The Application Of CRISPR-dcas9 System In Myxobacteria To Inhibit Genes Expression

Myxobacteria is a kind of bacterium that have the most complex social movement in prokaryotes.It has complex cell behavior and a variety of signal regulation pathways,also produce a large number of secondary metabolites which have drug activity,but its generation is so long that cultivation and genetic manipulation are difficult.The function of genes and production of compounds can not be effectively explored and developed.A inactivated Cas9(dCas9)unable to break DNA is used to control gene expression,the dCas9-sgRNA complex represses transcription either by occluding RNA polymerase binding to promoter or by generation a steric block to transcription elongation.CRISPRa enhances transcription of target genes by using a modified dCas9-sgRNA complex containing activator domains to recruit RNA polymerase to promoter DNA.CRJISPR-cas9 has the advantages of no species restriction,gene control in various ways(such as knockout,insertion,inhibition,activation)and multi-gene can be controlled at the same time.Therefore,it is desirable to introduce this system into myxobacteria for gene function exploration.The cas9 gene of CRISPR-cas9 system was derived from Streptococcus pyogenes,it GC content was only 31%,while the average GC content of the DK1622 genome was 67%,the corresponding transgenic RNA of cas9 gene is low frequency.So the codon should be optimized,the GC content of cas9 gene was increased to 61%,and the frequency of codon corresponding to transgenic RNA was improved(mxdcas9).The nuclease site of mxcas9 gene was mutated by Red/et technique,and then uses the integrated plasmid to insert it into the bacterial genome.The sgRNA was expressed by using the pZJY41 plasmid,which was engineered from the PMF1 plasmid.When the constructed CRISPR-dcas9 system was used to inhibit the pilA and difA genes,the phenotype of the strain was significantly different from the wild type strain.When the 0049 gene was inhibited,no boundary was observed between the inhibited strain and the wild strain.CRISPR-dcas9,which is constructed in myxobacteria,inhibits the expression of target genes in some way,but doesn't completely silence genes.To improve the efficiency of the CRISPR-dcas9 system,the CRISPR-dcas9 system should be optimized.To improve the efficiency of the target gene repression,we assume that increase the concentration of sgRNA and dcas9 in the cells can improve efficiency,therefore turn their promoters into more strong promoters(pili as a strong promoter of sgRNA,only need to replace dcas9's promoter).The expression of pilA gene was not significantly decreased after replaced promoter was used to inhibit the pilA gene.After the use of sgRNA targeting different parts of the same gene,found closer to the end of the gene,the lower the inhibitory efficiency.In general,the CRISPR-dcas9 system constructed in myxobacteria reduced the expression of target genes about 2/3.Propionyl coenzyme A and Methylmalonyl Coenzyme A are the substrates of epothilone A and epothilone B,respectively,which maintain a low level in the cells?useing the CRISPR-dcas9 system to inhibit their other branches to improve the concentration of substrates.Propionyl coenzyme A is an important substrate for the synthesis of fatty acids.Useing CRISPR-dcas9 inhibits the translation initiation site of the fatty acid synthase gene,and the yield of epothilone A is higher than control strain,The total yield of the epothilone was reduced for the growth of the strain was significantly affected.The yield of epothilone A was significantly increased when repress the end of ORF of the fatty acid synthase gene by CRISPR-dcas9 which was almost the same as that of epothilone B(ezetimycin A:epothilone B ? 1:3 in ZE9 strain).Methylmalonyl Coenzyme A can be converted to other substances by Methylmalonyl Coenzyme-A mutase and methylmalonyl-CoA carboxytransferase.inhibiting the gene of Methylmalonyl Coenzyme-A mutase,the production of epothilone decreased;inhibiting the gene of methylmalonyl-CoA carboxytransferase,the production of epothilone decreased increased.The dcas9 protein fuses the ? or ?subunit of the RNA polymerase to activate the transcription of the target gene.When the system activates the epothilone gene cluster,the former increases the yield of epothilone,while the latter reduce total yield of epothilone.