Date of Award


Degree Name

Doctor of Philosophy


Molecular Biology, Microbiology and Biochemistry

First Advisor

Gagnon, Keith


Clustered regularly interspaced palindromic repeats (CRISPR) and their associated (Cas) proteins co-opted as biotechnological tools have improved the simplicity and accessibility of gene editing for fields ranging from crop science to the treatment of human disease. These technologies, however, come with an inherent degree of risk associated with off-target events or direct misuse, accidental or intentional, leading to permanent genetic damage to ecosystems, livestock, or people. Naturally occurring anti-CRISPR proteins have been described, as well as synthetic small molecule inhibitors, but each of these approaches, while suitable for certain applications, leaves something to be desired in deliverability or efficacy in the face of many possible adverse CRISPR-related events. Inspired by strides in the field of oligonucleotide therapeutics, we developed the first reported anti-CRISPR nucleic acids for Streptococcus pyogenes (Sp)Cas9 to address the critical need for fail-safe inhibitors of Cas enzymes. These inhibitors, termed small nucleic acid-based inhibitors of Cas9 (SNuBs), comprise two modules which act in tandem to bind and disable the SpCas9 RNP. We have demonstrated that SNuBs inhibit Cas9 in vitro and in human cells. Successive rounds of optimization on our initial designs have yielded inhibitors capable of carrier-free uptake into human cells, high nuclease resistance, and robust inhibition at low stoichiometric concentrations relative to Cas9 and its RNA guide. In their current form, SNuBs quite possibly present the most tenable approach to inhibiting Cas9 in a variety of contexts including therapeutic applications in the near future.




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