Sullenger, Bruce ABush, Korie B2023-03-282022https://hdl.handle.net/10161/26837<p>While much of the current focus on advancing CRISPR-Cas9 editing revolves around the engineering of Cas9, the interrogation and evolution of sgRNA scaffold, in addition to novel Cas9 binding RNAs, represent another echelon of development and therapeutic potential. Currently, the majority of research utilizes a singular guide RNA scaffold sequence (the sgRNA) for a given Cas protein (e.g., the Streptococcus pyogenes Cas9 and associated guide RNA). This sequence inflexibility makes many sites within the genome intractable to CRISPR/Cas editing, often due to undesirable intramolecular interactions that result in undesirable secondary structures. Additionally, given the electrostatic potential of Cas9, it may be possible to generate additional Cas9 binding RNA molecules.Here, we use utilize SELEX to both identify novel Cas9 binding RNAs and interrogate the sequence depth of the sgRNA scaffold. First, a SELEX scheme utilizing a nitrocellulose filter binding assay was utilized to identify modified RNA aptamers that bind to Cas9 with specificity and an affinity rivaling that of the sgRNA. The aptamer was shown to tolerate truncations and sequence additions, demonstrating an ability to localize oligonucleotide-based therapeutics to the Cas9 protein. We believe that this aptamer can be expanded upon to incorporate novel functions instead of altering the sgRNA . Second, we use a novel combinatorial approach that utilizes a functional SELEX (Systematic Evolution of Ligands by Exponential Enrichment) to identify numerous, diverse sgRNA variants that bind S. pyogenes Cas9 and support DNA cleavage. These variants demonstrate surprising malleability in the sgRNA sequence and are utilized in a combinatorial approach to identify scaffolds that enhance editing efficiencies when paired DNA-binding antisense domains. Using molecular evolution, guide RNA scaffolds can be generated for specific targets and optimized to ensure that secondary structure is maintained. This selection approach should be valuable for generating gRNAs with a range of new activities. </p>GeneticsAptamerCRISPRGene editingRNA TherapeuticsSELEXDissertation