The Structural Characterization of CRISPR-Cas13a along its Mechanistic Pathway

Ribonucleic acids are an integral part for life on earth. In addition to their function as short-term storage of genetic information and transport, they are an integral component in the CRISPR-Cas (clustered regulatory interspaced short palindromic repeats – CRISPR associated protein) immune system. In synergy with the Cas proteins, the crRNA (CRISPR RNA) recognises foreign genetic material and initiates its cleavage. The Cas protein Cas13a differs from the other members of the CRISPR-system in that it targets exclusively RNA instead of DNA. It is capable of maturing precursor crRNA (pre-crRNA) into crRNA and cleaving invasive RNA. The nucleic acid degradation is facilitated both in cis and in trans, the latter of which has been harnessed for the development of detection platforms for viral RNA. Despite the RNA–protein complex possessing medical relevance and its utilization in diagnosis and therapy, many structural and mechanistic insights into the system are still lacking. Whilst static structures have been determined along the mechanistic pathway, these originate from varying organisms and partly exhibit different extents of truncation. The aim of this work was thus to map the entire mechanistic pathway of Cas13a from the organism Leptotrichia buccalis. Primarily, EPR spectroscopy was used to characterise the dynamic behaviour of the complex. In the course of this work, PELDOR (pulsed electron double resonance) distance measurements were carried out throughout all functionally relevant structures of the protein, as well as cw EPR measurements to characterise the dynamics of the RNA. Regarding the protein, it was found that the unresolved apo conformation in the REC lobe exhibits flexibility, whilst the NUC lobe of the protein, apart from the helical-2 domain, is already predefined. The pre-crRNA-bound structure was determined to be identical to the crRNA-bound structure, both of which adopt a rigid conformation. The conformational changes in the protein required for the binding of the target RNA were also confirmed. In the absence of protein, the crRNA adopts two conformations, of which one binds quantitatively. The binding between target RNA and crRNA without protein is highly condition-dependent, while the presence of protein enables a near quantitative binding, independent of experimental conditions. This work thus made it possible to map the entire mechanism of Cas13a of one organism, including insight from both the protein and the RNA.