Interaction between Cas12g and sgRNA.

(A) Ribbon diagram of sgRNA (left panel) and its interaction with the Helical 1, Helical 2 and RuvC domains (right panel). Close-up view of the opening on Cas12g guiding the guide crRNA into the central channel. (B) Recognition of the stem1 of sgRNA by the Helical 1 domain of Cas12g. (C) Recognition of the R:AR duplex 2 of sgRNA by the Helical 2 and RuvC domain of Cas12g. (D) RNA cleavage assay using wild-type Cas12g and Cas12g mutants in combination with sgRNA.</p

RNA transcription template used in this study.

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.</div

SDS-PAGE analysis of the wild-type and mutants of Cas12g protein.

SDS-PAGE analysis of the wild-type and mutants of Cas12g protein.</p

Schematic of sgRNA recognition by Cas12g.

Domains and residues are colored according to Fig 1(A). (TIF)</p

Circular dichroism (CD) spectra of wild-type and mutants of zinc finger motifs in Cas12g.

Circular dichroism (CD) spectra of wild-type and mutants of zinc finger motifs in Cas12g.</p

Structures of individual domains of Cas12g.

(A) Overall structure of apo-Cas12g. (B) Overall structure of the Cas12g binary complex. (C) The structure of Helical 1 domain (right). The SAD map around Helical 1 subdomain II discussed in article shown in left. (D-G) Structures of Helical 2, Nuc, WED and RuvC domains of Cas12g. Domains are colored according to Fig 1(A). (TIF)</p

X-ray crystallography data collection and refinement statistics.

X-ray crystallography data collection and refinement statistics.</p

Detailed cryo-EM density map of the Cas12g-sgRNA complex with final atomic model fitted in.

(A) Cryo-EM map and model of the Cas12g-sgRNA complex with each domain of Cas12g color coded as in Fig 1(A). (B) Fitting of nucleic acids to the corresponding cryo-EM map. The atomic models are shown in stick with crRNA and tracrRNA. The crRNA strand and tracrRNA colored in orange and sky blue, respectively. (C) Fitting of the Helical 1 domain. Despite the unresolvable structure Helical 1 subdomain II (aa 232–355) region, its position in the Cas12g-sgRNA complex can be determined based on the density. (D-G) Fitting of the Helical 2 (D), WED (E), RuvC (F) and, Nuc domain (G). (TIF)</p

The lid motif of Cas12g.

(A) The alignment of the amino acid sequences of Cas12g from different bacterial strains. The amino acids related to the zinc finger motifs are marked with black boxes. (B) Circular dichroism (CD) spectra of wild-type and mutants of zinc finger motifs in Cas12g. (C) Collateral cleavage of unrelated ssDNA (left) and ssRNA (right) by Cas12g. The results shown are representative of three experiments. (TIF)</p

Structure of the Cas12g-sgRNA binary complex.

(A) Cryo-EM map of the Cas12g binary complex shown in one view and rotated through 180°, with each domain of Cas12g color-coded as in Fig 1A. (B) Atomic model of the Cas12g binary complex displayed in two views. (C) Surface representations of the Cas12g binary complex shown in the same views as in (B). (D) Structure of sgRNA in the Cas12g binary complex. (E) Schematic representation of sgRNA, with the disordered region enclosed in the gray box. The ellipsis indicates the invisible region of crRNA.</p