NEW YORK (GenomeWeb) – A team of researchers at the University of California, Berkeley has developed a tool that it says can facilitate the rapid evolution of biotechnologically useful phenotypes in cells.
As they wrote today in Nature, they created a system called EvolvR that can continuously diversify all nucleotides within a tunable window at user-defined loci by generating mutations using engineered DNA polymerases targeted 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," the authors wrote. "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."
DNA polymerases can create substitutions as well as deletions in the genome and vary in the average number of nucleotides they incorporate after each binding event, in their fidelity, and in their substitution bias. In particular, nick-translating DNA polymerases are able to initiate synthesis from a single-stranded break in double-stranded DNA while displacing downstream nucleotides. The researchers hypothesized that recruiting an error-prone, nick-translating DNA polymerase with a nicking variant of Cas9 (nCas9) could offer an ideal targeted mutagenesis tool that is independent of homology-directed repair, calling this system EvolvR.
In assays using E. coli and experimenting with resistance to the antibiotic spectinomycin, the researchers tested coupling EvolvR-mediated mutagenesis to a genetic screen of a non-selectable phenotype in order to broaden the tool's utility. They found that EvolvR could diversify chromosomal loci by increasing the fraction of the population resistant to spectinomycin approximately 16,000-fold after targeting a Cas9 complex to the endogenous ribosomal protein subunit 5 gene of E. coli, which has mutations that are known to confer resistance to spectinomycin.
They also tested whether EvolvR avoids the toxicity associated with non-targeted mutagenesis systems and found that EvolvR does not impede cell viability or growth rate. EvolvR could also simultaneously diversify distant genomic loci through co-expression of multiple guide RNAs. "This capacity to simultaneously diversify multiple loci will be useful for identifying epistatic interactions," the authors noted.
Next, the team evolved resistance to both spectinomycin and streptomycin, using EvolvR for continuous directed evolution to allow adaptation to modulated selection pressures. Because spectinomycin is clinically useful as a broad-spectrum antibiotic, researchers have been motivated to characterize genomic mutations conferring resistance to it. The team used EvolvR to diversify the rpsE gene in E. coli to identify novel mutations that confer spectinomycin resistance by disrupting the spectinomycin-binding pocket of the 30S ribosomal subunit. After several experiments, they discovered novel mutations that confer spectinomycin resistance.
"This rapid method for discovering genotypes conferring antibiotic resistance will be generally useful for improving the effective use of antibiotics," the authors wrote. "EvolvR offers the first example of continuous targeted diversification of all nucleotides at user-defined loci, which will be useful for evolving protein structure and function, mapping protein–protein and protein–drug interactions, investigating the non-coding genome, engineering industrially relevant microbes, and tracking the lineage of cell populations that cannot tolerate double-stranded breaks."