High-throughput Characterization of Genetic Mechanisms of Traditional and Paired Prime Editing - Abstract The development of prime editing has allowed for genome manipulation with user-defined insertions, deletions, or single-nucleotide conversions, without installing harmful double-stranded breaks in the genome.5 This has revolutionized the genomic medicine field, with companies like Prime Medicine, Inc. now using prime editing in human clinical trials. Despite its versatility, prime editing often results in lower editing efficiencies for larger edits, and the nuances of how insertion and deletion edits are installed by prime editors are not well understood. 5,6,7,8 Past genomic mechanistic studies of prime editing have identified DNA mismatch repair (MMR) as a negative regulator of point mutation introduction9; however, MMR does not process loops or bulges larger than 16 nucleotides, which would be utilized to install large insertions and deletions by prime editing.10,11,12 Therefore, the mechanism(s) by which the cell processes prime edits larger than 16-nt is unexplored. While traditional prime editing often becomes hindered by longer edits, “paired” prime editing techniques can facilitate larger genome modifications by replacing a sequence between two nick sites. 8,13 More thorough understanding of the DNA repair pathways will improve both traditional and paired prime editing. Aim 1 of this proposed project will develop a “dead” fluorescent reporter that is corrected by a large genomic modification by either traditional or paired prime editing. The reporter will be utilized in Aim 2, in which a high-throughput Cas12 knockout screen will be used to identify the repair pathways involved in processing large edits by traditional and paired prime editing. DNA damage repair genes will be knocked out, and K562 cell lines constitutively expressing the developed reporter and prime editor system will be separated by editing efficiency via fluorescent activated cell sorting (FACS), high-throughput sequencing (HTS), and large-scale bioinformatics data processing. In Aim 3, the results from the genomic screen will be validated through testing at multiple endogenous sites and in various cell types to ensure reproducibility and rigor in identifying hit genes. With an increased understanding of traditional and paired prime editing, the resulting gene hits will be incorporated into novel prime editor systems to increase editing efficiency for longer edits, which can fill gaps on treating all genetic disease. Additional knowledge of what repair pathways process large edits will also add to the DNA damage repair field, enriching understanding on structure and biology of genomes. Ultimately, the proposed training plan is set to advance the PI’s technical foundation of genomic, high-throughput screening methods, as well as provide steps to develop professional skills, leadership, and scientific independence for a future in academic research. It will be supported by the sponsor Dr. Alexis Komor and UCSD’s Chemistry and Biochemistry department, in consortium with the many UCSD and Sanford core research facilities that will provide the necessary FACS, HTS, and supercomputing instruments.
