Jiangsu Key Laboratory for Microbes and Functional Genomics, College of Life Sciences, Nanjing Normal University, No.1 Wenyuan Rd., Xixia District, Nanjing, 210023, Jiangsu Province, People's Republic of China.
Biotechnol Lett. 2023 Jun;45(5-6):629-637. doi: 10.1007/s10529-023-03363-1. Epub 2023 Mar 17.
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.
定点和饱和突变是研究蛋白质结构和功能的关键 DNA 方法。对于基于质粒的基因突变，PCR 和重叠延伸 PCR 涉及繁琐的克隆步骤。当质粒尺寸较大时，PCR 产量可能太低而无法进行克隆；而对于单个密码子的饱和突变，一次实验可能不足以产生所有二十种编码变体。寡核苷酸介导的重组通过突变寡核苷酸与其靶位点之间的同源重组绕过了复杂的克隆过程。然而，低重组效率和无法选择重组体使得有必要筛选大量克隆。在这里，我们描述了两种基于质粒的诱变策略：CRISPR/Cas9 辅助 ssDNA 重组进行定点突变（CRM）和饱和突变（CRSM）。CRM 和 CRSM 涉及将靶质粒、sgRNA 表达质粒和诱变寡核苷酸共电穿孔到大肠杆菌细胞中，同时诱导表达 λ-Red 重组酶和 Cas9，然后提取和表征质粒。我们通过氨苄青霉素抗性基因修复和 N-乙酰-D-神经氨酸醛缩酶的突变建立了 CRM 和 CRSM。突变效率在 80%到 100%之间，通过单次 CRSM 策略在一个靶位点获得了所有二十种氨基酸编码变体。CRM 和 CRSM 有可能成为通用的基于质粒的基因诱变工具。
