Investigating the Conformational Landscape of Cas13a
Creators: name: Vicino, Maria Francesca
Publisher: name: Universitäts- und Landesbibliothek Bonn
Year: 2025
Subjects: subject: CRISPR-Cas
subject: Cas13a
subject: EPR spectroscopy
subject: PELDOR
subject: DEER
subject: spin-labelling
subject: conformational changes
subject: ddc:540

description: Bacteria and bacteriophages are in a constant arms race to develop strategies to coexist. One adaptive defense system, which bacteria and archaea have developed against phages is the CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats – CRISPR Associated proteins) system. Out of a variety of known CRISPR-Cas types, the type VI CRISPR effector protein Cas13a (Cas-associated 13a), composed of a recognition (REC) lobe and a nuclease (NUC) lobe, has unique properties. In contrast to the most prominent Cas protein representative Cas9, Cas13a binds and cleaves RNA and not DNA. In addition, it catalyzes the maturation of a precursor-CRISPR RNA (pre-crRNA) and the cleavage of the target RNA, which activates a unique sequence non-specific collateral RNA cleavage. This collateral cleavage was harnessed as nucleic acid detection tool, to detect viral RNA, causing human diseases. Further Cas13a has therapeutic applications for example in inhibiting cancer cell growth. <br> Despite the high medical relevance and application, the underlying coordinating mechanism and its structure-dynamics-function relationship was not investigated. Structures from all functionally relevant complexes are described (<em>apo</em>, pre-crRNA bound, crRNA bound, and cr- and target RNA bound), but they origin from proteins of different organisms and contain different extends of truncation. Additionally, it is unknown how the structures relate to functional aspects of this system. Thus, to identify and follow conformational changes on the molecular level, information of all states from one organism are needed. <br> In this work, mainly Pulsed Electron-Electron Double Resonance (PELDOR) spectroscopy was used to analyze conformational changes during the functional pathway of Cas13a. In the first part of this thesis, basic biochemical techniques were used to optimize the expression and purification approach for Cas13a from <em>Leptotrichia buccalis</em> (<em>Lbu</em>) and to optimize RNA cleavage assays in our laboratory. The second part of this thesis focuses on the development of active double labelled protein constructs, for PELDOR studies. A mutational analysis was used, to find replacements for three native cysteines, generating a protein construct that retained cleavage activity after spin labelling. The third part of the thesis focuses on validating the structures of <em>Lbu</em>Cas13a and following its conformational changes through the entire functional pathway. <br> The PELDOR measurements showed a high flexibility in the <em>apo</em> state of the REC lobe. This flexibility was not described by any known structure or prediction. In contrast, the <em>apo</em> state of the NUC lobe is rigid, with the helical-2 domain being the exception. The REC and NUC lobes are flexible towards each other. The pre-crRNA bound structure is not known. We reveal this structure to match the crRNA bound state. The expected conformational changes from the experimentally known binary crRNA-bound complex to the experimentally known ternary complex were found to occur in frozen solution. Interestingly, by using a combination of PELDOR spectroscopy and AFM measurements, a previously unknown dimeric structure of the protein complex with cr- and target RNA was found. This structure is different than the structure observed in the asymmetric unit of the cr- and target RNA bound <em>Lbu</em>Cas13a. <br> By summing up, for the first time, the entire functional cycle of the Cas13a protein from one organism is structurally and dynamically investigated and new details are added.