CRISPR-guided DNA polymerases enable diversification of all nucleotides in a tunable window.

The capacity to diversify genetic codes advances our ability to understand and engineer biological systems1,2. 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. Here we present EvolvR, a system that can continuously diversify all nucleotides within a tunable window length at user-defined loci. This is achieved by directly generating mutations using engineered DNA polymerases targeted to loci via CRISPR-guided nickases. We 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. We used EvolvR to identify novel ribosomal mutations that confer resistance to the antibiotic spectinomycin. Our 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.