Low immunogenicity is one of the major problems limiting the clinical use for DNA vaccines, which makes it impossible to obtain a strong protective immune response after vaccination. In order to explore whether Ag85A DNA vaccine could mount more efficiently protective immune response through new RNA sensor and its signal transduction pathway of antigen presentation we designed and synthesized Ag85A gene fragment containing multiple points mutations and transfected the gene fragment into the dendritic cell line (DC2.4) by CRISPR/Cas9. Subsequently, we focused on the changes of RNA sensors RIG-I, Mda-5, and the downstream adaptors MAVS, IRF3, IRF7 and IFN-β. The results indicated the significant increases in the mRNA and protein expression of RNA sensors RIG-I, Mda-5 and related adaptors MAVS, IRF3, IRF7, and IFN-β in the mutant DC 2.4 cells. The flow cytometry results demonstrated that the expression of MHC II on the surface of DC 2.4 significantly increased when compared with that in control. Therefore, it is suggested that Ag85A mutant DNA could release immunogenic message through RNA sensors and related adaptors via non protein pathway. There is at least one RNA signal transduction pathway of Ag85A DNA in DC2.4 cell. The work provides a new mode of action for nucleic acid vaccine to improve immunogenicity and meaningful data for the better understanding of the mechanisms of DNA vaccine.