Research On Improving The Editing Efficiency Of CRISPR System In Saccharomyces Cerevisiae

CRISPR/Cas9 is an adaptive immune defense developed by bacteria and archaea during long-term evolution and can be used to fight invading viruses and foreign DNA.Since 2012,CRISPR/Cas9 has been transformed into a gene editing tool,and a series of efficient and convenient gene editing tools have been derived,which are rapidly used in basic research,genetic diagnosis and clinical treatment research fields.However,there are still some urgent problems to solve in CRISPR/Cas9,such as low editing efficiency,cytotoxicity and off-target,which limits the application of CRISPR/Cas9 to a certain extent.Compared with the CRISPR/Cas9 system,the molecular weight of a new generation of genome editing system CRISPR/Cpf1 needs only one cr RNA and Cpf1 enzyme is smaller than the standard Cas9 recognition,Cpf1 complex recognize PAM 5'T-rich sequence,the cutting site is far from the recognition site and generate cohesive ends,let gene insertion more manageable.Taking Saccharomyces cerevisiae as the research object,we explored the technical system to improve the efficiency of CRISPR editing system in Saccharomyces cerevisiae.and a technological platform for CRISPR/Cpf1 editing system of Saccharomyces cerevisiae has been set up.The main results are as follows:1.The effect of the target gene expression on the efficiency of the CRISPR/Cas9 editing system was explored.According to the characteristics of the codon usage of Saccharomyces cerevisiae,the Cas9 gene was optimized,and sg RNA and its corresponding components were rationally designed.Li AC method was used to transform yeast cells under different culture conditions.(1)The effect of the expression of the target gene on the efficiency of the CRISPR/Cas9 editing system was explored.With hygromycin gene(Hyg)as the object of study,we investigated whether the target gene's expression on plasmid influences the editing efficiency of CRISPR/Cas9 system: The Hyg gene and Hyg gene with terminators were connected to the expression plasmid p ESC-His respectively.The g RNA component of Hyg gene was connected with p CRCTG418 plasmid to get recombinant plasmid.Using these two recombinant plasmids to co-transform Saccharomyces cerevisiae and the results show that the CRISPR system has edited the genes expressed on plasmid,but has not edited the genes that are not expressed.Taking Saccharomyces cerevisiae Ado1 gene as the research object,we explored whether the target gene was expressed on chromosome or not on the efficiency of CRISPR/Cas9 editing system: Recombination of Ado1 with terminator on chromosome,and PCRCTG418 plasmid with Ado1 gene g RNA component was transformed into original strain and Ado1 mutant.The results show that the CRISPR system has edited the genes expressed on chromosomes,but has not edited the genes that are not expressed.(2)The effect of the expression level of the target gene on the efficiency of the CRISPR/Cas9 editing system was explored.Based on SAM1 and SAM2 gene as research objects,the g RNAs components for the target genes were designed and connected to the p CRCTG418 plasmid.By controlling the composition of the medium to control the expression level of SAM1 and SAM2,the recombinant plasmid was used to edit the Saccharomyces cerevisiae under different culture conditions.The results showed that increasing the expression level of target gene could improve the efficiency of CRISPR/Cas9 editing system.2.The effect of VPR on the efficiency of CRISPR/Cas9 editing system is explored.We use the three party activating factor VP64-p65-Rta(VPR)that can specifically improve the expression of the target gene to the C-terminal of the Cas9 protein.p CRCTG418 plasmid and p CRCTG418-VPR plasmid were used to edit the Gal7 gene of Saccharomyces cerevisiae,and the editing efficiency of the two were compared.The results showed that the Gal7 gene was edited in the CRISPR system during the fourth generation,and the CRISPR system fused with VPR was edited for Gal7 gene in the second generation.It is proved that VPR can improve the efficiency of CRISPR/Cas9 system.It provides a new way of thinking for the better application of the system in the treatment of genetic diseases and other diseases.3.The CRISPR/Cpf1 editing system suitable for Saccharomyces cerevisiae was established.The Cas9 protein of p CRCTG418 plasmid was replaced by Cpf1 protein and the DR sequence was replaced.A CRISPR/Cpf1 editing system p CRCTG418-Cpf1 suitable for Saccharomyces cerevisiae was obtained.The ADH7 gene of Saccharomyces cerevisiae was edited by p CRCTG418 plasmid and p CRCTG418-Cpf1 plasmid respectively,and the editing efficiency of the two was compared.The results showed that the p CRCTG418-Cpf1 plasmid was suitable for Saccharomyces cerevisiae,and its editing efficiency was equivalent to that of p CRCTG418 plasmid.The establishment of this system broadens the scope of gene editing in the CRISPR system in Saccharomyces cerevisiae.