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

Abstract

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.

Fig 1. Crystal structure of apo-Cas12g.

(A) Domain organization of Cas12g. The amino acid segment marked by the solid gray line represents the unresolved region. (B) Overall structure of apo Cas12g shown in one view (left) and rotated through 180° (right). (C) Surface representations of apo Cas12g in the same views as in (B).

Fig 2. 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.

Fig 3. 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.

Fig 4. Zinc finger motifs of Cas12g.

(A) A zinc ion chelated by four residues (H248, C252, C323 and C330) in the Helical 1 subdomain II. (B) A zinc ion chelated by four residues (C228, C240, H335 and C339) in the Helical 1 subdomain II. (C) A zinc finger motif with four cysteines (C711, C714, C735 and C738) in the Nuc domain. (D) Fitting of the unsharpened cryo-EM map with the structure of Cas12g binary complex. Helical 1 domain is colored cyan, and the remaining domains are colored gray. The cryo-EM map color zone with the structure of Cas12g and the density of Helical 1 domain represented by the cyan mesh. (E) Superposition of the Helical 1 domain between apo-Cas12g (gray) and Cas12g binary complex (cyan). The EM density of the Helical 1 is shown as cyan mesh. (F) RNA substrate cleavage assay using wild-type Cas12g and Cas12g with mutations, as indicated.

Fig 5. Domain rearrangement of Cas12g.

(A) Superimposition of apo-Cas12g (gray) and the Cas12g binary complex (pale yellow). (B) Superimposition of the Cas12g binary (pale yellow) and ternary (PDB code: 6XMG) (orange) complexes. (C-E) Formation of the central channel induced by conformational changes. The aa 655–680 of the RuvC domain constitute the lid motif and are colored in blue. The central channel widens as Cas12g transitions from apo-Cas12g (C) to the Cas12g binary complex (D) upon sgRNA binding. The central channel further expands with the binding of target RNA (E).

Fig 6. Conformational rearrangement of the lid motif.

(A-C) The conformations of the lid motif (colored in blue) in apo-Cas12g, the Cas12g binary and ternary (PDB code: 6XMG) complexes. Active residues are colored red. (D) Interactions associated with the lid motif in the apo-Cas12g. (E) Interactions associated with the lid motif in the Cas12g-sgRNA complex. (F) The Nuc domain is attached to the active site, with multiple positively charged residues pointing to the active site. (G-H) The substrate RNA cleavage assay using wild-type Cas12g and Cas12g with mutations, as indicated. Alanine substitution of the Asp and Glu residues in the RuvC domain (G) and the alanine substitution of the key residues in the lid motif and Nuc domain (H).