Iranian Research Organization for Science and Technology (IROST) Microbiology, Metabolites and Biotechnology 2980-8855 8 2 2025 11 01 Efficient in vivo Directed Evolution in E. coli using a Gibson Assembly-Adapted EvolvR System 65 72 1596 10.22104/mmb.2025.7751.1177 EN Yousef Vatanparast Department of Microbiology and Microbial Biotechnology, Faculty of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran Gholamhossein Ebrahimipour Department of Microbiology and Microbial Biotechnology, Faculty of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran Mohammad Yaghoubi-Avini Department of Microbiology and Microbial Biotechnology, Faculty of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran 0000-0001-5761-1062 Journal Article 2025 07 18 Genetic diversity is vital for species adaptation and evolution, enhancing resilience to environmental changes and improving desirable traits. Directed evolution simulates natural selection in labs to engineer proteins and microorganisms, utilizing iterative cycles of genetic variation to achieve desirable characteristics. EvolvR is a system that can continuously diversify all nucleotides during an adjustable window at user-defined locations. It is achieved by mutagenesis using engineered DNA polymerases directed to the target site via CRISPR-directed nickases. Although the typical plasmid assembly and gRNA insertion method is Golden Gate cloning, the aim of this study was to set up EvolvR according to our equipment and conditions. The gRNA targeting rpsE in E. coli DH5α was selected from a previous study. Specific primers that included the gRNA sequence and provided homology on one side were inserted into the EvolvR plasmid using the Gibson assembly method. The constructed plasmid was chemically transformed into E. coli DH5α. Bacterial resistance was evaluated by colony counting on culture media containing 50, 100, and 500 µl/ml of spectinomycin. Results showed that the number of E. coli DH5α cells in an antibiotic-free medium was 11×108 CFU.mL-1, while no growth was observed at any antibiotic concentration. The non-induced EvolvR cells did not grow in a medium containing 100 and 500 μg.mL-1 spectinomycin, but grew at 20×106 CFU.mL-1 in a medium with 50 μg.mL-1 antibiotic. Induction of the EvolvR system resulted in a dramatic increase in spectinomycin-resistant mutants, yielding up to 4×108 CFU.mL-1 on 100 μg⋅mL−1 spectinomycin and a resistance frequency order of magnitude higher than previously reported. Our findings validate Gibson assembly as a robust and accessible alternative to Golden Gate for constructing EvolvR systems and emphasize the high efficacy achievable with a strategically targeted single gRNA.

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