UNDERSTANDING THE STRUCTURE-FUNCTION RELATION OF CRISPR-CAS ENDONUCLEASES

Abstract

Originally identified as an adaptive immune system in bacteria, CRISPR–Cas has been repurposed into a transformative platform for genome engineering. This technology, now approved as a therapy for sickle cell anemia and in clinical trials for conditions such as cystic fibrosis, holds substantial promise for treating previously incurable genetic diseases. CRISPR–Cas systems are classified into two classes (Class 1 and Class 2), which are further divided into Types I-VI based on differences in composition and mechanism of action. This dissertation focuses on Class 2 systems, specifically Type II-A (Cas9), Type V-A (Cas12a), and Type V-J (Cas12j).The CRISPR-Cas machinery functions as a programmable molecular tool in which Cas proteins act as nucleases, while CRISPR RNAs (crRNAs) guide them to target DNA through sequence complementarity. By altering the crRNA sequence, Cas proteins can be directed to virtually any matching DNA locus, enabling precise genome modification. Despite its transformative potential, CRISPR technology faces key challenges that limit its effectiveness for gene editing. Two common challenges are unintended “off-target” DNA cleavage, which occurs when the crRNA binds to sequences that are similar, but not identical, to the intended target, potentially leading to harmful genetic changes. And, the large size of commonly used Cas proteins, which complicates efficient delivery into human cells. This dissertation focuses on these challenges by investigating the structure–function relationships of Class 2 CRISPR-Cas nucleases using an integrated approach combining cryo-electron microscopy and biochemical assays. A central focus is the bridge helix (BH), a conserved structural element that connects the recognition and nuclease lobes and regulates conformational transitions during target engagement. Structural and biochemical analyses of a Francisella novicida Cas12a BH variant (FnoCas12aKD2P) show that the BH acts as an allosteric regulator of RNA-DNA hybrid propagation and catalytic activation. Cryo-EM structures reveal intermediate states along the activation pathway and suggest that perturbation of BH dynamics delays progression toward cleavage, providing a mechanistic basis for reduced off-target activity. Complementary studies of mismatch-bound FnoCas12a wild-type and FnoCas12aKD2P complexes further suggest that off-target substrates may trap the nuclease in early RNA-DNA hybrid states, delaying activation. To extend these findings to Cas9, sample preparation conditions were systematically optimized to improve stability of a BH-modified SpyCas9 variant (SpyCas92Pro) for cryo-EM analysis. Although the resulting reconstructions are limited in resolution, these efforts provide a basis for generating stabilized ternary complexes for future structural studies. Finally, biochemical characterization of a structure-guided, computationally designed Cas12j2 variant (RCas-480) was performed to evaluate whether nuclease activity can be retained following domain minimization. The results show that RCas-480 retains measurable DNA cleavage activity, supporting the computational design strategy while also highlighting constraints associated with reducing protein size. Together, this work provides a structural framework for understanding how conformational dynamics govern CRISPR–Cas function and offers guiding principles for designing nucleases with improved specificity and reduced size.