Ubiquitin pathway modulation to enhance prime editing efficiencies.

• Applicants: Dr.-Ing. Koraljka Husnjak, Ph.D.; Professor Manuel Kaulich, Ph.D.
• Subject Area: General Genetics and Functional Genome Biology
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 529014374

After more than a decade of CRISPR-based functional genomics in human cells, studying and understanding gene variants in their endogenous context still remains a major challenge. This is in large part due to technological constraints, statistical outcomes of gene edits, and the lack of unbiased phenotypic readouts. While recent work established prime editing to generate desired point mutations in human cells, generating larger sets of mutations to enable the unbiased testing of gene variants at scale has so far lacked feasibility. This is mainly due to (i) the large number of possible gene variants, and (ii) limitations inherent to prime editing techniques. Exploiting our technological and computational expertise, we here propose to develop experimental approaches, enhance prime editing efficiencies, and establish unbiased gene variant testing. All of these, plus implementing computational frameworks for prime editing screening analysis, rely on key areas of expertise in our laboratories. We will harness prime editing phenotypes for biological discovery at multiple levels. First, the exploitation of ubiquitin system genes (USGs) in their regulatory role for prime editing efficiencies will inform about the biological constraints enabling prime editing in human cells. Moreover, they will inform about the regulatory role of USGs in DNA mismatch repair and will be applied to unbiased gene variant testing. Second, we will perform USP1 prime editing variant testing to provide a comprehensive USP1 mutational landscape from which we will extract amino acids relevant to USP1 function. The mutational landscape will be paired with structure predictions to elucidate functionally relevant domains at amino acid resolution. Third, coupling USP1 variant testing with available USP1 inhibitors (iUSP1) will provide much needed insights into possible iUSP1 resistance-causing mutations and generate the first resistance landscape generated by gene variant testing in human cells. Lastly, techniques and methodologies developed here will, at a broader scale, serve as blueprints to generate gene-specific mutational and resistance landscapes in human cells. Our proposed project represents our first joint experimental effort merging arrayed CRISPR and prime editing screens with unbiased gene variant testing in human cells to date. It promises to produce innovative approaches and provide multifaceted biological insights into the regulation of prime editing and DNA repair by ubiquitin system genes.

• DFG Programme: Research Grants