Myhrvold, Cameron A.Guo, Andrew2025-08-082025-08-082025-04-18https://theses-dissertations.princeton.edu/handle/88435/dsp01qv33s111sCRISPR-Cas13 is an endonuclease system capable of precise recognition and cleavage of RNA. Since its discovery in 2015, it has been harnessed for a wide range of applications, including RNA editing, tracking, detection, imaging, and targeted transcript regulation. This thesis explores the use of CRISPR-Cas13 for two primary applications: (1) the development of diagnostic tools for detecting zoonotic influenza A viruses, and (2) the creation of Cas-seq, a novel high-throughput platform for scRNA-seq. For the first application, we adapted mCARMEN – a highly multiplexed microfluidic platform that harnesses CRISPR-Cas13’s trans-cleavage activity for simultaneous RNA virus detection across multiple samples – to target nine avian influenza viruses (Avian mCARMEN) and ten swine-origin influenza viruses (Swine mCARMEN) of pandemic potential. In vitro validation demonstrated that crRNAs on both panels reliably detect their respective targets with limits of detection ranging from 10²-10⁴ copies/μl. However, notable cross-reactivity was observed between several assays and off-target samples, largely attributable to reduced discriminatory power when distinguishing genetically similar viral strains. We also adapted SHINE, a Cas13-based diagnostic assay suitable for point-of-care testing, for detection of the emerging H5N1 avian influenza virus by developing two assays targeting both general and clade-specific H5 sequences. Both SHINE assays demonstrated limits of detection between 10–100 copies/μl. Notably, the clade-specific assays exhibited strong specificity, detecting off-target samples only at high input concentrations of 10⁴ copies/μl. Altogether, this project sets the groundwork for CRISPR-Cas13-based approaches for zoonotic IAV surveillance and point-of-care detection. For the second application, we sought to develop and optimize Cas-seq, a novel platform that combines CRISPR-Cas13 systems with viral delivery to enable efficient and cost-effective preparation of high-throughput scRNA-seq libraries from large-volume samples. Cas-seq functions by expressing barcoded crRNA constructs within cells designed with spacers complementary to mRNA 3’ poly-A tails. These crRNAs serve as primers for in situ hybridization and barcoded cDNA synthesis, ultimately enabling single-cell transcriptome profiling. Initial Cas-seq sequencing runs revealed low barcode incorporation into the final libraries. Suspecting that low intracellular crRNA expression or suboptimal crRNA priming contributed to poor barcode incorporation, we initiated a series of in vitro optimization experiments, modifying factors such as the RNA polymerase used for transcription and the design of crRNA spacer sequences to enhance both expression and priming efficiency. Optimized crRNA constructs were then cloned and delivered into HEK293T cells via lentiviral transduction, with protocol steps refined to enhance delivery efficiency. Finally, a complete sequencing experiment with the optimized Cas-seq workflow was performed. The experiment again showed limited barcode inclusion, with sequencing analysis indicating several potential failing points, including incomplete crRNA processing within cells and excessive tagmentation during library prep. Future efforts will focus on addressing these technical challenges to enhance Cas-seq performance.en-USPrinceton University Senior Theses