CRISPR‐DNA Polymerase Assisted Targeted Mutagenesis for Regulable Laboratory Evolution

Targeted hypermutation tools are useful for engineering proteins and pathways, and exploring the evolutionary landscapes. However, existing targeted hypermutation tools for genomic loci mostly exhibit restricted mutation windows and limited mutational types. Here, by integrating mutagenic, high‐processivity bacteriophage T5 or T7 DNA polymerases (DNAPs) with CRISPR‐Cas9, the study develops an in vivo mutagenesis system that enables all possible types of nucleotide substitutions and an expanded mutation window of up to 2 kilobases, achieving a maximum mutation rate 1.1 × 10 6 ‐fold higher than wild‐type Escherichia coli . Through MS2‐mediated recruitment of T5 or T7 DNAP for co‐localization with nickase nCas9, off‐target rate is reduced by up to 96.8% without compromising on‐target rate. Further benefiting from the dTnpB‐based transcriptional repression system, the mutagenesis process can be properly regulated during continuous evolution. Finally, the CRISPR‐TDNAP‐assisted targeted mutagenesis for regulable laboratory evolution (CTRLE) confers cellular triple‐antibiotic resistance in 8 days, and enhances the efficiency of the twin‐arginine translocation pathway by over threefold in 6 days. Furthermore, CTRLE proves effective in Bacillus subtilis and Kluyveromyces lactis , yielding targeted mutation rates 1.2 × 10⁵‐fold and 5 × 10⁷‐fold higher than host backgrounds, respectively. Collectively, CTRLE provides an efficient and universal way to accelerate the continuous evolution of different microbial cells.