Study On The Genomic Editing Of CRISPR/Cpf1 System In Corynebacterium Glutamicum ATCC 14067

Corynebacterium glutamicum(C.glutamicum)has great potential in the microbial fermentation industry.It can produce a variety of amino acids through metabolic engineering,and genome editing technology plays an important role in its metabolic engineering.In C.glutamicum,traditional genome editing techniques have the disadvantages of low recombination efficiency and long time,which is difficult to meet the needs of industrial strain transformation.In addition,due to genomic differences between different strains,the applicability of genome editing techniques is also limited.For example,the SacB reverse screening system is not suitable for C.glutamicum ATCC 14067,so further research on new genome editing techniques is needed.Based on this,this study used C.glutamicum ATCC 14067 as the research object,and initially established a CRISPR/Cpf1-based genome editing system.The application of this system in C.glutamicum was further optimized by the exploration of different PAM and editable loci ranges.It was then successfully applied to the simultaneous editing of multiple genes of C.glutamicum and the deletion of large DNA fragments.This technology effectively improved the genome modification system of C.glutamicum ATCC 14067 and laid a solid foundation for the transformation of its metabolic pathway.The specific research contents and results are as follows:(i)Establishment and optimization of CRISRP/Cpf1-based genome editing technology in C.glutamicum ATCC 14067.First,the genome editing technique based on CRISPR/Cpf1 system was established by testing the suitability of the CRISPR/Cpf1 system corresponding to the double plasmid(pJYS1Ptac,pJYS2_crtYf)in C.glutamicum ATCC 14067.Then the PAM(5'-TTTN-3',5'-NTTC-3',5'-NTTG-3')and the bias selection of ssDNA further improved the editing efficiency of the CRISPR/Cpf1 system to 92%-100%.Finally,the point of editing of the CRISPR/Cpf1 system in C.glutamicum was determined by point mutation at different sites within the range of 7 nt at the 5' end of the crRNA spacers and PAM.(ii)Simultaneous editing of multiple genes in the CRISPR/Cpf1 system in C.glutamicum ATCC 14067.The Cpf1 protein recognizes and cleaves DNA with only 42–44 bp of single-stranded RNA,which simplifies the experimental design steps and facilitates multi-gene editing.Based on this advantage,this study completed the simultaneous editing of three genes in C.glutamicum ATCC 14067 using a combination of Cpf1 and crRNA arrary.The efficiency of editing two genes was as high as 81%,while the efficiency of editing three genes was 3.69%.Simultaneous editing of the four sites on the crtB gene was performed in the C.glutamicum ATCC 14067 using the CRISPR/Cpf1 system,and the efficiency of editing four sites simultaneously was 21.80%.(iii)DNA fragment deletion application of CRISPR/Cpf1 system in C.glutamicum ATCC 14067.The CRISPR/Cpf1 combined with RecET recombination system was successfully applied to the double plasmid system(pJYS1-Cpf1-Ptac-RecET,pJYS3-crRNA-UD)for large DNA deletion,and the trace deletion of crtB gene was completed.The editing efficiency was as high as 79.63%.It indicated that the introduction of RecET recombination system can effectively improve the editing efficiency of CRISPR/Cpf1 system for gene deletion in C.glutamicum ATCC 14067.The deletion of large DNA fragments was efficiently performed by the CRISPR/Cpf1-RecET system,and the deletion efficiency of 20 kb was 27.78%.It is a simple and efficient tool for knockout of C.glutamicum.