Date of Award

January 2024

Document Type

Thesis

Degree Name

Medical Doctor (MD)

Department

Medicine

First Advisor

Peter Glazer

Abstract

RNA guided CRISPR endonucleases are a revolutionary technology that has the potential to address many genetic diseases. However, there are limitations in which Cas enzymes have difficulty discriminating between similar genomic sequences, resulting in off-target editing and genotoxicity. The differentiation of single base pair mismatches between wild-type and mutant sequences can pose challenges for the Cas9 system, especially depending on the mismatches proximity to the Protospacer Adjacent Motif (PAM) site. To address this limitation, we have designed synthetic peptide nucleic acids (PNAs) that selectively bind to spacer sequences within the single guide RNA (sgRNA). The binding adds an extra energetic step in R-Loop initiation, resulting in an improved level of specificity. We demonstrate that PNAs binding over the PAM-distal region of the sgRNA facilitate the retention of on-target editing while concurrently reducing off-target effects. Furthermore, our investigation has unveiled that PNAs exhibiting perfect complementarity to the PAM-proximal region effectively inhibit Cas9 activity. As such, we have developed a platform technology that has the potential to improve specificity and control across gene editing systems utilizing a sgRNA. Our hypothesis posits that the strategic incorporation of distal PNA designs holds the potential to enable precise allele-specific discrimination. This would be especially useful in the treatment of autosomal dominant diseases, where knockout of the mutant allele and preservation of the wild-type allele is essential. As a proof of concept, we developed a corneal dystrophy reporter system, where we tested varying lengths and positions of PNAs annealed to the gRNAs and complexed with Cas9 to measure on target and off target editing of WT or mutant TGFBI sequences. We found that a 14mer PNA bound to the 7-20 position of the guide resulted in a 10-fold increase in specificity compared to traditional cas-9 editing (Figure 4.4C). These data provide compelling initial evidence to advance PNA modulation of CRISPR-Cas9, as a therapeutic platform to address autosomal dominant disease.

Comments

This thesis is restricted to Yale network users only. This thesis is permanently embargoed from public release.

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