Liu Tao, Li Yingjun, Wang Xiaodi, Ye Qing, Li Huan, Liang Yunxiang, She Qunxin, Peng Nan
State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, P. R. China.
State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, P. R. China Hubei Collaborative Innovation Center for Industrial Fermentation, Wuhan 430070, P. R. China.
Nucleic Acids Res. 2015 Jan;43(2):1044-55. doi: 10.1093/nar/gku1383. Epub 2015 Jan 7.
Acquisition of de novo spacer sequences confers CRISPR-Cas with a memory to defend against invading genetic elements. However, the mechanism of regulation of CRISPR spacer acquisition remains unknown. Here we examine the transcriptional regulation of the conserved spacer acquisition genes in Type I-A of Sulfolobus islandicus REY15A. Csa3a, a MarR-like transcription factor encoded by the gene located adjacent to csa1, cas1, cas2 and cas4 cluster, but on the reverse strand, was demonstrated to specifically bind to the csa1 and cas1 promoters with the imperfect palindromic sequence. Importantly, it was demonstrated that the transcription level of csa1, cas1, cas2 and cas4 was significantly enhanced in a csa3a-overexpression strain and, moreover, the Csa1 and Cas1 protein levels were increased in this strain. Furthermore, we demonstrated the hyperactive uptake of unique spacers within both CRISPR loci in the presence of the csa3a overexpression vector. The spacer acquisition process is dependent on the CCN PAM sequence and protospacer selection is random and non-directional. These results suggested a regulation mechanism of CRISPR spacer acquisition where a single transcriptional regulator senses the presence of an invading element and then activates spacer acquisition gene expression which leads to de novo spacer uptake from the invading element.
获得新生间隔序列赋予CRISPR-Cas系统一种记忆，以抵御入侵的遗传元件。然而，CRISPR间隔序列获得的调控机制仍然未知。在此，我们研究了冰岛硫化叶菌REY15A的I-A型中保守间隔序列获得基因的转录调控。Csa3a是一种由位于csa1、cas1、cas2和cas4基因簇相邻但反向链上的基因编码的MarR样转录因子，已证明它能特异性结合具有不完全回文序列的csa1和cas1启动子。重要的是，已证明在csa3a过表达菌株中，csa1、cas1、cas2和cas4的转录水平显著提高，而且该菌株中Csa1和Cas1蛋白水平也增加。此外，我们证明在存在csa3a过表达载体的情况下，两个CRISPR位点内独特间隔序列的摄取会过度活跃。间隔序列获得过程依赖于CCN PAM序列，原间隔序列的选择是随机且无方向性的。这些结果提示了一种CRISPR间隔序列获得的调控机制，即单个转录调节因子感知入侵元件的存在，然后激活间隔序列获得基因的表达，从而导致从入侵元件中新生摄取间隔序列。
