Optimization Of Multiplex Base Editing In Corynebacterium Glutamicum

As a new type of genome editing tool,the base editing technology combines the positioning function of the CRISPR/Cas system and the editing function of base deaminase,which can realize the base mutation at a specific site,and has no double-stranded DNA breaks and no need for external The source template does not rely on the advantages of homologous recombination of chromosomal DNA.At present,researchers have developed a variety of base editing tools in the important industrial production strain Corynebacterium glutamicum,and realized simultaneous editing of two genes and three genes.In this study,aiming at the shortcomings of the CRISPR/Cas9-based multi-site base editing system in Corynebacterium glutamicum(multiple sg RNA structure is cumbersome,repetitive sequence interference,difficulty in replacing targets,etc.),a variety of strategies are used to optimize,and Compare base editing efficiency:1.Optimize multiple sg RNA expression cassettes based on individual promoters/terminators.By constructing framework plasmids and combining the Golden Gate connection method,target replacement is accelerated and repeated sequence interference is avoided.Although this method is cumbersome in sg RNA structure,it has the highest editing efficiency.Compared with previous reports,after changing the culture medium to a more nutritious LBHIS medium,the simultaneous inactivation efficiency of both upp and rfp genes was as high as(93.05±4.81)%;the simultaneous inactivation efficiency of upp,rfp and ald genes was(69.4±10.49)%.In addition to the three targets of upp,rfp and ald,this study chose cit B and Cgl0121 as the fourth target to edit the four genes simultaneously.The results showed that the efficiency of simultaneous inactivation of the four genes upp,rfp,ald and cit B was(50.00±11.02)%,while the efficiency of simultaneous inactivation of the four genes upp,rfp,ald and Cgl0121 was(16.67±11.03)%.The main reason for the large difference between the two situations is that the cit B and Cgl0121 genes differ in their individual inactivation efficiency.It is inferred that the individual editing efficiency of different genes has a greater impact on the multi-gene editing efficiency.2.Develop an expression cassette based on the Type II CRISPR cr RNA array.This format requires only one promoter and one terminator sequence,and the length of the co-repetitive sequence and the spacer sequence in the cr RNA array are shorter.This strategy is applied to multiple sites When editing,the PCR process of the target sequence will be omitted,which not only simplifies the plasmid construction process,but also saves costs.However,compared with the single promoter/terminator sg RNA expression cassette,the inactivation efficiency of this strategy is significantly reduced.The simultaneous inactivation efficiency of upp and rfp is only(23.20±9.20)%,and the upp,rfp and ald genes are lost at the same time.The living efficiency is only(4.20±0.00)%.3.Construct a multiple sg RNA expression cassette based on t RNA^Glyprocessing to further optimize the multiple sg RNA structure.This strategy combines the endogenous t RNA^Glyprocessing system in the bacterial cell with multiple sg RNA structures,using a promoter and terminator to transcribe an RNA sequence,inserting t RNA^Glysequences between sg RNAs containing different targets,and endogenous Sexual RNase will specifically recognize the t RNA^Glysequence and cut it to release different sg RNAs.This method does not require the introduction of foreign proteins,and the t RNA^Glysequence is small(about 70 bp),which not only simplifies the plasmid structure,but also realizes the rapid and efficient construction of the plasmid.Compared with the single promoter/terminator sg RNA expression cassette,the inactivation efficiency of upp and rfp genes is similar,as high as(91.67±4.15)%,while the efficiency of simultaneous inactivation of upp,rfp and ald genes has decreased,Is(33.33±7.22)%.4.Construct multiple sg RNA expression cassettes based on Csy4 processing.This form is similar to the multiple sg RNA expression cassettes processed by t RNA^Gly.Both use a promoter and terminator to form an RNA sequence.The difference is that the strategy is at different targets.Csy4 protein cleavage sequence is inserted between sg RNAs,and different sg RNAs are released after cutting.The inactivation efficiency of this strategy is compared with the single promoter/terminator sg RNA expression cassette.The inactivation efficiency of the upp and rfp genes is slightly reduced,which is(76.39±8.67)%,and the upp,rfp and ald genes are lost.The activation efficiency decreased more,and the deactivation efficiency was only(6.94±6.36)%.This research has provided altemative genome editing tools for Corynebacterium glutamicum and enabled rapid genetic engineering of this strain.