Over billions of years, Darwinian evolution has led to the development of remarkable biological systems through nature’s selective pressures on genetic variation. Today, the ability to employ repeated rounds of synthetic genetic diversification and artificial selection in the wet-lab – termed directed evolution - has created powerful and novel molecular and cellular functionalities. However, current genetic diversification techniques are either limited by a narrow editing window length and nucleotide substitution restrictions, stringent host cell and culture conditions, or a dependency on transformation and oligonucleotide integration efficiency. Last year, our research group developed EvolvR, a modular, HDR-independent, multiplexable genetic diversifier. EvolvR can generate all four nucleotide substitutions within a tunable window by localizing error-prone DNA Polymerases to user-defined loci via CRISPR-guided Cas9-nickases. By developing a fluctuation analysis assay, we screened EvolvR variants that showed editing window lengths of up to 350 nucleotides and targeted mutation rates up to six orders of magnitude greater than mutation rates in wild type E. coli cells. Currently, EvolvR generates mutations within its gRNA complementary region, limiting its capacity for continuous genetic diversification over extended periods of time. Through a modified architecture, we are continuing to develop a fully continuous EvolvR system, which will expand EvolvR’s utility in the directed evolution of biotechnologically useful phenotypes.

Towards engineering a fully continuous CRISPR diversifier

2019 Spring National Meeting

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