UC San Diego

All organisms have mechanisms for degrading macromolecules that are critical for homeostasis and immunity. The ubiquitin proteasome system (UPS) and CRISPR-Cas System both implement targeting capabilities to degrade specific nucleic acids or proteins in response to their environment. In the UPS, an enzymatic cascade of E1 activating enzymes, E2 conjugating enzymes, and E3 ligases facilitate the post-translational modification of target proteins with ubiquitin (Ub), leading to the eventual formation of polyubiquitin chains which signal for proteasomal degradation and the immune response. CRISPR-Cas Systems utilize effector proteins that first bind CRISPR RNA (crRNA) that recognize specific stretches of nucleic acids and ultimately cleave these sequences to defend against invading viral species.In Chapter II, histones H3 and H4 are identified as novel substrates for the ASB9-CUL-RING E3 ubiquitin ligase. In vitro assays confirmed specific ubiquitylation towards histones H3 and H4 in a DNA-free form and that ASB9-CRL did not require supplementary E3 ligases to ubiquitylate histones, unlike other substrates. Additionally, specific modified lysines were detected by mass spectrometry. Chapter III investigates the structure and dynamics of the type III-E CRISPR-Cas effector, DiCas7-11 in different its apo, binary crRNA-bound state, and ternary crRNA-target bound state. Using HDX-MS, the structure and dynamics of the apo state is revealed for the first time elucidating a highly dynamic and solvent exposed structure that is heavily remodeled for target cleavage upon crRNA binding. The study also posits new hypotheses for the crRNA processing mechanism and the function of the insertion domain. Chapter IV leverages the specific and non-specific cleavage activities of the type VI CRISPR-Cas effector, RfxCas13d, to develop an CRISPR-based diagnostic to detect SARS-CoV-2. Development of a high-throughput fluorescence assay and lateral-flow strip assay are described with efficacy confirmed on SARS-CoV-2 positive patient samples.