Research On CRISPR/Cas9 Technology In Pichia Pastoris And Bacillus Subtilis

CRISPR(Clustered Regularly Interspersed Short Palindromic Repeats)/Cas is a genetic system which is used by bacteria to resist virus and gives rise to immune response.The working principle of this system is crRNA(CRISPR-derived RNA)pairing with tracrRNA(trans-activating RNA)to form the tracrRNA/crRNA complex,which recruits an nuclease(Cas9)to cleave genome DNA in the position where crRNA complemented with,resulting a DSB(double stranded break).By artificial designation,these two RNA can be replaced by a sgRNA(single guide RNA).Once the damage occurred,the DNA repair mechanisms are adopted in the cell.CRISPR-Cas9 technology is another effective gene-knockout method with high efficiency,after the zinc finger nuclease(ZFN)and transcription activator-like effector nuclease(TALEN)technologies.In addition CRISPR/Cas9 system is economic and easy to be designed.In terms of genetic manipulation CRISPR/Cas9 system is an extremely flexible tool that mutated Cas9 protein-dCas9(D10A and H840A)retained the ability to bind to DNA while lose the ability to cut the target DNA.It has also been used to down regulate gene expression.1.Research on CRISPR/Cas9 technology in Pichia pastorisPichia pastoris is one of the most widely used host for foreign protein expression,but the genetic improvement of strains are barely reported,the reason probably is that comparing with the brewing yeast(Saccharomyces cerevisiae),homologous recombination efficiency in Pichia pastoris is much lower,which for the engineering strain is extremely unfavorable.Therefore developed a new method for genome modification and transformation is more and more arouse people's interest.This study intends to build CRISPR/Cas system in Pichia pastoris.First we cloned the cas9 gene(from streptococcus pyogenes,two NLSs were added at both ends)into the vector pPICZA,and obtained pPICZA-SpCas9,then we transformed the plasimid into GS115,induced with methanol,we detected Cas9 expression through SDS-PAGE,we designed the target gene sequences in HIS4 and artificial synthesis the sequence,we put these sequences under the control of RNA polymerase ? promoter SNR52,then we insert the sgRNA expression unit into plasmid pPICZA-SpCas9 at Bam HI site,we named the plasmid pPICZA-SpCas9HIS1-2.Last we analyzed the Cas9 cutting efficiency,we chose 3,6,9 hours three different induction time,choose 10 different transformation respectively for targeted HIS4 gene sequencing analysis,the sequencing results show that the induced in only six hours of samples,a point mutation,the rest of the samples had not been changed.2.Research on CRISPRi technology in Bacillus subtilisBacillus subtilis are GARS industrial strains,which is widely used in biological medicine and food industry,such as the strain itself can produce riboflavin,purine nucleotides,etc.but these metabolites often must have a lot of genes involved in,Using the method of gene knockout metabolic pathways tend to cause growth defects and other problems.CRISPRi(CRISPR interference)technology provides a new method of metabolic engineering.This work intends to establish a bacillus subtilis CRISPRi system.DCas9 is obtained by PCR amplification(D10A and H840A)gene,the clone to bacillus subtilis expression vector pHT01,obtained the plasmid pHT01-dCas9.at the same time we put sgRNA under the control of p43 promoter constructed a self-replication plasmid pBE980B-sgRNA,we transformed the plasmid pHT01-dCas9 into bacillus subtilis,after IPTG induction,we detected Cas9 expression through SDS-PAGE.We designed the target of an exogenous gene GFP,we constructed pBE980B-sgRNAGFPgPT1-3?pBE980B-sgRNAgT1-3?pBE980B-sgRNAgNT 1-3.We put these plasmids co-transformation into Bacillus subtilis,with fluorescence enzyme standard instrument analysis showed in targeted promoter GFP promoter regions fluorescence value compared with the control level has fallen by 70.25%,63.14%and 34.41%,using the qRT-PCR detection mRNA expression level detection,transcription results compared with control group the three sites fell 53%,90%and 67%.In targeted GFP ORF regions targeted results showed that the best template strand of 52.42%,found a 86%drop in mRNA level detection.The next work targeting endogenous genes in B.subtilis Amylase,same design promoter region and ORF region.Designed the endogenous gene AMY sgRNA sequence,pBE980B-sgRNAapT1-3?pBE980B-sgRNAaT1-3?pBE980B-sgRNAaNT1-3.by measuring the activity of amylase compared with control group,the targeting effect of promoter regions can inhibit the enzyme activity of 42.62%,and in the targeting of amylase ORF region inhibition effect in 19.6%?62.3%,the mRNA level inhibition in 21%?84%.