胞嘧啶单碱基编辑技术与λ-Red同源重组技术构建鼠伤寒沙门氏菌SlyA敲除菌株的比较研究

Comparative study on construction of SlyA knockout strain of Salmonella typhimurium using CRISPR/Cas9-guided-Cytidine Base Editor and λ-Red homologous recombination technology

摘要:

由食源性致病菌引发的食品安全问题是威胁人类健康的重要因素。因此，研究食源性致病菌的感染机理对于控制病原菌危害具有重要意义。[目的] 以常见的食源性致病菌——鼠伤寒沙门氏菌为研究对象，以其发挥重要致病性的转录调控因子SlyA为靶标，比较胞嘧啶单碱基编辑技术（CRISPR/Cas9-guided-Cytidine Base Editor，CBE）和λ-Red同源重组技术在构建鼠伤寒沙门氏菌SlyA敲除菌株方面的方法差异，为鼠伤寒沙门氏菌的基因编辑技术应用提供数据。同时，也为其他类型病原菌的基因编辑技术开发提供有力参考。[方法] 采用PCR、Golden Gate、Sanger测序等方法完成CBE系统以及λ-Red系统的构建以及敲除结果的验证，采用Editor-R软件分析CBE系统的单碱基编辑效率，采用Western blotting在蛋白表达层面对敲除结果进行验证。此外，本研究还结合了表型鉴定的方法验证了基因敲除结果。[结果] 经PCR产物测序鉴定、Western blotting分析及溶血素活性鉴定等结果表明，本研究成功将CBE系统应用于鼠伤寒沙门氏菌slyA的单碱基编辑中，应用前述两种方法构建了鼠伤寒沙门氏菌SlyA敲除菌株。[结论] CBE系统虽然以其操作的简便性在基因编辑中优势明显，但同λ-Red系统相比，该方法需要设立特定的gRNA及PAM位点，在非模式菌株中的普适性较低，且在进行编辑时，CBE系统存在不稳定的问题。尽管如此，但CBE系统在鼠伤寒沙门氏菌中的成功建立，为进一步拓展与完善该菌的基因编辑系统提供了基础。

Abstract:

Food safety issues caused by food-borne pathogens have become a critical factor threatening human health. Therefore, studying the infection mechanism of food-borne pathogens is of great significance to control the harm caused by pathogens. [Objective] Taking the common food-borne pathogenic bacteria Salmonella typhimurium as the research object, we studied the genetic manipulation methods for knocking out its pathogenicity associated transcriptional regulator coding gene SlyA by systematically compared the technical differences between CRISPR/Cas9-guided-Cytidine Base Editor (CBE) and λ-Red homologous recombination methods. The work present here not only provides detailed data for the application of gene editing technology for S. typhimurium, but also provides a useful reference for the development of gene editing technology for other pathogen species. [Methods] The PCR, Golden Gate, and Sanger sequencing were used to construct CBE and λ-Red gene editing systems and verify the gene edit results. Editor-R was used to analysis base edit efficiency of CBE system. Western blot was used to verify the results of gene editing at the protein expression level. In addition, phenotypic identification was used to verify the knockout results. [Results] The PCR product sequencing, Western blot verification results and hemolysin activity identification showed that the CBE gene editing system has been successfully applied to S. typhimurium. The SlyA null mutant of S. typhimurium was constructed based on the two above-mentioned technology, respectively. [Conclusion] Although the CRISPR/Cas9-guided Cytidine Base Editor has the advantages of simpler operation and more efficient editing than the λ-Red system, the system still has several shortcomings, such as the need to establish a specific gRNA sequence and PAM locus which are not so common in non-model strains. In addition, the CBE system has the problem of instability during gene editing. Nevertheless, CBE was successfully applied to Salmonella typhimurium for the first time, which will provide a basis for optimizing and improving the gene editing of this strain.