Abstract
Site-directed and saturation mutagenesis are critical DNA methodologies for studying protein structure and function. For plasmid-based gene mutation, PCR and overlap-extension PCR involve tedious cloning steps. When the plasmid size is large, PCR yield may be too low for cloning; and for saturation mutagenesis of a single codon, one experiment may not enough to generate all twenty coding variants. Oligo-mediated recombineering sidesteps the complicated cloning process by homologous recombination between a mutagenic oligo and its target site. However, the low recombineering efficiency and inability to select for the recombinant makes it necessary to screen a large number of clones. Herein, we describe two plasmid-based mutagenic strategies: CRISPR/Cas9-assisted ssDNA recombineering for site-directed mutagenesis (CRM) and saturation mutagenesis (CRSM). CRM and CRSM involve co-electroporation of target plasmid, sgRNA expression plasmid and mutagenic oligonucleotide into Escherichia coli cells with induced expression of λ-Red recombinase and Cas9, followed by plasmid extraction and characterization. We established CRM and CRSM via ampicillin resistance gene repair and mutagenesis of N-acetyl‑D‑neuraminic acid aldolase. The mutational efficiency was between 80 and 100% and all twenty amino acid coding variants were obtained at a target site via a single CRSM strategy. CRM and CRSM have the potential to be general plasmid-based gene mutagenesis tools.
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Acknowledgements
We thank for Dr. Frederick Blattner, Dr. Josef Altenbuchner, Dr. John Cronan, and Dr. Barry Wanner for providing the plasmids used in this research. We thank the two anonymous reviewers for their critical comment and insightful contributions to the article.
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Financial support was provided by National Natural Science Foundation of China (NSFC 82073742).
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GS conceptualized and designed the study, supervised the research and finalized the manuscript. GZ, JW and YL carried out the experiments, participated in data analysis and manuscript preparation. All authors read and approved the final manuscript.
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Zhang, G., Wang, J., Li, Y. et al. CRISPR/Cas9-assisted ssDNA recombineering for site-directed mutagenesis and saturation mutagenesis of plasmid-encoded genes. Biotechnol Lett 45, 629–637 (2023). https://doi.org/10.1007/s10529-023-03363-1
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DOI: https://doi.org/10.1007/s10529-023-03363-1