Nature 560, 248–252 (2018)

Directed evolution is an efficient approach to creating biomolecules with desired functions by applying an artificial selection pressure to screen libraries of genetic variants. The degree of diversity in the library can directly affect the screening results. However, diversifying all nucleotides within a defined DNA region in a cell remains a challenge. To overcome this problem and produce libraries with all possible nucleotide mutations, Halperin et al. developed a genomic-editing system called EvolvR by fusing a nicking variant of Cas9 (nCas9) to an error-prone variant of Escherichia coli DNA polymerase I (PolI3M), such that mutant nucleotides were introduced downstream of the nicks generated at target sites. By altering the processivity and fidelity of the PolI and the affinity of nCas9 to DNA, the targeted mutation rate could reach as high as 7,770,000-fold greater than that in wild-type cells with a mutagenesis window as long as 350 nucleotides. Using the EvolvR system, the authors identified novel ribosomal mutations in the E. coli rpsE gene that conferred spectinomycin resistance. This study provides a targeted mutagenesis tool independent of homology directed repair and adds a new element to the genome-editing toolbox.