Unraveling the Conformational Landscape of CRISPR/Cas13a during Function

The type VI CRISPR effector protein Cas13a is an RNA nuclease that catalyzes two RNA cleavage events: the maturation of pre-crRNA and the cleavage of target RNA, thereby activating nonspecific collateral cleavage. Although all four structures of Cas13a on route from apo, via the pre-crRNA (Cas13a/precr), the crRNA (Cas13a/cr), and the crRNA/target RNA bound complex (Cas13a/cr/target) are known, they originate from proteins of different organisms and contain different extents of truncation. To identify and track conformational changes, structures of Cas13a in all states from a single organism are needed. We aim at following conformational changes by site-directed spin labeling in combination with Pulsed Electron-Electron Double Resonance (PELDOR or DEER) spectroscopy, which enables measuring distance distributions between spin labels in e.g., biomolecules in the range of 1.6-16 nm. We choose Leptotrichia buccalis (lbu) Cas13a, for which the structures in complex with crRNA and crRNA/targetRNA are known, whereas the structures of the apo and precrRNA-bound complexes are unknown. We labelled lbuCas13a with pairs of nitroxide spin labels at different positions and compared, in collaboration with the group of Grubmüller, the resulting distance distributions with MD simulations. We show that the apo structure of lbuCas13a has a very flexible and wide-open recognition lobe (REC-lobe). In contrast, the nuclease lobe (NUC-lobe) is preorganized. Upon pre-crRNA addition, a rigidification and conformational selection of the Helical-2 domain in the NUC-lobe occurs. The structure of the Cas13a/precr complex is very similar to the known structure. The conformations and conformational changes between Cas13a/cr and Cas13a/cr/target fit the known structures. In addition, our data suggest Cas13a dimerization in the presence of RNA.