An evolved, orthogonal ssDNA generator for targeted hypermutation of multiple genomic loci

Achieving targeted hypermutation of specific genomic sequences without affecting other regions remains a key challenge in continuous evolution. To address this, we evolved a T7 RNA polymerase (RNAP) mutant that synthesizes single-stranded DNA (ssDNA) instead of RNA in vivo, while still exclusively recognizing the T7 promoter. By increasing the error rate of the T7 RNAP mutant, it generates mutated ssDNA that recombines with homologous sequences in the genome, leading to targeted genomic hypermutation. This approach, termed T7 RNAP mutant-assisted continuous evolution (T7ACE), functions effectively in both typical prokaryotic and eukaryotic microorganisms (Escherichia coli and Saccharomyces cerevisiae), achieving targeted hypermutations at rates 2800- and 1200-fold higher than the genomic mutation rates, respectively. Using T7ACE, we successfully evolved an eight-fold increase in tigecycline resistance within 7 days and doubled the efficiency of a xylose utilization pathway in 10 days, demonstrating the efficiency and broad applicability of this single-component tool for continuous evolution.