CRISPR/Cas9 Establishment And Large-scale Deletion Of Non-essential Genes In Chromosome ?? And ?? Of Saccharomyces Cerevisiae

Saccharomyces cerevisiae is a widely spread single cell eukaryotes.With the deep research on the yeast genome,a growing number of gene function and genetic redundant were found.The existence of redundant sequences would consume more resources,causing the substance and energy waste in metabolic pathway.Removing the genetic redundant could make more metabolic flux distribute to target pathway to improve yield of the target products,which have important research significance to construct stable high yield industrial strain.Latour and CRISPR/Cas9 system were used to edit the S.cerevisae genome in this paper,the results were showed as below:1.Establishment of S.cerevisiae genome editing using CRISPR/Cas9 systemThe non-essential DNA fragments were determined by analyzing S.cerevisiae genome,of which the gRNAs were designed according to the two ends sequence.The gRNAs were ligated to the plasmid containing the cas9 gene,and the ligation were transformed into S.cerevisiae competent cell.After confirming the plasmid in the cell,the recombinant strains were cultured several days to screen the mutant.2.The auxotrophic S.cerevisiae haploid strains JM(mat?,?ura3)preserved in our laboratory were used as test strains,two mutants deleted single-fragment were obtained based on Latour system.Strain JM-X?a L,chromosomes X? 99 794-111 550 nt(about 11 kb)were deleted,strain JM-XV4,chromosomes XV 472 597-503 553 nt(about 31 kb)were deleted.The auxotrophic S.cerevisiae haploid strains JM-XV2-1(mat?,?ura3)preserved in our laboratory were used as test strains,two mutants deleted three-fragment and four-fragment respectively were obtained.Strain JM-XV2-1-3,chromosomes XV 142555-181 682 nt?202 696-226 611 nt and 371 685-411 459 nt(about 102 kb)were deleted,strain JM-XV2-1-3-4,chromosomes XV 142 555-181 682 nt?202 696-226 611 nt,371685-411 459 nt and 472 597-503 553 nt(about 133 kb)were deleted.3.The auxotrophic S.cerevisiae haploid strains JM(mat?,?ura3)were used as test strains,three mutants deleted single-fragment were obtained based on CRISPR/Cas9 system.Strain JM-X?a C,chromosomes X?99 794-111 550 nt(about 11 kb)were deleted,strain JM-Va,chromosomes V 25 635-42 593 nt(about 17 kb)were deleted.The auxotrophic S.cerevisiae haploid strains JM-X?1-2-3(mat?,?ura3)were used as tested strains,two four-fragment deletant were obtained respectively.Strain JM-X?-1-2-3-4a,chromosomes X? 298 868-327 482 nt?339 301-352 281 nt?706 921-722 613 nt and 99 794-111 550 nt were deleted(about 68 kb),strain JM-X?-1-2-3-V4 a,chromosomes X? 298 868-327 482nt?339 301-352 281 nt?706 921-722 613 nt and V 25 635-42 593 nt(about 74 kb)were deleted.It was showed that CRISPR/Cas9 system can be used to multi-area deletion of large fragments in yeast.4.Cas9/sgRNA plasmid used in S.cerevisiae diploid,named pCRCTG418,was constructed using the seamless cloning technology.The S.cerevisiae diploid strains W303 were used as test strains,the gRNA and donor DNA sequence were designed according to ade2 gene sequence,and 7 bp deletion mutant was obtained.The mutant showed adenine auxotroph and was unable to grow in the absence of adenine medium.It was showed that pCRCTG418 plasmid can be applied to genome editing in S.cerevisiae diploid.