[CRISPR/Cas9-mediated knockout of the Soc gene in T4 bacteriophage and mutant construction]

To address the antigen display limitations of current vaccine carriers, we engineered the T4 bacteriophage into a high-capacity platform. The T4 bacteriophage has advantages such as structural stability, high loading capacity, and easy production. However, the presence of high-copy endogenous Soc protein on its capsid surface severely restricts the effective display of exogenous proteins. To overcome the aforementioned spatial limitations, we employed the CRISPR/Cas9 system to precisely knockout the Soc gene of T4 bacteriophage. We successfully achieved the knockout of the Soc gene by co-transferring the three plasmid systems-pCas, pTargetF-sgRNA, and pMD19-T-Soc-arm-into Escherichiacoli TG1, inducing the expression of Cas9 with L-arabinose, and then infecting the engineered bacteria with the wild-type T4 bacteriophage (named T4 WT). The PCR, SDS-PAGE, and sequencing results confirmed a Soc gene-deficient T4 bacteriophage mutant strain (named T4ΔSoc) was successfully constructed. This mutant strain had comparable growth, thermal stability, and pH stability to the wild type, and maintained complete infectivity. After continuous passage for five generations, it remained stable in terms of genome, protein composition, and phage plaque phenotype, with no occurrence of revertant mutations. In conclusion, T4ΔSoc is stable both genetically and phenotypically, effectively breaking through the limitation of the restricted display space of T4 WT. This study provides a reliable vector tool and technical foundation for constructing an efficient and stable antigen display and vaccine delivery platform and has good application potential in vaccine research and the construction of targeted delivery systems.