CRISPR-guided DNA polymerases for targeted genetic diversification
The capacity to diversify genetic codes advances our ability to understand and engineer biological systems. A method for continuously diversifying user-defined regions of a genome would enable forward genetic approaches in systems that are not amenable to efficient homology-directed oligonucleotide integration. It would also facilitate the rapid evolution of biotechnologically useful phenotypes through accelerated and parallelized rounds of mutagenesis and selection, as well as cell-lineage tracking through barcode mutagenesis. Programmable nucleases, such as CRISPR/Cas9, have revolutionized our ability to easily disable targeted genetic elements; however, substituting nucleotides in user-defined regions of a genome using programmable nucleases remains inefficient in many contexts due to the need for homology-directed repair (HDR), donor templates, cytotoxic double-stranded breaks, and competition with non-homologous end-joining (NHEJ). Here I will present EvolvR, the first system that can continuously diversify all nucleotides within a tunable window length at user-defined loci without relying on HDR, donor templates, double-stranded breaks, or NHEJ. This is achieved by enzymatically generating mutations using engineered DNA polymerases targeted to loci via CRISPR-guided nickases. I identified nickase and polymerase variants that offer a range of targeted mutation rates that are up to 7,770,000-fold greater than rates seen in wild-type cells, and editing windows with lengths of up to 350 nucleotides. I used EvolvR to identify novel ribosomal mutations that confer resistance to the antibiotic spectinomycin and adapted EvolvR for use in human cells. My results demonstrate that CRISPR-guided DNA polymerases enable multiplexed and continuous diversification of user-defined genomic loci, which will be useful for a broad range of basic and biotechnological applications.