Development Of CRISPR-Cas9-mediated Genome-editing Vector In Brucella

Brucellosis is a zoonotic infectious diseases caused by brucella.The main manifestations of infected animals characterized by genital inflammation,abortion,infertility and multiple organs lesions.The infection of brucella hamper the animal husbandry greatly,and cause huge economic losses.Humans can also be infected by consuming infected animal products or direct contact with infected animals,which threaten human life and health greatly.Thus the prevention of brucellosis and the research of pathogenesis of burcella are extremely urgent.However,the existing brucellosis vaccines are not enough protective,and have the risk of virulence backing strong.Moreover,these vaccines will disturb the distinction between naturally infected animals and immuned animals.Furthermore,the study on pathogenesis of brucella are still to be improved,which restricts the prevention and control of brucellosis greatly.Studies have shown that brucella gene-deficient strains can solve some problems in the clinical prevention and control of brucellosis and the research of brucella pathogenesis.However,the traditional homologous recombination methods to prepare brucella gene-deficient strains still have some problems to be solved,such as not easy to operate,time-consuming and low success rate.CRISPR-Cas9-mediated gene editing system is an efficient and accurate gene editing system developed in recent years.The target gene sequence is identified and cleaved by a short RNA molecule(sg RNA)and Cas9 endonuclease,respectively.This system has a better specificity,efficiency and simplicity.Notablely,CRISPR-Cas9-mediated gene editing system has been widely used in eukaryotic cells and has achieved very significant results,which provides a good theoretical and practical basis for the using of CRISPR-Cas9 system in prokaryotic gene editing.From the above,this study intends to develop a CRISPR-Cas9-mediated gene editing vector for brucella.In order to solve the problem exist in gene complementation,and to ensure the correct construction of CRISPR-Cas9-mediated editing vector of brucella gene,this study constructed an expression vector for brucella base on the p BBR1MCS-5-EGFP.Then,a CRISPR-Cas9-mediated gene editing vector for brucella was constructed on the basis of the constructed expression vector.The results are as follows:(1)The promoter from the gentamicin resistance gene(Gm R)was inserted into the broad host cloning vector p BBR1MCS-5-EGFP between the Nsi I and Xba I restriction enzyme sites,which contains the Gm R promoter and a Flag-tag.Then,an appropriate multiple cloning sites(MCS)was added to the downstream of the Flag-tag using the Xba I and Sac I restriction enzyme sites.Eventually,the brucella expression vector p BBGm R-Flag-MCS was established,and Western blot analysis showed that the expression vector could express foreign genes in brucella stably.(2)The Cas9 gene was inserted into the p BBGm R-Flag-MCS between the Sal I and Eco R I restriction enzyme sites,and then the optimized g RNA scaffold was into the p BBGm R-Flag-MCS between the Kas I and Bsp H I restriction enzyme sites,consequently,the recombinant gene editing vector p BBGm R-sg RNA-Cas9 mediated by CRISPR-Cas9 was constructed.In this study,the expression vector p BBGm R-tag-MCS and CRISPR-Cas9-mediated gene editing vector p BBGm R-sg RNA-Cas9 for brucella were constructed.The expression vector constructed here simplifies the gene complement in the brucella gene-deficient strain.The CRISPR-Cas9-mediated editing vector is expected to greatly improve the efficiency and accuracy of the preparation of brucella gene-deficient strains.