Spacer acquisition from RNA mediated by a natural reverse transcriptase-Cas1 fusion protein associated with a type III-D CRISPR-Cas system in Vibrio vulnificus
Spacer acquisition from RNA mediated by a natural reverse transcriptase-Cas1 fusion protein associated with a type III-D CRISPR-Cas system in Vibrio vulnificus
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DOI：
10.1093/nar/gkz746
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发表时间：
2019-11-04
影响因子：
14.9
通讯作者：
Toro, Nicolas
中科院分区：
文献类型：
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作者：
Gonzalez-Delgado, Alejandro;Rodriguez Mestre, Mario;Toro, Nicolas
逆转录酶（RT）与CRISPR-Cas系统的关联最近引起了人们的兴趣，因为RT活性似乎有助于从RNA分子中获得依赖于RT的间隔物。然而，我们对这种间隔器获取过程的理解仍然有限。我们鉴定了创伤弧菌YJ016 III-D型系统中RT-Cas1融合蛋白介导的间隔物在体内的获取，并证明了由RT-Cas1融合、两种不同的Cas2蛋白（a和B）和两种CRISPR阵列中的一种组成的适应模块在异源宿主中完全起作用。我们发现，RT结构域活性位点的突变显著减少了新间隔序列的获取，并表明这种RT- cas1相关的适应模块能够将RNA分子中的间隔序列整合到CRISPR阵列中。我们证明了适应模块的两个Cas2蛋白是获得间隔物所必需的。此外，我们发现一些序列特异性特征是获取和整合来自基因组任何区域的间隔物所必需的，在编码序列的5‘和3’端没有偏倚。这项研究为RT-Cas1融合蛋白介导的从RNA分子中获取间隔物提供了新的见解。
The association of reverse transcriptases (RTs) with CRISPR-Cas system has recently attracted interest because the RT activity appears to facilitate the RT-dependent acquisition of spacers from RNA molecules. However, our understanding of this spacer acquisition process remains limited. We characterized the in vivo acquisition of spacers mediated by an RT-Cas1 fusion protein linked to a type III-D system from Vibrio vulnificus strain YJ016, and showed that the adaptation module, consisting of the RT-Cas1 fusion, two different Cas2 proteins (A and B) and one of the two CRISPR arrays, was completely functional in a heterologous host. We found that mutations of the active site of the RT domain significantly decreased the acquisition of new spacers and showed that this RT-Cas1-associated adaptation module was able to incorporate spacers from RNA molecules into the CRISPR array. We demonstrated that the two Cas2 proteins of the adaptation module were required for spacer acquisition. Furthermore, we found that several sequence-specific features were required for the acquisition and integration of spacers derived from any region of the genome, with no bias along the 5' and 3' ends of coding sequences. This study provides new insight into the RT-Cas1 fusion protein-mediated acquisition of spacers from RNA molecules.