Multiplexed Locus-Specific Hypermutation for Functional Characterization of the Coding and Noncoding Genome

SUMMARY While laboratory directed evolution has become a workhorse for generating novel therapeutics and synthetic biomolecules, few tools exist for directly evolving the endogenous mammalian genome. Most tools have extremely broad or narrow targeting windows (e.g., alkylators that introduce mutations genome-wide2 or CRISPR base-editors that target narrow windows of 4-10 nts3,4). To systematically evolve entire gene regulatory networks and query gene functions with base-level resolution, we must develop targeted long-range approaches that can quickly introduce variants in gene sets and in multiple regulatory elements that contribute to gene expressions. Here, we will develop a novel platform for performing multiplexed locus-specific hypermutation within the mammalian genome called helicase-assisted continuous evolution (HACE). HACE utilizes a nick-directed helicase fused to a deaminase domain to perform continuous mutagenesis in a large (>1000nt) genomic target. This helicase-deaminase fusion can be directed to specific genomic regions of interest using a Cas9 nickase (nCas9) with loci-specific sgRNAs. We will apply HACE to functionally characterize both non-coding regulatory elements, as well as enable multiplexed probing of the MEK signaling pathway for small molecule resistance.