Figure 1.

NgAgo-assisted gene editing in P. multocida and E. coli. (A) Schematic construction of the isogenic lyi (lysozyme inhibitor) gene mutant for P. multocida. An NgAgo gene with a ribosome binding sequence (rbs) was introduced upstream of the kanamycin resistance gene (kan^r) in the temperature-sensitive P. multocida-E. coli shuttle vector pSHK5(TS) to generate the pSHK5(TS)-NgAgo plasmid. The homologous arms of the lyi gene were then inserted into the pSHK5(TS) or pSHK5(TS)-NgAgo plasmid to yield pSHK5(TS)-lyiLR or pSHK5(TS)-NgAgo-lyiLR, respectively. Subsequently, the two plasmids were electro-transformed into the P. multocida strain GX-PM with guide DNA (gDNA) to isolate the isogenic mutants by PCR with the lyi-ID1F/R primers. (B) Construction efficiency of the avian P. multocida strain GX-PM with deletion of the lyi, opa (Opacity-associated protein), pilia (type IV fimbrial subunit protein), fhgb (Flavohemoglobin), hfq (RNA chaperone Hfq), or hyae (hydrogenase-1 operon protein) gene with or without NgAgo system. (C) Representatives of whole genome sequencing for the P. multocida strain GX-PM with the deletion of the lyi gene with or without gDNA. (D) Comparison of the virulence of the avian P. multocida strain GX-PM with the constructed isogenic hyae mutant in chickens. Infection of chickens with the avian P. multocida strain GX-PM caused mortality of 100% in the third day of post-infection, while infection of the constructed isogenic hyae mutant could not cause any death during the trial (n = 5). (E) Severe damage to several vital organs in response to infection with the avian P. multocida strain GX-PM was not observed in the chickens infected with the constructed isogenic hyae mutant. (F) Construction efficiency of the rabbit P. multocida strain C51-17 with deletion of the opa (Opacity-associated protein) gene or insertion of a vp60 gene from rabbit haemorrhagic disease virus; and the E. coli strain with the aste (succinylglutamate desuccinylase) gene deletion with or without NgAgo system.