Construction Of Corynebacterium Glutamicum Gene Inactivation Library And Screening Of Functional Genes Based On Base Editing Technology

Corynebacterium glutamicum is an important industrial workhorse used for the production of many valuable compounds.However,more than 40% of its genes are still have unknown functions or lack of experimental evidence,which limits the artificial design or engineering of Corynebacterium glutamicum.Through the construction and screening of genome-scale perturbation libraries,elements with new biological functions can be discovered,and the correspondence between genotypes and phenotypes can be systematically studied.Until now,there is no comprehensive and efficient method for constructing a full-genome-scale perturbation library of Corynebacterium glutamicum.Recently,base editing and multiple genome editing technology has been developed and applied in a variety of microorganisms due to its simplicity,high efficiency,and rapidity,as well as the advantages of no double-stranded DNA breaks,no need for foreign templates,and no dependence on homologous recombination,providing new tools for the construction and screening of genome-scale perturbation libraries.Therefore,in this study,we first optimized the CRISPR/Cas9-based multiplex base editing system in Corynebacterium glutamicum,established a multiple g RNA expression cassette based on t RNA processing,which enriched the genome editing tools of Corynebacterium glutamicum.Then calculated,selected,and synthesized sg RNAs for inactivating genes in the whole genome,constructing a whole genome-scale single gene inactivation library,and the library was screened for functional genes.The specific research content and results are as follows:(1)Optimization of CRISPR/Cas9-based multiplex base system in Corynebacterium glutamicumDeveloped a multiple g RNA expression cassettes based on cell endogenous t RNA processing.This form requires only one promoter and one terminator sequence,which greatly simplifies the structure of the expression cassette and enables simultaneous editing of two genes or three genes.Among them,the efficiency of simultaneous editing of two genes was(91.67±4.15)%,and the efficiency of simultaneous editing of three genes was(33.33±7.22)%.(2)Construction and screening of C.glutamicum genome-scale single-gene inactivation libraryFirst,we choose sp Cas9 and sp Cas9-VQR base editors and calculated,selected and synthesized 11647 sg RNAs in the target C.glutamicum genome,with a genome-wide gene coverage as high as 98%(3041/3099).After that,the sg RNA plasmid library and the genome-scale single gene inactivation library were constructed respectively.We then calculated the sg RNA and gene coverage,and the removal efficiency of Cas9-AID protein expression plasmid.The gene editing ratio of the library were also evaluated during the construction of the library.Finally,the genome-scale single gene inactivation library was used for screening under the culture conditions of adding 5-FU or 5-FOA,and the enriched genes were individually inactivated to verify the phenotype-related functional genes.