Keywords: 
• CPRISPR /
• out membrane vesicles（OMVs） /
• Streptococcus agalactiae /
• elimination of pathogenic bacteria /
• plasmid transformation

Abstract: As bacterial unique immune system, clustered regularly interspaced short palindromic repeats（ CRISPR）/Cas9 is the most effective gene editing tool at present. Outer membrane vesicles（ OMVs） are the physiological structures of active secretion and absorption of bacteria,and have the ability to carry DNA,protein and some other substances. To investigate the possibility of OMVs transporting CRISPR/Cas9 and eliminating pathogenic bacteria,the plasmid pCas9 was reconstructed to develop shuttle plasmid pCas9-2. 0（ which can be replicated in E. coli and Streptococcus agalactiae or Group B Streptococcus,GBS）,and p Cas9-cfb( which is derived by inserting crRNA targeting cfb gene in GBS E0. The E. coli X6097（ Δasd） was used for harboring pCas9-2. 0 or pCas9-cfb. The OMVs of recombinant X6097 were extracted,purified and observed by electron microscope. The pCas9-2. 0 or pCas9-cfb was detected from OMVs by PCR. Recombinant X6097 and GBS E0 strains were mixed and co-cultured,and finally counted on BHI medium by using the colony count method. The results showed that the OMVs were secreted by the recombinant X6097 as a carrier for shipping the plasmid from the X6097 to the GBS E0. The secretion and transfer efficiency of OMVs were enhanced by prolonging co-culture time with low-level kanamycin. The GBS E0 was killed by absorbing pCas9-cfb. Compared with the traditional prophylactic vaccine,this study provides a new research strategy for the control of pathogenic bacteria in natural environment. Compared with the current phage delivery systems,this method is more convenient and lower in cost. In addition,this study developed a new plasmid transformation method for some strains that are hard to be electroporated or some labs without electroporation apparatus.