Development and Application of a Dual-Targeting CRISPR System Reveals Intron-Level Gene Functions
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| Veröffentlicht in: | ProQuest Dissertations and Theses (2025) |
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| Abstract: | Alternative precursor (pre-)mRNA splicing vastly expands transcriptome and proteome diversity. Yet, the specific functions of most coding and non-coding transcript isoforms remain unknown. For a long time, it has been challenging to functionally investigate transcript isoforms in a high- throughput manner. During my PhD, I contributed to the development of ‘Cas Hybrid for Multiplexed Editing and Screening Applications’ (CHyMErA), a dual-targeting CRISPR system designed for the functional interrogation of sub-genic elements. I applied this platform to screen the functions of 2,600 intronic regions in cell growth, including highly conserved intronic sequences, a subset of which overlap frame-disrupting alternative exons. This screen identified a set of ‘fitness’ introns and premature termination codon (PTC)-introducing exons. Through extensive validation, I assessed the performance of the CHyMErA platform for isoform-level screening and validated observed cell growth phenotypes. Perturbations of the ‘fitness’ sequences often impact intron retention and/or expression levels of their host genes. Follow-up investigations uncovered that deletions in nuclear speckle-associated retained introns of the FNBP4 and DDX5 genes are linked to downstream effects on cell growth-related genes and intron retention, respectively. Specifically, deletion of sequences in the DDX5 intron, which leads to increased retention of a subset of speckle-proximal introns, also results in the accumulation of R-loops at these sites. Overall, these findings highlight critical and multifaceted roles of highly conserved intronic sequences in gene regulation, R-loop resolution, and cell growth. |
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| ISBN: | 9798265447012 |
| Quelle: | ProQuest Dissertations & Theses Global |