Towards a functional investigation of human DNA mismatch repair variants

Abstract

Despite DNA mismatch repair (MMR) being closely related to human diseases, most MMR gene variants continue to have unknown functional consequences. Here, I develop the tools necessary to build a novel high-throughput compatible, SaCas9 prime editing-based MMR readout reporter assay that can assess the functionality of MMR variants in a sensor-specific manner. The reporter leverages the inhibitory effect MMR has on prime editing, such that a successful prime edit indicates MMR deficiency, and failure to install the edit indicates MMR proficiency. Specifically, I have designed libraries of SaCas9-based pegRNA-target pairs, as well as engineered an optimized SaCas9-based prime editor, SaPE7. Together, these components can be combined to assemble the MMR reporter and can be used in a mutational scanning approach to uncover the structure-function relationship between MMR gene variants and its phenotypic outcomes.