CRISPR-Cas12a has revolutionized genome editing, offering precise control over genetic modifications while also serving as extraordinarily rapid and reliable diagnostic tools. This thesis provides comprehensive insights into Cas12a through advanced molecular dynamics simulations and experimental approaches. The second chapter involves multi-microsecond molecular dynamics simulations to reveal the allosteric switches governing conformational activation in Cas12a. It demonstrates how target DNA binding activates the complex, marked by a significant increase in the coupled dynamics between the REC2 and Nuc domains. Taking the investigation forward, the third chapter addresses the broader question of how Cas12a generates double-strand DNA breaks using its single RuvC nuclease domain through sequential cleavage of the non-target strand (NTS) followed by target strand (TS). Here, continuous tens of microsecond- long molecular dynamics and free-energy simulations uncovers the pivotal role of an α-helical lid within the RuvC domain. This lid anchors the crRNA:target strand duplex and guides the target strand toward the RuvC core, a mechanism corroborated by DNA cleavage experiments. The fourth chapter further investigates the role of the α-helical lid by examining R-loop formation using cryo-electron microscopy and advanced free-energy simulations. Structural and dynamic insights reveal that the lid assumes an unstructured loop at the 5-bp seed state, accompanied by distinct REC domain rearrangements. As the R-loop progresses to the 16-bp and 20-bp states, the lid resets into an α-helical structure, aiding in the accommodation of the non-target strand (NTS) followed by the target strand (TS). These structural insights rationalize Cas12a’s specificity and highlight mechanistic comparisons to other class 2 effectors. The fifth chapter focuses on the trans-cleavage property of Cas12a, which is the basis for nucleic acid detection. Kinetic studies show that the trans-cleavage activity rate of Cas12a is significantly enhanced due to its improved affinity (Km) for hairpin DNA structures, also providing mechanistic insights through molecular dynamics simulations. This enhanced signal transduction enables faster detection of clinically relevant double-stranded DNA targets with improved sensitivity and specificity. Finally, the sixth chapter investigates CRISPR-Cas9-based Adenine Base Editors (ABEs), which hold significant promise for addressing human genetic diseases caused by point mutations. We identify critical residues and demonstrate that the dimerization of TadA8e (the deaminase domain) and its unique juxtaposition to Cas9 are pivotal for efficient DNA deamination by ABE8e, the most efficient ABE to date. Overall, this thesis advances our understanding of CRISPR-based (Cas12a and Cas9) genome-editing tools, providing mechanistic insights into critical processes that will enrich fundamental knowledge and facilitate further engineering strategies for genome editing and diagnostic applications.

Molecular dynamics (MD) is a simulation technique that has been utilized to analyze biomolecular systems. Classical MD simulations and constant pH MD simulations have been applied to observe the behavior of components involved in two biomolecular systems of interest, the complement system and CRISPR-Cas9. The complement system is a defense mechanism part of the innate immune system. Complement associated diseases, such as paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome, are treated with complement inhibitors. Monoclonal antibody, eculizumab, is used as treatment for these diseases and functions as an inhibitor of complement component 5 (C5). A next generation version of eculizumab has also been developed known as ravulizumab, resulting from mutations within the heavy chain of eculizumab. MD simulations elucidated key residues involved in intermolecular interactions between complement inhibitors, eculizumab and ravulizumab, and C5. CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 is part of the bacterial adaptive immune system that functions as a genome editing tool. The HNH domain of CRISPR-Cas9 is involved in DNA cleavage. Through classical MD simulations, residues near the catalytic center of the HNH domain were observed. Mutations were applied to several residues within the HNH domain. The wild type structure was compared to different mutated structures to analyze the effect of the mutations. Distances between residues and RMSD were calculated. Constant pH MD simulations determined pKa values for histidine for the wild type and mutated structures. Taken together, our simulations clarified mechanisms and function of the complement and CRISPR-Cas systems, helping fundamental understanding and engineering.