Study Of A Gene-Editing Technique-CRISPR-Cas9 In Brucella Melitensis

Brucellosis is a zoonotic disease caused by Brucella which is gram-negative bacteria.The pathogen can cause abortion and orchitis in animals.For humans,Brucella can cause heat waves,arthritis,loss of labor ability.It is really a great threat to human health and animal husbandry.There is no brucellosis vaccine for human,and most animal brucellosis vaccine were live attenuated.However,after immuning the susceptible herd with attenuated live brucellosis vaccine,it cann't be distinguished between natural infection and artificial immol/Lunity.At the same time,some vaccines still have virulence,causing abortion in pregnant animals.The mechanism of Brucella's intracellular survival and pathogenicity is still unknown.Brucella survives and reproduces in the host relying on its virulence genes.With the development of molecular biology,the gene deletion vaccine is expected to be a new type of safe and effective attenuated vaccine which is becoming a hot research area.At present,the development of the gene deletion vaccine produced by traditional homologous recombination technology is limited.In recent years,CRISPR-Cas9,a new efficient technique for gene editing with high accuracy and efficiency,has been widely used for gene editing both in plants and animals and partly in prokaryotes bacteria such as Escherichia coli and Agrobacterium tumefaciens.However,it has not been reported to be used for gene editing in Brucella.The aims of this study are to explore whether CRISPR-Cas9 can edit gene in Brucella,to provide a platform for the research of gene function in Brucella and to provide a convenient theoretical basis for the development of virulence gene editing in Brucella.In this study,firstly,the basic components such as pBBR1 replicon,pcat for CRISPR-Cas9 carried by the vector plasmid were verified that they can work in Brucella.Then,CRISPR-Cas9 systerm was constructed to the vector plasmid and tested to make sure that it can kill Brucella by causing DSBs.The donor sequence was cloned into the CRISPR-Cas9 vector because of the lack of NHEJ in Brucella.At the same time,the sacB gene was also introduced in order to overcome the problems such as identification interference,DNA pollution and biology safety caused by the introduction of the resistant gene into Brucella.However,there was no gene deletion mutant bacteria identified after plasmid elimination using 10% sucrose solution.The false positive gene deletion bacterias were called‘escapers'.1.pBBR1 replicon and GmR gene were cloned from plasmid pBBR1MCS-5.pcat,E.coli ColE1 replicon,rrnB T1 terminator were cloned from plasmid pZK001-szk001.lacI gene was cloned from plasmid pET28 a.Then the inducible shuttle vector plasmid was constructed.After that,the lacZ&gfP gene cloned from plasmid pXG-10 sf was introduced into the inducible shuttle vector plasmid to test wether the pcat can express in Brucella.2.CRISPR-Cas9 system was cloned from plasmid pZK35 and then introduced into the inducible shuttle vector plasmid.The sgRNA targeting hfq designed by biology software with Brucella melitensis 16 M acting as template was introduced into the gRNA scaffold present in the inducible shuttle vector plasmid.The plasmid expressing CRISPR-Cas9 with sgRNA targeting hfq and the plasmid expressing CRISPR-Cas9 with nonspecific sgRNA were transformed into Brucella competent cells respectively to verify whether CRISPR-Cas9 works in Brucella.The results of PCR and western blot show that CRISPR-Cas9 with sgRNA targeting hfq can lead most of the bacteria to death.3.The sacB gene and hfq donor sequence cloned from plasmid pK18 mobSacB and the genome of Brucella melitensis respectively were constructed into CRISPR-Cas9-sgRNA expressing plasmid to construct the CRISPR-Cas9 based knockout system for Brucella melitensis 16 M with Donor sequence to homologus repair.Then,the plasmid was introduced into Brucella competent cell by electric transformation with 1mmol/L IPTG to induce the expression of CRISPR-Cas9-sgRNA(targeting hfq)and then eliminating plasmid with 10% sucrose solution.There was no gene deletion bacteria identified by PCR and Sanger sequencing.According to the result,the survival clones were all ‘escapers'.At the same time,it was strange that the number of ‘escapers' rises significantly after CRISPR-Cas9 expressing plasmid was transformed with linear DNA fragment or spiral type plasmid DNA.In this case,anti-CRISPR-Cas9 system is presumed to exist in Brucella.