EvolvR, a tool developed in UC Berkeley and the Innovative Genomics Institute (IGI), set out to solve one grand challenge – the ability to completely diversify all of the nucleotides at a user-defined location in an organism’s genome in a continuous fashion . EvolvR relies on a fusion of two proteins. The first, nCas9 creates a single-stranded nick in an “editing window” and the second enzyme, a nick-translating low-fidelity DNA polymerase I (PolI3M), then proceeds to diversify the “editing window” through substitution, deletion, and insertions of bases. The work focused on engineering and optimizing the components of the protein chimera such that the mutation rate and base mutation window were increased, as well as identifying mutations that enable improved dissociation of nCas9 from its DNA target .
The ability to generate a completely diversified library in a sixty nucleotide window using a single microliter of a saturated Escherichia Coli culture containing the EvolvR system, opens the door to exciting engineering opportunities in the field of synthetic biology. Specifically, Dueber and Coworkers were able to screen for novel mutations in antibiotic resistance genes and generated new ribosomes that were found to be resistant to Spectinomycin. Future applications to EvolvR include screening for novel ribosomal functions, RNA based drug screening, as well as the development of new nucleic acid-based catalysts .
- Halperin SO, Tou CJ, Wong EB, Modavi C, Schaffer DV, Dueber JE. (2018) “CRISPR-guided DNA polymerases enable diversification of all nucleotides in a tunable window.” Nature. doi:10.1038/s41586-018-0384-8