Cytosolic sensing of nucleic acids initiates tightly regulated programs to limit infection. Oocyte fertilization represents a scenario when inappropriate responses to exogenous yet non-pathogen-derived nucleic acids would have negative consequences. We hypothesized that germ cells express negative regulators of nucleic acid sensing (NAS) in steady state and applied an integrated data-mining and functional genomics approach to identify a rheostat of DNA and RNA sensing – the inflammasome component NLRP14. We demonstrated that NLRP14 interacted physically with the nucleic acid sensing pathway and targeted TBK1 (TANK Binding Kinase 1) for ubiquitination and degradation. We further mapped domains in NLRP14 and TBK1 that mediated the inhibitory function. Finally, we identified a human nonsense germline variant associated with male sterility that results in loss of NLRP14 function and hyper-responsiveness to nucleic acids. This discovery points to a mechanism of nucleic acid sensing regulation that may be of particular importance in fertilization.

CRISPR/Cas13 systems are increasingly used for programmable targeting of RNAs. While Cas13 nucleases are capable of degrading both target RNAs and bystander RNAs in vitro and in bacteria, initial studies fail to detect collateral degradation of non-target RNAs in eukaryotic cells. Here we show that RfxCas13d, also known as CasRx, a widely used Cas13 system, can cause collateral transcriptome destruction when targeting abundant reporter RNA and endogenous RNAs, resulting in proliferation defect in target cells. While these results call for caution of using RfxCas13d for targeted RNA knockdown, we demonstrated that the collateral activity can be harnessed for selective depletion of a specific cell population defined by a marker RNA in an in vitro setting.

In eukaryotes, sequences that code for the amino acid structure of proteins represent a small fraction of the total sequence space in the genome. These are referred to as coding sequences, whereas the remaining majority of the genome is designated as noncoding. Studies of translation, the process in which a ribosome decodes a coding sequence to synthesize proteins, have primarily focused on coding sequences, mainly due to the belief that translation outside of canonical coding sequences occurs rarely and with little impact on a cell. However, recently developed techniques such as ribosome profiling have revealed pervasive translation in a diverse set of noncoding sequences, including long noncoding RNAs (lncRNAs), introns, and both the 5' and 3' UTRs of mRNAs. Although proteins with amino acid sequences derived partially or entirely from noncoding regions may be functional, they will often be nonfunctional or toxic to the cell and therefore need to be removed. Translation outside of canonical coding regions may further expose the noncoding genome to selective pressure at the protein level, leading to the generation of novel functional proteins over evolutionary timescales. Despite the potentially significant impact of these processes on the cell, the cellular mechanisms that function to detect and triage translation in diverse noncoding regions, as well as how peptides that escape triage may evolve into novel functional proteins, remain poorly understood. This thesis will describe novel findings that offer new insight into the process of noncoding translation mitigation revealed by a combination of high-throughput systems-based approaches and validated by biochemical and genetic approaches. Chapter 1 will discuss general

While single-cell sequencing has allowed rapid identification of novel cell types or states and associated RNA markers, functional studies remain challenging due to the lack of tools that are able to target specific cells based on these markers. Here we show that targeting a single marker RNA with CRISPR/RfxCas13d led to collateral transcriptome destruction in human cells, which can be harnessed to inhibit cell proliferation or to suppress cell state transition.

Despite extensive efforts to characterize the transcriptional landscape of pancreatic ductal adenocarcinoma (PDA), reproducible assessment of subtypes with actionable dependencies remains challenging. Systematic, network-based analysis of regulatory protein activity stratified PDA tumours into novel functional subtypes that were highly conserved across multiple cohorts, including at the single cell level and in laser capture microdissected (LCM) samples. Identified subtypes were characterized by activation of master regulator proteins representing either gastrointestinal lineage markers or transcriptional effectors of morphogen pathways. Single cell analysis confirmed the existence of Lineage and Morphogenic states but also revealed a dominant population of more differentiated Oncogenic Precursor (OP) cells , present in all sampled patients, yet not apparent from bulk tumor analysis. Master regulators were validated by pooled, CRISPR/Cas9 screens, demonstrating both subtype-specific and universal dependencies. Conversely, ectopic expression of Lineage MRs, such as OVOL2, was sufficient to reprogram Morphogenic cells, thus providing a roadmap for the future targeting of patient-specific dependencies in PDA.