Fig. 4: Mechanism of RNA target-activated collateral nuclease activity in Cas12a2.
From: RNA targeting unleashes indiscriminate nuclease activity of CRISPR–Cas12a2
In the binary complex (Cas12a2–crRNA), the active site is occluded. The 3′ end of the crRNA is a pre-ordered seed, and the PI domain is flexible. Binding of a complementary RNA target strand (TS) containing a PFS triggers significant conformational changes within the REC lobe, exposing the RuvC active site to facilitate TS RNA trimming in cis and nuclease activity in trans. This enables binding of dsDNA in trans, resulting in duplex kinking and local melting. The duplex is held open through the action of two pairs of aromatic clamp residues (Fig. 3), enabling nicking by RuvC.
