Utilizing The CRISPR-Cas9 System In Corynebacterium Glutamicum For Genome Editing

Corynebacterium glutamicum as a high-value model of microorganism,is widely used in large-scale production of amino acids,important organic acids and bioenergy.With the rapid development of metabolic engineering and the application of genome sequencing technology,the research of using genetic engineering to reconstruct C.glutamicum is becoming more and more attentional;Thus,gene editing technology as an effective genetic engineering tool,its development and application will also accelerate the progress of synthetic biology and genomics.The efficient gene editing technology,represented by CRISPR-Cas9,single-stranded recombination,can be used to modify the gene expression,regulate the metabolic flux,and ultimately regulate the overall metabolism by site-directed modification and genetic transformation of DNA sequences at the genomic level.The aim of this research is to develop genomic editing tools suitable for C.glutamicum.Based on the widely used CRISPR-Cas9 technique,we preliminarily explored the application of two cases about non-homologous end joining(NHEJ)repair system and single-chain recombination.The main results were divided into two parts.The first part was to explore the application of CRISPR-Cas9 assisted NHEJ(CA-NHEJ)with one-step deletion in C.glutamicum.First,the function of the CRISPR-Cas9 system was validated.The vector pECXK99E-Cas9-ffl was constructed with designating the target site on the plasmid.Then,the expression vector was transferred into wild-type C.glutamicum,the results verified that CRISPR-Cas9 can effectively play the fixed-point cutting function by indicating the expression of Cas9 protein.Next,the expression vector of NHEJ system pXMJ19-NHEJ was constructed to repair the double strand breaks(DSB)of the linearized plasmid.The experimental results showed that the system could not only repair the DSB effectively with gene deletion,but also found that there is an endogenous repair system in C.glutamicum that could secure the DSB of the exogenous linear fragment.In the case of verifying that both systems were effective in the genome,it was desirable to use CA-NHEJ for deletion of genomic fragments.Unfortunately,we did not get the ideal results,possibly due to insufficient expression of NHEJ or interference of endogenous fidelity repair system.However,the technology needs further exploration.The second part of this paper was to use the ability of DSBs generated by CRISPR-Cas9 to screen the mutant recombinants.According to the design concept of multiple automated genome engineering(MAGE),this study aims to use the synthetic ssDNA to regulate the expression of interfering genes in C.glutamicum.First,the vector pXMJ19-RecT,which expressed the single-stranded recombinant protein,was established.We selected a higher efficient recombinase by comparing the efficiency of them from different sources with strains.Then,we proved the operability of ssDNA recombineering by using the marker gene to verify the nucleotide homologous mutation experiments of multiple sites.Next,ssDNA with 75 nt was synthesized for the selected target site.At the same time,the spacer site was designed in the mutation site of ssDNA.And we successfully constructed the vector pECXK99ES-Cas9-32k.For the first step,inducing the expression of RecT protein,and then according to the co-transformation strategy of ssDNA and screening plasmid,we can screen the mutation after the completion of ssDNA recombination finally.After comparing with resistance in the plate and sequencing,the correct rate of recombination reached to 60%.At last,we optimized the design of the CRISPR-Cas9 system,so that the single guide RNA(sgRNA)site linked became more easily by the method of Gloden Gate Assembly.It is useful that making the screening work more efficiently and quickly,and improving the work efficiency as well as changing the sequence genomic gene.Based on the current research focus,using CRISPR-Cas9,ssDNA recombineering in C.glutamicum,we have successfully established an efficient,rapid,and recyclable genomic editing technique.Thereby,we provided a set of valuable genetic operation tools allows for rapid metabolic pathway optimization in the genome.Although the NHEJ system did not successfully mediate the DSB repair of the genome.It would have the desired effect with further exploration.In addition,we also found the endogenous fidelity repair system in the study,which provided a theoretical platform for the next work.