Structural transitions upon guide RNA binding and their importance in Cas12g-mediated RNA cleavage

Liu, Mengxi1; Li, Zekai1; Chen, Jing1; Lin, Jinying1; Lu, Qiuhua1; Ye, Yangmiao1; Zhang, Hongmin2; Zhang, Bo1; Ouyang, Songying1

2023-09-01

10.1371/journal.pgen.1010930

PLOS GENETICS   Impact Factor & Quartile

1553-7404

["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 is an RNA-guided ribonuclease (RNase) that displays both collateral RNase and single-stranded DNase activities distinguishing it from other Cas12 proteins discovered so far that targeting DNA. RNA-targeting CRISPR-Cas effectors have been developed into various tools for manipulating specific RNA molecules. Currently, RNA-targeting applications primarily rely on the RNA-specific type VI systems, particularly Cas13a-13d, which possess HEPN domains that are exclusively involved in RNA cleavage. Although both Cas12g and Cas13 effector proteins possess the ability to cleave RNA, they exhibit significantly different characteristics in terms of structure and molecular mechanisms. To harness Cas12g and apply it as an effective RNA editing tool, it is important to understand the molecular basis for sgRNA recognition as well as target RNA recognition and cleavage by Cas12g. Our findings not only improve understanding of Cas12g at molecular level but also provide useful information for rational engineering of the CRISPR-Cas12g system and its application in RNA editing and other fields."]

SCI

English

Others

National Key Research and Development Program of China[2021YFC2301403] ; National Nature Science Foundation of China grants["82225028","82172287"] ; Special Funds of Fuzhou Science and Technology Bureau[2022-ZD-019] ; High-level personnel introduction grant of Fujian Normal University[Z0210509]

Genetics & Heredity

Genetics & Heredity

WOS:001071174100002

PUBLIC LIBRARY SCIENCE

Web of Science

1.Fujian Normal Univ, Key Lab Innate Immune Biol Fujian Prov, Key Lab OptoElect Sci & Technol Med,Minist Educ, Biomed Res Ctr South China,Coll Life Sci,Key Lab M, Fuzhou 350117, Peoples R China
2.Southern Univ Sci & Technol, Dept Biol, Shenzhen, Guangdong, Peoples R China

Liu, Mengxi,Li, Zekai,Chen, Jing,et al. Structural transitions upon guide RNA binding and their importance in Cas12g-mediated RNA cleavage[J]. PLOS GENETICS,2023,19(9).

Liu, Mengxi.,Li, Zekai.,Chen, Jing.,Lin, Jinying.,Lu, Qiuhua.,...&Ouyang, Songying.(2023).Structural transitions upon guide RNA binding and their importance in Cas12g-mediated RNA cleavage.PLOS GENETICS,19(9).

Liu, Mengxi,et al."Structural transitions upon guide RNA binding and their importance in Cas12g-mediated RNA cleavage".PLOS GENETICS 19.9(2023).