UC Berkeley

Directed evolution has long been impactfully leveraged to engineer therapeutics, industrial enzymes and other revolutionary biotechnologies. Unfavorably, the application of directed evolution towards engineering complex phenotypes in mammalian cells, such as kinase signaling cascades or antitumor T cell cytotoxicity for adoptive cell therapies has been limited by a lack of appropriate biomolecular tools for continuous targeted genomic diversification in mammalian cell genomes. Most critically, existing gene editing modalities for continuous diversification in mammalian cells rely exclusively on targeting deaminases to target loci to generate transition mutations (A to G, G to A, C to T, T to C), limiting the nucleotide substitutions that can be generated to a small minority of possible missense mutations. To enable the full diversification of all four nucleotides within native mammalian genomic loci for the first time, I engineered and characterized the use of RNA-guided error-prone DNA polymerases (EvolvR) in mammalian cells. Using a cell-based fluorescent reporter fluctuation assay and next-generation sequencing, I demonstrated that EvolvR can diversify all four nucleotides throughout a gRNA spacer to generate both transition and transversion substitutions. Then, I implement EvolvR to diversify a native genomic locus and select for previously unidentified selumetinib-resistant MAP2K1 variants in melanoma-derived A375 cells. Additionally, I elucidate the role of nickase fidelity and gRNA length as critical features determining the diversity of EvolvR’s editing outcomes and expand the EvolvR’s ease of use by identifying a compatible compact, PAM-flexible nuclease the cDNA for which can be packaged into a lentiviral vector to initiate directed evolution in human cell lines with a single transduction. Together, my data show that RNA-guided DNA polymerases can access all 12 nucleotide substitutions to identify gain of function missense mutations not reachable by deaminase-based diversifiers in mammalian cells and establish a novel state-of-the-art modality for genetically engineering mammalian cells via directed evolution.