32 results about "Yeast genome" patented technology

The invention belongs to the field of biosynthesis, and in particular relates to LBD (Levandanenediol Pyrophosphate) synthase and engineered yeast for producing sclareol. In order to solve the problem that in the prior art, the sclareol biosynthesis yield is generally low, LSLPPs mutant containing at least one mutation of D730L, D374L, N674L or G379M is obtained by performing mutation and optimization on LSLPPs pyrophosphate synthase, and the mutant is used for sclareol synthesis. Meanwhile, in order to solve the problem of genetic instability caused by free plasmid expression of part of saccharomyces cerevisiae strains, a sclareol synthesis pathway is constructed in a yeast genome, and meanwhile, an acetyl coenzyme A synthesis pathway, an MVA pathway and a sclareol synthesis pathway are enhanced; chassis bacteria for synthesizing sclareol are modified in a manner of knocking out part of transcription inhibition factors, and the yield of a 5L fermentation tank reaches 26.11 g / L and is greatly improved compared with the prior art.

The invention belongs to the field of biosynthesis, and particularly relates to sclareol synthase and engineered yeast for producing sclareol. In order to solve the problem that in the prior art, sclareol biosynthesis yield is generally low, sclareol synthase is mutated and optimized, an SsScs mutant containing at least one mutation of Y491F, G307L or N269F is obtained, and the mutant is used for sclareol synthesis. Meanwhile, in order to solve the problem of genetic instability caused by free plasmid expression of part of saccharomyces cerevisiae strains, a sclareol synthesis pathway is constructed in a yeast genome, and meanwhile, an acetyl coenzyme A synthesis pathway, an MVA pathway and a sclareol synthesis pathway are enhanced; chassis bacteria for synthesizing sclareol are modified in a manner of knocking out part of transcription inhibition factors, and the yield of a 5L fermentation tank reaches 21.13 g / L and is greatly improved compared with the prior art.

The invention discloses a yeast engineering bacterium for producing apigenin and application thereof. The yeast engineering bacterium comprises a yeast genome, a whole-cell catalytic synthesis apigenin module, a heterologous apigenin biosynthesis path and an NADPH supply module, the whole-cell catalytic synthesis apigenin module comprises an FNSI coding gene and an FNSII coding gene; the heterologous apigenin biosynthesis path comprises a TAL coding gene, a 4CL coding gene, a CHS coding gene, a CHI coding gene and an FNSII coding gene; the NADPH supply module comprises a G6PD coding gene and a PGD coding gene; the method disclosed by the invention has the characteristics of greenness and safety, and lays a foundation for synthesizing apigenin in a yeast cell factory.

The invention belongs to the technical field of bioengineering, and particularly relates to kluyveromyces marxianus engineering bacteria for synthesizing eicosapentaenoic acid and application of the kluyveromyces marxianus engineering bacteria. According to the engineering bacterium, pyruvate decarboxylase PDC1 genes, alcohol dehydrogenase ADH1 genes and alcohol dehydrogenase ADH2 genes are sequentially knocked out from a kluyveromyces marxianus FIM1 delta ura3 strain; meanwhile, a citrate lyase ACL gene, a pyruvate oxidase PO gene and a phosphate transacetylase PTA gene are inserted into the loci respectively, so that synthesis of acetyl coenzyme A is enhanced; then a plurality of genes are sequentially inserted into a Kluyveromyces marxianus genome, and then a vector of a delta 8 desaturase delta 8D gene is transferred into the Kluyveromyces marxianus genome. The invention also provides a method for efficiently preparing the EPA, namely the Kluyveromyces marxianus engineering strain is used as a fermentation strain, glucose, a yeast extract, peptone and other components are used as a culture medium, and the yield of the EPA produced by culturing and fermenting reaches 8.71 mg / g of dry cell weight.

The invention discloses pichia pastoris for efficiently synthesizing lactoferrin as well as a construction method and application of the pichia pastoris, and belongs to the technical field of biosynthesis. The pichia pastoris for efficiently synthesizing the lactoferrin expresses at least one of KAR2, PDI1 and ERO1 in molecular chaperones in a host in an enhanced manner and carries an optimized secretory signal peptide, and the secretory signal peptide is obtained by replacing a leading peptide of alpha-factor with any signal peptide in LacZ, PelB, FLO and alpha-factor. The yield of LF can be effectively increased by enhancing expression of at least one of KAR2, PDI1 and ERO1 molecular chaperones in molecular chaperones in a host; when the lactoferrin expression cassette is integrated into the recombinant pichia pastoris X-33 genome and the copy number is increased to 2-6, the LF yield can be further increased, and when the copy number is 4, the LF yield reaches a peak value.

The invention discloses an adenosine deaminase mutant. The adenosine deaminase mutant is a C141R mutant and an L144P mutant based on a wild type. The invention also discloses a pichia pastoris base editor containing the adenosine deaminase mutant, the pichia pastoris base editor can realize accurate editing of specific base sites of a pichia pastoris genome, and a gene editing tool is provided for basic and application research based on pichia pastoris.

The present invention discloses an adenosine deaminase mutant, which is a C141R and L144P mutant based on the wild type. The present invention also discloses a Pichia pastoris base editor containing the adenosine deaminase mutant. The Pichia pastoris base editor can achieve precise editing of specific base sites in the Pichia pastoris genome, providing a gene editing tool for basic and applied research based on Pichia pastoris.

The invention relates to the technical field of bioengineering, and discloses a yeast engineering bacterium for producing squalene and application thereof, the yeast engineering bacterium comprises yeast chassis cells, a heterologous squalene biosynthesis module, an NADPH supply module, a precursor supply module and a grease body engineering module, the specific technological process comprises the following steps: integrating a key enzyme coding gene in a heterologous squalene biosynthesis module into a yeast chassis cell to obtain a yeast genome containing a squalene biosynthesis pathway; integrating the NADPH supply module, the precursor supply module and the grease body engineering module into a yeast genome to obtain the yeast engineering bacteria for producing squalene; and fermenting the yeast engineering bacteria by taking a YYG culture medium as a substrate to obtain squalene. The yeast engineering bacterium constructs and optimizes a biosynthesis integration module required by squalene, realizes adaptation of a squalene biosynthesis pathway and yeast chassis cells, can efficiently and stably produce squalene, and also has the characteristics of greenness and safety.

Described herein are recombinant Kluyveromyces lactis (K. lactis) yeasts which are capable of the highly efficient expression of one or more foreign proteins and are suitable for use as a vaccine for generating a protective immune response against pathogens. The invention provides in particular K. lactis strains for the targeted cloning of foreign antigen-coding nucleic acids into the yeast genome of the K. lactis strain, which is characterized in that the K. lactis strain has integrated expression cassettes for foreign antigens as an alternative or in addition to the KILAC4 locus on the KIURA3-20 locus (KLLA0E22771g) and / or on the KIMET5-1 locus (KLLA0B03938g). The invention further relates to integrative expression vectors and to methods for producing the K. lactis strains of the invention as well as to the use thereof as vaccines.

The present invention discloses flavonoid 4'-O-methyltransferase and its application in the production of hesperetin, belonging to the field of genetic engineering and bioengineering technology. The present invention screened and identified new flavonoid 4'-O-methyltransferases derived from tea-branch orange and Huazhou pomelo; the flavonoid 4'-O-methyltransferases CrcOMT-2 and CgtOMT-3 obtained by screening were integrated and expressed in the multi-copy site rDNA and ZETA site on the genome of Yarrowia lipolytica YE26, respectively, to achieve the de novo synthesis of hesperetin. The Yarrowia lipolytica Z02 constructed by the present invention achieved the de novo synthesis of 130.2 mg / L hesperetin in a 5L fermentor, which is also the highest yield of hesperetin de novo synthesis in microorganisms reported, laying the foundation for the subsequent biosynthesis of flavonoids.

The invention discloses a recombinant yeast engineering bacterium for secretory expression of recombinant bovine lactoferrin as well as a construction method and application of the recombinant yeast engineering bacterium, and belongs to the technical field of genetic engineering. According to the invention, a CRISPR / CAS9 gene editing technology is utilized to construct sgRNA expression vectors and bovine lactoferrin homologous recombination gene expression cassettes targeting multiple sites of yeast genomes AOX1, AOX2, FLD1, DAS1 and DAS2, and the sgRNA expression vectors and the bovine lactoferrin homologous recombination gene expression cassettes are transformed into yeast host bacteria to obtain recombinant yeast engineering bacteria with bovine lactoferrin genes inserted at multiple sites. The yield of the bovine lactoferrin of the recombinant yeast engineering bacterium is remarkably increased, heterologous secretion and efficient expression of the bovine lactoferrin in pichia pastoris are achieved, industrial production and construction of the bovine lactoferrin are promoted, and the recombinant yeast engineering bacterium has wide application value.

The application discloses a Yarrowia lipolytica strain for synthesizing (2S)-naringenin and application thereof, and belongs to the field of genetic engineering and bioengineering technology. The application improves the yield of (2S)-naringenin by 60% through semi-rational modification of a key enzyme CHS, and further integrates the modified CHS into the yeast genome. In addition, the application promotes the conversion of coumaric acid to (2S)-naringenin by strengthening the shikimic acid pathway and enhancing the supply of auxiliary factors, so that the yield of (2S)-naringenin reaches 776 mg / L after 96 hours of fermentation of the constructed strain in a 24-hole plate, and the yield reaches 8.65 g / L in a 5L fermenter. The application lays a foundation for subsequent biosynthesis of flavonoids, and has potential value and significance for the development of synthetic biology.

This invention belongs to the field of biosynthesis, specifically relating to a variant of lysine pyrophosphate diol synthase and the constructed engineered yeast strain for perillaldehyde synthesis. To address the generally low yield of perillaldehyde biosynthesis in existing technologies, this invention mutates and optimizes the lysine pyrophosphate diol synthase to obtain an SsLPPs mutant containing the G379M mutation, which is then used for perillaldehyde synthesis. Simultaneously, to address the genetic instability issues associated with free plasmid expression in some Saccharomyces cerevisiae strains, this invention constructs a perillaldehyde synthesis pathway within the yeast genome. Furthermore, by enhancing the acetyl-CoA synthesis pathway, the MVA pathway, and the perillaldehyde synthesis pathway, and by knocking out some transcriptional repressors, the substrate strain synthesizing perillaldehyde is modified, resulting in a significantly increased yield compared to existing technologies.

The application discloses a kind of production cordycepin yeast engineering bacteria and application.The yeast engineering bacteria is inserted in the genome of yeast cordycepin biosynthesis module, and purine metabolic pathway overexpression module or ATP and precursor supply module;The cordycepin biosynthesis module is composed of Cns1 gene and Cns2 gene;The purine metabolic pathway overexpression module is composed of ADE4 gene and Cns3-NK gene;The ATP and precursor supply module is composed of PGK gene and Cns3-NK gene.The application is according to the biosynthesis pathway of cordycepin and its metabolic regulation, constructs and optimizes the biosynthesis integrated module required in yeast in vivo cordycepin, creates yeast cell factory, and establishes microbial fermentation production cordycepin technical system with cheap carbon source sugarcane molasses as raw material, realizes the efficient, green, safe production of cordycepin.

The invention belongs to the field of gene engineering and enzyme engineering, and relates to lipase, a coding gene Lip1.1, a genetically engineered bacterium of the lipase and application of the lipase and the coding gene Lip1.1. The invention aims to provide lipase with a specific nucleotide sequence (SEQ ID NO. 1), and further provides a lipase gene, an extracellular secretion expression engineering bacterium and application of the lipase gene in the field of foods or medicines. Particularly, the invention emphasizes the application potential of the lipase gene in the aspect of fish oil fatty acid. The invention provides a lipase gene sequence which is integrated into a yeast genome through a plasmid vector, so that the high expression stability and the exocytosis capacity of target encoding protein are ensured. The recombined target gene fragment can be transferred to any strain with strong lipase production capacity, and the strong expandability of the recombined target gene fragment is embodied. The characteristic not only enhances the utilization efficiency of gene resources, but also provides powerful support for popularization of lipase in various industrial applications.

The invention discloses a preparation method of a protease overexpression yeast strain, the protease overexpression yeast strain and application of the protease overexpression yeast strain, and relates to the technical field of molecular biology.According to the technical scheme, a first expression plasmid of protease A is provided; carrying out one-step assembly on the first expression plasmid, a second expression plasmid containing the TPI1 gene and a third basic vector, and screening and verifying to obtain a third expression plasmid; the third expression plasmid is transferred into a yeast strain, a protease overexpression yeast strain is obtained through screening and verification, the yeast strain is a TPI1 gene knockout strain, a TPI1 gene transcription unit and a protease A gene transcription unit are connected in series and introduced into the yeast strain with the TPI1 gene knockout in a plasmid form, and overexpression of protease is achieved. The yeast genome does not need to be directly edited. The method not only simplifies the construction process, but also possibly improves the genetic stability of the engineering strain, and reduces the potential risk caused by genome integration.

Provided is a method for constructing an engineered yeast for de novo synthesis of mogroside. The method comprises: inserting constructed cucurbitadienol synthase gene expression cassette Ptef1-cds-Tcyc1, epoxide hydrolase gene expression cassette Ptef1-eph3-Tcyc1, cytochrome P450 enzyme-encoding gene CYP and cytochrome P450 enzyme reductase gene expression cassette Ttdh3-cyp87d18-P gal1 / 10-atcpr2-Tcyc1, and glycosyltransferase-encoding gene expression cassette Tadh1-ugt74ac1-Pgal1 / 10-ugtms1-Tcyc1 into a yeast genome, so as to obtain an engineered yeast for mogroside production. The engineered yeast can be used for de novo synthesis of mogroside by using glucose as a substrate for fermentation.

The invention belongs to the field of biosynthesis, and in particular relates to an improved variant of libdanenediol pyrophosphate synthase and engineered yeast for producing sclareol. In order to solve the problem that in the prior art, the sclareol biosynthesis yield is generally low, LsLPPs mutant containing N674L mutation is obtained by performing mutation and optimization on LsLPPs pyrophosphate synthase, and the mutant is used for sclareol synthesis. Meanwhile, in order to solve the problem of genetic instability caused by free plasmid expression of part of saccharomyces cerevisiae strains, a sclareol synthesis pathway is constructed in a yeast genome, and meanwhile, an acetyl coenzyme A synthesis pathway, an MVA pathway and a sclareol synthesis pathway are enhanced; and the chassis bacteria for synthesizing sclareol are modified in a manner of knocking out part of transcription inhibition factors, so that the yield is greatly improved compared with the prior art.

The invention discloses a saccharomyces cerevisiae genetically engineered bacterium for efficiently synthesizing mogroside V through multi-dimensional cytoplasm adaptability and a construction method of the saccharomyces cerevisiae genetically engineered bacterium. The construction method of the saccharomyces cerevisiae genetically engineered bacterium comprises the following steps: integrating truncated 3-hydroxy-3-methylglutaryl coenzyme A reductase tHMG1, pentenyl pyrophosphate isomerase IDI1, squalene synthetase ERG9, epoxy squalene cyclization enzyme ERG1, choline kinase ScCK, isopentenyl phosphokinase AtIPK, phosphoketolase BbPK, phosphotransacetylase PTA and dihydroxyacetone hydrolase EcHAD at a site of a saccharomyces cerevisiae genome GAL80, and carrying out enzymolysis, so as to obtain the saccharomyces cerevisiae genetically engineered bacterium. The invention relates to a preparation method of a cytochrome P450 enzyme, which comprises the following steps of: preparing a cycloepoxide hydrolase SgEPH3, a cytochrome P450 enzyme CYP87D18 and a cytochrome P450 enzyme reductase AtCPR1; an ABC efflux protein PDR11, a glycosyl transferase UGTMG1, a sucrose synthase Susy and a glycosyl transferase SgUGT94-289-3 are integrated at a site Exg1 of a saccharomyces cerevisiae genome. The saccharomyces cerevisiae genetically engineered bacterium constructed by the invention realizes a higher level of mogroside V yield synthesized from the beginning in microorganisms, and has important industrial application potential.

The invention relates to the technical field of saccharomyces cerevisiae genetic engineering transformation, in particular to a high-throughput saccharomyces cerevisiae transformation method. The method comprises the following steps: screening an IGR sequence on a saccharomyces cerevisiae genome through bioinformatics, respectively taking 45bp from upstream and downstream of the IGR sequence as genome homologous sequences for primer design, designing and subpackaging a PCR premixed solution and the designed primer by using an automatic platform script for PCR amplification, adding an obtained PCR product into competent cells, adding a conversion solution, and carrying out PCR amplification on the competent cells to obtain the saccharomyces cerevisiae gene. Performing heat shock; washing and resuspending the cells subjected to heat shock, and then coating the cells on a screened solid culture medium for culturing; the conversion process is carried out on an automatic platform island. According to the method, the transformation efficiency is improved, the operation time of high-throughput transformation is shortened, the intelligence and stability of each transformation are improved, and the method is suitable for large-scale yeast genome editing, functional gene screening and saccharomyces cerevisiae genome site screening.

The present application belongs to the field of biosynthesis, and particularly relates to a sclareol synthase and a yeast engineering bacterium for producing sclareol. In order to solve the problem of low yield of sclareol biosynthesis in the prior art, the sclareol synthase is mutated and optimized in the present application, a SsScs mutant containing at least one mutation of Y491F, G307L or N269F is obtained, and the mutant is used for sclareol synthesis. Meanwhile, in order to solve the problem of genetic instability existing in the expression of free plasmid in some Saccharomyces cerevisiae strains, the present application constructs a sclareol synthesis pathway in the yeast genome, strengthens the acetyl coenzyme A synthesis pathway, the MVA pathway and the sclareol synthesis pathway, and knocks out part of the transcriptional repressor to modify the chassis bacterium for synthesizing sclareol. The yield in a 5 L fermenter reaches 21.13 g / L g / L, which is greatly improved compared with the prior art.

The invention discloses a qPCR (quantitative polymerase chain reaction) method for detecting saccharomyces cerevisiae, Wickerhamomyces anomalus and zygosaccharomyces bayer and application of the qPCR method, and belongs to the technical field of bioengineering. The invention establishes a method for screening specific gene segments and designing qPCR primers based on multi-yeast genome comparison, successfully identifies the specific genes of saccharomyces cerevisiae, Wickerhamomyces anomalus and zygosaccharomyces bayer, verifies high specificity in non-target yeast, establishes qPCR standard curves of the three yeasts, and provides a basis for screening the specific genes of the saccharomyces cerevisiae, the Wickerhamomyces anomalus and the zygosaccharomyces bayer. The linear range is 103-108 copies / [mu] L, the detection limit reaches 10 copies / [mu] L, and the quantification limit reaches 103 copies / [mu] L. The method realizes quantitative and qualitative detection of saccharomyces cerevisiae, Wickerhamomyces anomalus and zygosaccharomyces bayer in a pure bacterium sample and a simulated fermentation sample, has the characteristics of high sensitivity, high specificity, high stability and the like, and is accurate and reliable in detection result.

The invention discloses a method for rapidly and quantitatively detecting the number of saccharomycetes cells in honey. The method comprises the following steps: (1) separating the saccharomycetes cells in the honey; (2) extracting a yeast genome; (3) carrying out real-time fluorescent PCR (Polymerase Chain Reaction) (Taqman qPCR) amplification; and (4) constructing a standard curve. According to the method, accurate quantification of the total number of saccharomycetes in the honey is realized through a specific molecular detection technology, and reliable technical support is provided for quality control, safety evaluation and decay early warning of honey products. The method successfully provides a first specific quantitative solution for yeast pollution in honey, and has high sensitivity, rapidity and universality, so that the method has important application value in the aspects of honey production quality monitoring, storage and transportation process risk early warning and food safety standard upgrading.

The present application belongs to the field of biosynthesis, and particularly relates to pyrophosphate lyddane diol ester synthase and yeast engineering bacteria for producing sclareol. In order to solve the problem of generally low yield of sclareol biosynthesis in the prior art, the pyrophosphate lyddane diol ester synthase is mutated and optimized in the present application, and a SsLPPs mutant containing at least one mutation of D730L, D374L, N674L or G379M is obtained, and the mutant is used for sclareol synthesis. Meanwhile, in order to solve the problem of genetic instability existing in the expression of free plasmid in part of saccharomyces cerevisiae strains, the present application constructs a sclareol synthesis pathway in the yeast genome, and at the same time, the acetyl coenzyme A synthesis pathway, the MVA pathway and the sclareol synthesis pathway are strengthened, and part of the transcriptional repressor is knocked out, so that the bottom bacteria for synthesizing sclareol are modified. The yield in a 5 L fermenter reaches 26.11 g / L, and the yield is greatly improved compared with the prior art.

The invention discloses a yarrowia lipolytica promoter library responding to pH and application, and belongs to the technical field of biology. A bioinformatics method is utilized to predict a promoter sequence of a key gene in a central carbon metabolism pathway of a lipolytic yeast genome, mCherry red fluorescent protein is utilized as a reporter gene, expression intensity change of each promoter in a pH range of 2.0-7.0 is analyzed, a pH-responsive yarrowia lipolytica promoter library is constructed, and a Yarrowia lipolytica promoter library is constructed. A plurality of pH-responsive constitutive promoters are screened, and available promoter elements are provided for constructing yarrowia lipolytica cell factories.

This invention belongs to the field of biosynthesis, specifically relating to a variant of lysine pyrophosphate diol ester synthase and engineered bacteria for producing perillaldehyde. To address the generally low yield of perillaldehyde biosynthesis in existing technologies, this invention mutates and optimizes lysine pyrophosphate diol ester synthase, obtaining an SsLPPs mutant containing the D374L mutation, and uses this mutant for perillaldehyde synthesis. Simultaneously, to address the genetic instability issues associated with free plasmid expression in some Saccharomyces cerevisiae strains, this invention constructs a perillaldehyde synthesis pathway in the yeast genome. Furthermore, it modifies the substrate bacteria synthesizing perillaldehyde by enhancing the acetyl-CoA synthesis pathway, the MVA pathway, and the perillaldehyde synthesis pathway, and by knocking out some transcriptional repressors, resulting in a significant increase in yield compared to existing technologies.

The invention discloses a method for constructing a vitamin E production strain by synchronously editing and recombining three sites of a yeast genome and application, and relates to the technical field of synthetic biology and metabolic engineering. According to the invention, a helper plasmid for expressing CRISPR-Cas9 protein and three targeted specific site guide RNA (sgRNA) is constructed, and the helper plasmid and three vitamin E pathway gene donor fragments which are assembled and cloned in escherichia coli in advance are co-transformed into saccharomyces cerevisiae, so that efficient editing and large-scale gene fragment integration can be simultaneously carried out on three chromosome sites of HO, GAL80 and ARO10. The three-site synchronous editing and recombination efficiency of the method exceeds 80%. By utilizing the method, an engineering strain of a complete vitamin E synthesis route can be rapidly constructed at one time, and fermentation verifies that the yield of the vitamin E can reach 5.9 g / L. The invention provides an efficient and universal tool for quickly constructing complex metabolic pathways in yeast.

The present invention discloses adenosine deaminase mutants, including wild-type adenosine deaminase mutants M61V, V69A, M70T, and Y73C. The present invention also discloses a Pichia pastoris base editor containing the adenosine deaminase mutants. The Pichia pastoris base editor is capable of efficiently editing bases in the Pichia pastoris genome, with an editing efficiency of 90-100%, providing a gene editing tool for basic and applied research based on Pichia pastoris.

The invention relates to preparation and application of a kit for efficiently extracting yeast genome DNA. The kit comprises a lysate prepared from chaotropic salt with the final concentration of 1-5 mol / L, a nonionic surfactant with the mass-volume percent of 0.5%-10%, an anionic surfactant with the mass-volume percent of 0.1%-10%, a buffer solution with the final concentration of 10-100 mmol / L and an ionic chelating agent with the final concentration of 0.1-20 mmol / L, and the lysate is matched with digestive juice, RNase A, a protease K solution, a washing solution 1, a washing solution 2 and an eluent. According to the method, the yeast genome can be effectively extracted without using toxic reagents such as phenol and chloroform, the operation is simple and convenient, and the extracted genome is high in content and good in quality and can be directly used for subsequent experiments.

The invention belongs to the field of gene engineering, and particularly relates to a non-interval loop iteration yeast genome editing system. The system is composed of Cas and gRNA double plasmids, wherein the gRNA plasmids comprise auxotrophy gRNA-URA3, gRNA-HIS3 and gRNA-LEU2, and the gRNA plasmids comprise gRNA-URA3, gRNA-HIS3 and The resistance is gRNA-BleoR, gRNA-HygR, gRNA-NrsR, gRNA-BleoR, gRNA-HygR and gRNA-NrsR; through coordinated use with a Cas selection marker, the method realizes interval-free, cyclic and iterative efficient editing operation on a yeast genome, and can be used as a practical tool for multi-gene continuous iterative editing of saccharomyces cerevisiae and other fungi. The method has extremely high editing efficiency, an experimental terminal eliminates Cas and gRNA plasmids to realize traceless operation, and the method is low in off-target rate, universal, accurate and easy to operate and can be applied to large-scale gene knockout, knockin, site-specific mutagenesis and the like.