Gene Editing In The Corynebacterium Glutamicum Genome Based On The Subtype I-B-Svi CRISPR-Cas System

Corynebacterium glutamicum is a kind of The important industrial microorganisms without the CRISPR-Cas system that produce amino acids and many useful metabolites during fermentation,such as glutamic acid,butadiene,etc.,which have a very important position in industrial production.With the rapid development of genetic engineering and gene editing technology,many researchers try to artificially control the growth and metabolism of microorganisms at the genetic level to maximize the benefits.However,due to the biocompatibility problem between Corynebacterium glutamicum and Cas9(the strain expressing Cas9 cannot grow),the current CRISPR/Cas9 system cannot be directly applied to the gene editing in Corynebacterium glutamicum.Genome editing technology(referred to as gene editing technology)is the same as genetic engineering,which is said to change the genetic characteristics of cells,but the difference lies in the fact that gene editing technology directly targets the chromosomes of cells,so that its function is far greater than traditional genetic engineering relying on vectors.CRISPR-Cas is an adaptive immune system in the prokaryotic domain.It is a front-end genetic editing technology that can delete,insert,and replace genes in prokaryotic and eukaryotic genomes with a distinct,rapid,and effective advantage.The methods reported in the literature for gene editing mainly include Type 2 Type ?(CRISPR/Cas9)and Type V(Cpfl/Cas12a),especially the Type II CRISPR/Cas9 system in Streptococcus pyogenes.But the system also has its limitations,such as off-target and biocompatibility.The Type I CRISPR-Cas system is the most common type in nature.Cas3 in this system usually degrades DNA rather than directly produces double-strand breaks.This may be why the Type I CRISPR-Cas system is the most natural in nature.Common reasons.However,to date,except for the laboratory,gene editing tools derived from the Type I CRISPR-Cas system have not been reported.The laboratory isolated an actinomycete that can grow with a steroidal compound as the sole carbon source from the activated sludge of a chemical pharmaceutical factory.We named it:Streptomyces virginiae IBL14,The whole genome sequencing analysis of the strain revealed that there is an IB-Svi type CRISPR-Cas system on the genome.Fortunately,the Cascade and single SviCas3 based on this system have already implemented gene editing in the strain itself,E.coli and Saccharomyces cerevisiae.The purpose of this paper is to study the biocompatibility between SviCas3 and Corynebacterium glutamicum,and to develop a gene editing tool based on this strain.In the experiment,we constructed two protein expression plasmids(pEC-XK99E-cas753;the initiation codon in pEC-XK99E-cas3 is ATG)and three gene editing plasmids(pXMJ19-t/g-?ldh;pXMJ19-t/g-?ldh::egfp;pXMJ19-t/g-?ldh::cat),and the protein expression plasmid and the gene editing plasmid were separately electroporated into the electroporation of Corynebacterium glutamicum by two-step electrotransformation.The results showed that:(1)The protein expression plasmid pEC-XK99E-cas753,which is directly derived from the gene cas7,cas5 and cas3 of the Streptomyces elegans IBL14 chromosome,can be successfully used to the genome of Corynebacterium glutamicum(the internal lactate dehydrogenase gene knocked out 615 bp).(2)The protein expression plasmid pEC-XK99E-cas3 binding gene editing plasmid containing only a single cas3 gene directly derived from the S.cerevisiae IBL14 chromosome can also successfully perform genome editing on Corynebacterium glutamicum(successful The egfp and cat genes are inserted into the lactate dehydrogenase).(3)No off-target events were found in off-target analysis studies.(4)The transformant of Corynebacterium glutamicum was not obtained in the gene editing based on Cas9 and was used as a control experiment.In summary:This paper(1)demonstrates for the first time that an editing tool developed directly from the SviCas7-5-3 gene in the I-B-Svi-type CRISPR-Cas system can perform genome editing of the strain,indicating the base preference of the gene in the prokaryotic microorganism requirements were not strict;(2)It was first confirmed that a single unoptimized SviCas3 could edit the genome of Corynebacterium glutamicum,indicating that SviCas3 is more biocompatible than SpCas9 in Corynebacterium glutamicum;(3)No off-target phenomenon was found in the potential target sites by sequencing analysis,and the research viewpoint that the published Cas3 did not participate in the target sequence localization was hidden,and important application prospects were hidden.