To construct wild-type E. coli irp2 gene deletion strains, CRISPR/Cas9 gene editing technology was used, and the difficulty and key points of gene editing of wild-type strains were analyzed. Based on the resistance of the CRISPR/Cas9 system expression vector, 4 strains of 41 E. coli strains isolated from Saba pigs were selected as the target strains for the deletion of the irp2 gene, which were sensitive to both ampicillin and kanamycin. Then, CRISPR/Cas9 technology was combined with homologous recombination technology to construct recombinant vectors containing Cas9, sgRNA and donor sequences to knock out the irp2 gene. Finally, the absence of the irp2 gene in E. coli was further verified by iron uptake assays, iron carrier production assays and growth curve measurements. The results showed that three of the selected strains showed single base mutations and deletions (Δirp2-1, Δirp2-2 and Δirp2-3). The deletion of the irp2 gene reduced the ability of E. coli to take up iron ions and produce iron carriers, but not affect the growth characteristics of E. coli. It is shown that the CRISPR/Cas9 knock-out system constructed in this study can successfully knock out the irp2 gene of the wild-type E. coli. Our results providing new insights into genome editing in wild-type strains, which enable further functional studies of the irp2 gene in wild-type E. coli.

利用CRISPR/Cas9基因编辑技术构建野生型大肠杆菌irp2基因缺失菌株，并分析了野生型菌株基因编辑的难点和要点。基于CRISPR/Cas9系统表达载体的抗性，选取沙巴猪分离的41株大肠杆菌菌株中的4株作为irp2基因缺失的目标菌株，对氨苄西林和卡那霉素均敏感。然后，将CRISPR/Cas9技术与同源重组技术相结合，构建含有Cas9、sgRNA和供体序列的重组载体，敲除irp2基因。最后，通过铁摄取测定、铁载体产生测定和生长曲线测量进一步验证了大肠杆菌中是否缺乏irp2基因。结果显示，所选菌株中有3个显示出单碱基突变和缺失（Δ irp2-1 、Δ irp2-2和Δ irp2-3 ）。 irp2基因的缺失降低了大肠杆菌摄取铁离子和产生铁载体的能力，但不影响大肠杆菌的生长特性。表明本研究构建的CRISPR/Cas9敲除系统能够成功敲除野生型大肠杆菌的irp2基因。我们的结果为野生型菌株的基因组编辑提供了新的见解，从而能够对野生型大肠杆菌中的irp2基因进行进一步的功能研究。