The Application Of CRISPR/Cas9 Technology For AroA Gene Knockout In Escherichia Coli

As multiple species of whole-genomes have been analyzed by people, studies on genic functions will be of great importance. From the rise of gene-targeting techno-logy in the late 1980s, more and more gene-editing technologies have been created and applied to genic function studies. CRISPR/Cas9 knockout system is a recently developed manner for genetic manipulation, which enables accurate and efficient gene knockout or modification to better accomplish genes-associated functional studies.5-enolpyruvylshikimate-3-phosphate acid synthase (EPSPS) is encoded by aroA gene and plays a great role in aromatic amino acid metabolism of gram positive and negative bacteria and deletion of aroA gene obviously impairs virulence of bacteria. This study is aimed to construct CRISPR/Cas9-based knockout system for E. coli aroA gene and analyze knockout efficiencies in different E. colis. The established system will provide a novel and effective tool for functional studies on aroA genes of other pathogenic microorganisms and further development of attenuated vaccine.In the present study, based on the comparative analysis of aroA genes among several E. coli strains, aroA gene-targeting short guide RNA (sgRNA) was designed and constructed together with Donor sequence for homologous repair, then this resulting vector and pEwt/Cas9 vector co-constituted the CRISPR/Cas9 system. Preliminary application was respectively performed on E. coli DH10B, DH5a and JM109 strain through stepwise transformation and selection of double resistant positive strains. Knockout efficiencies of aroA gene were analyzed by PCR amplification, TA cloning and subsequent DNA sequencing. Finally, growth curves of the obtained gene-deleted strains were determined for metabolic analysis. The restriction enzyme digestion analysis and sequencing results showed that homologous repair vector was successfully constructed and double resistant positive strains were screened out and termed as QDH10B(1)、QDH5a(1) and QJM 109(1) strain, respectively. PCR results indicated that the established CRISPR/Cas9 system could be used for aroA gene knockout in multiple E. colis with efficiency ranging from 46% to 50%. Sequencing results further confirmed the successful and accurate knockout of aroA gene. Growth curves analysis showed that proliferation between normal and variant bacteria was indistinctive, especially in lag and stationary phases, indicating that artificial mutation of aroA gene did not have much effect on bacterial proliferation. This non-lethal mutation of bacteria would provide potential candidate strains in further development of genetically engineered vaccine.