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.

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