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Vanessa, a rising senior at Colorado College majoring in molecular biology and minoring in biochemistry, investigated the use of engineered suppressor tRNAs (sup-tRNAs) in combination with prime editing to promote readthrough of premature stop codons caused by nonsense mutations, with the goal of exploring their potential as a therapeutic strategy for genetic diseases.

Nonsense mutations, which introduce premature stop codons that often lead to nonfunctional proteins, are responsible for 11% of all genetic diseases. Engineered suppressor transfer RNAs (sup-tRNAs), which recognize premature stop codons, are one approach to bypass early stop signals and restore the production of full-length proteins, potentially treating nonsense-associated genetic diseases. My summer at the Ó³»­´«Ã½ and participation in BSRP were challenging in the best ways, deeply gratifying, and profoundly inspiring. Being immersed at the forefront of cutting-edge research pushed me to grow as a scientist and further solidified my vision of pursuing a future in academia. The intentional, patient mentorship I received helped build both my skills and confidence in ways I’ll carry with me throughout my journey in science. The science communication course reminded me how vital it is to make science accessible, and the unwavering support from the BSRP staff shaped how I see myself as a future leader in this field. I’m leaving this summer with greater confidence, curiosity, and clarity about the impact I hope to make in research.
Despite the promise of sup-tRNAs as a mutation-agnostic approach in reading through premature stop codons across many genetic diseases, current methods to deliver sup-tRNAs into cells is transient, leading to reduced protein restoration and limited long-term therapeutic effects. In this project, our goal is to leverage prime editing (PE), a genome editing tool, to permanently install sup-tRNA sequences into the genome of mammalian cells for sustained therapeutic effect. We cloned pegRNAs that can install the sup-tRNAs into the genome into DNA plasmids and introduce them into HEK293T cells via transfection. Preliminary results using a GFP-based reporter system show that PE6c outperformed PEmax in restoring GFP expression, with a significantly higher percentage of GFP+ cells. These early findings suggest that PE6c may enable more efficient installation of functional sup-tRNAs and support further comparison of alternative editing strategies. We will use next generation sequencing to measure the efficiency of the prime edit installation. We expect that PE will lead to successful and lasting integration of sup-tRNAs in the genome. If successful, this strategy could offer a more reliable and mutation-agnostic treatment for genetic diseases caused by nonsense mutations. 

Project: Optimizing sup-tRNA genome integration for the treatment of nonsense mutations

Mentors: Sarah E. Pierce and Holt A. Sakai, Chemical Biology and Therapeutics Science