基因组的任意区域获取间隔序列，在编码序列的5'与3'端无偏好性，这使得该RT-Cas1系统有望成为生物技术应用的优质备选方案。本研究为RT-Cas1融合蛋白介导的RNA来源间隔序列获取过程提供了全新的认知视角。