Organic Chemistry Seminar
Currently, less than 1% of identified human single nucleotide variants (SNVs) have a defined clinical interpretation. New laboratory-based methods that facilitate the functional characterization of SNVs are needed to combat this interpretation issue. Genome editing technologies have the potential to enable the generation of cellular models of SNVs, but current tools suffer from key limitations related to efficiency, precision, and specificity. "Traditional" genome editing tools function by first introducing double-strand DNA breaks (DSBs) at a target locus using a programmable nuclease such as Cas9 and a guide RNA (gRNA). These methods are typically inefficient at point mutation correction and induce an abundance of random insertions and deletions (indels) at the target locus due to the cellular response to DSBs. Here I will describe our efforts to enhance the precision of DSB-reliant tools via the development of the "double tap" method. I will also describe our work to understand the enzymatic and cellular mechanisms by which "nontraditional" tools (such as base editors and prime editors) function, and our development of streamlined methods to multiplex these tools for modeling of polygenic disorders. Finally, I will detail our applications of base editors to the functional characterization of SNVs that currently lack clinical interpretations.