Towards a high-throughput small molecule screen for DNA mismatch repair inhibitors

Abstract

Prime editing (PE) is a highly versatile CRISPR-Cas-based gene editing tool designed to introduce specific edits of varying length and nucleotide concentration into the genome using a programmable reverse transcriptase. Although PE can introduce more complex, accurate edits with fewer off-target effects than other CRISPR-Cas-based tools, it still suffers from low editing efficiency. While our lab previously found that disabling mismatch repair (MMR) machinery increases PE efficiency, all methods to do so require genetic modulation of the cell. To perform a screen for small molecule inhibitors of MMR, Jun Yan designed a bicistronic Nluc-mCherry reporter, where Nluc cannot be translated since it lacks a start codon. I designed the AnyATG, AGG-PAM, and +5-MutS library generation pipelines to identify the top candidate target sites and accompanying start-codon-creating edits for use in the reporter, and successfully cloned lKW002, an AGG-PAM-generated library. The AGG-PAM-generated library insertions in lKW002 exhibited high rates of recombination between the guide, reverse transcriptase template, and target site loci. I additionally tested conditions to optimize K562 cell growth in the 384-well plates necessary for the small molecule screen. While I found that use of a plate-sealing sticker did not sufficiently reduce well volume loss due to evaporation, I confirmed that cells could be grown in media without pH indicator phenol red and that the optimal seeding density for cells in 384-well plates is at or below 31,250 cells per milliliter. This provides a solid foundation for the lab to continue work designing the small molecule screen for MMR inhibitors.