Date of Award
Doctor of Philosophy
CRISPR (clustered regularly interspaced short palindromic repeats)-guided nucleases such as Cas9 remain atop the most exciting biological systems in gene editing and therapeutic potential. The modern adaptation of the Streptococcus pyogenes (Spy) Cas9 machinery for directed DNA editing relies on two major components; a Cas9 protein, and an RNA guide. This study aims at modification of the guide RNA to probe structural requirements and modification tolerance while studying the biochemical properties of the ribonucleoprotein complex. We find that the SpyCas9-RNP is not only capable of heavy substitutions and modifications in the guide, but specific properties can be tuned by careful and directed modifications. DNA substitutions in the 3’ end of the guide RNA improves cleavage activity and efficiency in vitro. Various RNA chemistries such as 2’-fluoro and other RNA mimics maintain biochemical activity but can be used to improve stability against nucleases. Additionally, tethering of the tracrRNA and crRNA together into a sgRNA was found to affect the fundamental properties of the Cas9 protein, improving activity, but reducing target binding affinity and cleavage rate. Directed modification of the guide RNA can be used to exploit certain biochemical properties for effective therapeutic applications.
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