Extension Of CRISPR-Cas9 Base Editing Tools In Corynebacterium Glutamicum

In recent years,the base editing technology based on CRISPR/Cas9 has been gradually developed into a powerful genome editing tool,which has been widely applied in animals,plants,yeasts and bacteria because of its advantages of no DNA double strand break,no need of foreign DNA template and independence on the homologous recombination and repair of the host.It combines the targeting function of CRISPR/Cas system with the editing function of base deaminase,and can achieve base substitution at specific location.Previously,our research team has developed a multiplex automatic base editing technology(MACBETH)in Corynebacterium glutamicum,an important industrial model strain,to achieve C to T conversion on multiple genome loci simultaneously.However,the genome targeting range,editing window size and base conversion ability of this method are limited,which restrict its application in the basic research and industrial application of C.glutamicum.Therefore,it is of great significance to develop and optimize the base editing tools in C.glutamicum.In this study,we first expanded the base editing tools in C.glutamicum on the following three aspects:Introduce four Cas9 mutants that recognize different PAM sequences,and evaluate their base editing efficiencies,editing window and editing scope of the base editing tools that combine with each mutant under their own recognizable PAM sites.After comprehensive evaluation,we prove that our base editing tools can expand the limitation of PAM from NGG to NG.The number of possible target sites in gene inactivation in C.glutamicum genome increased 3.9 times.The original 5 bp editing window was extended to 7 bp by using the truncated or extended gRNA sequence.The adenine base editing tool of C.glutamicum was constructed to expand the types of base transformation.By introducing the adenosine deaminase mutant,the transformation of A to G in C.glutamicum was realized.The highest editing efficiency was 65.9%.Then,in order to further improve the editing efficiency of the base editing tool in C.glutamicum,the culture conditions,induction conditions,editing time and other factors in the editing process were optimized.Compared with the initial conditions,the editing efficiency after optimization was increased by 1.3 times.Finally,in order to prove the important application potential of the extended and optimized base editing tools in the metabolic engineering of strains,this study performed a T311I amino acid mutation of lysC gene through base editing,alleviated the feedback inhibition of aspartate kinase,and improved the production of lysine.In my research,we effectively expanded and optimized the base editing tools in C.glutamicum,provided a strong technical support for the metabolic engineering of C.glutamicum,and will contribute to the basic research and industrial application of C.glutamicum.