Key Laboratory of Microbial Pathogenesis and Interventions-Fujian Province University, the Key Laboratory of Innate Immune Biology of Fujian Province, Biomedical Research Center of South China, Key Laboratory of OptoElectronic Science and Technology for Medicine of the Ministry of Education, College of Life Sciences, Fujian Normal University, Fuzhou, China.
Department of Biology, Southern University of Science and Technology, Shenzhen, Guangdong, China.
PLoS Genet. 2023 Sep 20;19(9):e1010930. doi: 10.1371/journal.pgen.1010930. eCollection 2023 Sep.
Cas12g is an endonuclease belonging to the type V RNA-guided CRISPR-Cas family. It is known for its ability to cleave RNA substrates using a conserved endonuclease active site located in the RuvC domain. In this study, we determined the crystal structure of apo-Cas12g, the cryo-EM structure of the Cas12g-sgRNA binary complex and investigated conformational changes that occur during the transition from the apo state to the Cas12g-sgRNA binary complex. The conserved zinc finger motifs in Cas12g undergo an ordered-to-disordered transition from the apo to the sgRNA-bound state and their mutations negatively impact on target RNA cleavage. Moreover, we identified a lid motif in the RuvC domain that undergoes transformation from a helix to loop to regulate the access to the RuvC active site and subsequent cleavage of the RNA substrate. Overall, our study provides valuable insights into the mechanisms by which Cas12g recognizes sgRNA and the conformational changes it undergoes from sgRNA binding to the activation of the RNase active site, thereby laying a foundation for the potential repurposing of Cas12g as a tool for RNA-editing.
Cas12g 是一种属于 V 型 RNA 指导的 CRISPR-Cas 家族的内切酶。它以其使用位于 RuvC 结构域中的保守内切酶活性位点切割 RNA 底物的能力而闻名。在这项研究中，我们确定了 apo-Cas12g 的晶体结构、Cas12g-sgRNA 二元复合物的 cryo-EM 结构，并研究了 apo 状态到 Cas12g-sgRNA 二元复合物转变过程中发生的构象变化。Cas12g 中的保守锌指模体经历了从 apo 状态到 sgRNA 结合状态的有序到无序的转变，其突变对靶 RNA 切割产生负面影响。此外，我们在 RuvC 结构域中鉴定出一个盖子模体，它从螺旋到环的转变调节了对 RuvC 活性位点的进入以及随后对 RNA 底物的切割。总的来说，我们的研究提供了有关 Cas12g 识别 sgRNA 的机制以及从 sgRNA 结合到 RNase 活性位点激活过程中经历的构象变化的有价值的见解，从而为 Cas12g 作为 RNA 编辑工具的潜在重新利用奠定了基础。
