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.

中文翻译：

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指导 RNA 结合的结构转变及其在 Cas12g 介导的 RNA 切割中的重要性。

Cas12g 是一种核酸内切酶，属于 V 型 RNA 引导的 CRISPR-Cas 家族。它以其使用位于 RuvC 结构域的保守核酸内切酶活性位点切割 RNA 底物的能力而闻名。在本研究中，我们确定了apo-Cas12g的晶体结构、Cas12g-sgRNA二元复合物的冷冻电镜结构，并研究了从apo状态向Cas12g-sgRNA二元复合物转变过程中发生的构象变化。Cas12g 中的保守锌指基序经历了从 apo 到 sgRNA 结合状态的有序到无序的转变，它们的突变对靶 RNA 切割产生负面影响。此外，我们在 RuvC 结构域中发现了一个盖子基序，它经历了从螺旋到环的转变，以调节对 RuvC 活性位点的访问以及随后 RNA 底物的切割。总的来说，我们的研究为 Cas12g 识别 sgRNA 的机制及其从 sgRNA 结合到 RNase 活性位点激活所经历的构象变化提供了有价值的见解，从而为 Cas12g 作为 RNA 编辑工具的潜在重新利用奠定了基础。