Method for improving gene editing efficiency of saccharomyces cerevisiae

Abstract

The invention discloses a method for improving gene editing efficiency of saccharomyces cerevisiae. On the premise that a homologous recombination template is not added, based on non-homologous end connection repair, the CRISPR / Cas9 editing efficiency is improved by exogenously adding methionine or modifying an autophagy key gene. The method comprises the following steps: constructing an autophagy deficient strain, adding methionine, and constructing a sgRNA and CRISPR / Cas9 expression system of a targeted target gene. The CRISPR / Cas9 system comprises a nucleic acid positioning signal SV40 and nuclease Cas9, wherein the expression of the nuclease Cas9 is controlled by a galactose inducible GAL1 promoter and a CYC1 terminator. The improvement of the mutation efficiency shows that the method can effectively improve the mutation efficiency of the saccharomyces cerevisiae, can be widely applied to saccharomyces cerevisiae evolution, phenotype optimization and industrial strain construction, and provides an efficient strain improvement tool for the fields of food fermentation, biological energy, medicinal protein production and the like.

Description

Technical Field

[0001] This invention belongs to the field of microbial genetic engineering and breeding technology, specifically relating to a gene editing improvement method and its application for improving the mutant breeding of Saccharomyces cerevisiae. Background Technology

[0002] brewing yeast ( Saccharomyces cerevisiae Saccharomyces cerevisiae (Saccharomyces cerevisiae) is a commonly used model strain in the field of industrial biotechnology, widely applied in food fermentation, bioenergy, and pharmaceutical protein production. Its excellent genetic properties and production characteristics make it an ideal model microorganism for scientific research and a "super factory" in industrial production. Mutation breeding is the foundation and starting point for constructing Saccharomyces cerevisiae supercell factories. Its core function lies in breaking metabolic bottlenecks, expanding substrate utilization, enhancing environmental tolerance, and improving product synthesis efficiency through random or directed mutations. However, traditional mutation breeding methods (such as physical mutagenesis and chemical mutagenesis) suffer from low mutation efficiency, high randomness, and long screening cycles, making it difficult to meet the needs of precision breeding.

[0003] In recent years, the explosive development of Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) and its associated protein (Cas) genome editing technology provides a powerful DNA rewriting tool for the construction and genome synthesis evolution of Saccharomyces cerevisiae chassis cells. By constructing and optimizing the yeast chassis cell to improve cell tolerance to meet the actual requirements of industrial production, the maximum economic benefit is obtained. With the continuous development of gene editing technology, the use of non-homologous end joining (NHEJ) to introduce diversity random mutations at the DSB site can accelerate genome evolution and provide a new strategy for Saccharomyces cerevisiae mutation breeding. Unlike gene expression regulation based on dead Cas9 (dCas9) assisted, CRISPR editing mutation breeding based on NHEJ can form stable and heritable genome variation. Compared with single base editing technology assisted by dCas9 or nickase (Cas9 nickase, nCas9), CRISPR editing mutation breeding based on NHEJ produces more types of mutations, which is particularly important for obtaining a mutant library with diverse phenotypes. However, the low efficiency of NHEJ repair in Saccharomyces cerevisiae results in low mutation efficiency and diversity of mutation types after CRISPR editing, making it difficult to quickly construct a large-scale mutant library with rich genetic diversity during mutation breeding, significantly reducing the probability of screening strains with high yield, stress resistance, and broad substrate utilization; At the same time, it prolongs the breeding cycle and increases the screening cost, which ultimately restricts the directed improvement efficiency and innovation potential of Saccharomyces cerevisiae in the fields of industrial fermentation and biopharmaceuticals. Therefore, it is of great significance to develop an improved method that can improve the gene editing efficiency of Saccharomyces cerevisiae and simplify the screening process. SUMMARY

[0004] The present application aims at the technical bottleneck of low NHEJ repair efficiency in Saccharomyces cerevisiae leading to insufficient mutation rate of CRISPR editing, and realizes significant improvement of mutation efficiency through the strategy of autophagy factor modification and methionine exogenous intervention combined with optimized CRISPR / Cas9 system.

[0005] A method for improving the gene editing efficiency of Saccharomyces cerevisiae, comprising the following steps: (1) Knocking out the autophagy key gene in Saccharomyces cerevisiae by homologous recombination; (2) Designing the targeting site sequence of DNA of Saccharomyces cerevisiae and synthesizing gRNA; (3) Constructing pRS423-gRNA-Cas9 vector; transfecting pRS423-gRNA-Cas9 vector into Saccharomyces cerevisiae cells or autophagy-deficient strains, and screening positive transformants; (4) Under the culture condition of 10-30 g / L of galactose, inducing cell editing of wild-type or autophagy-deficient strain cells or directly culturing and inducing cell editing of autophagy-deficient strain cells by adding 100-200 mg / L of methionine exogenously; (5) Screening mutant strains by screening medium, sequencing, and verifying mutation effect.

[0006] In step (1), the autophagy key gene is ATG11 , the homologous recombination fragment is ATG11 upstream 500 bp homologous arm, KanMX label and ATG11 downstream 500 bp homologous arm.

[0007] The pRS423-gRNA-Cas9 vector comprises gRNA and CRISPR / Cas9 protein gene; the backbone plasmid is pRS423 which is multi-copy and carries HIS3 auxotrophic marker; the nuclease Cas9 gene and Cpf1 gene are respectively fused with SV40 nucleic acid positioning signal at the N terminal, and the expression thereof is controlled by galactose inducible GAL1 promoter and CYC1 terminator; the gRNA expression cassette is controlled by SNR52 promoter and SNR52 terminator.

[0008] In step (2), the targeting gene of Saccharomyces cerevisiae is URA3 , and the gRNA sequence is shown in SEQ ID NO: 1.

[0009] In step (3), the screening medium is SD-His auxotrophic medium.

[0010] In step (5), the screening medium is SD+Ura+5-FOA medium.

[0011] In step (5), the sequence of the sequencing primer is URA3-vf-F and URA3-vf-R; the nucleotide sequences are shown in SEQ ID NO: 2-3.

[0012] The Saccharomyces cerevisiae cell is BY4741a model strain.

[0013] Advantages of the present application: (1) Mutation efficiency is significantly improved: the mutation efficiency of Δatg11 strain is 1.54 times higher than that of wild type; the mutation efficiency of wild-type strain is increased by 2.12 times after adding Met, and the mutation efficiency of Δatg11 strain is increased by 2.57 times at most after adding Met; (2) Easy operation and controllable cost: using galactose inducible promoter, avoiding the toxicity of Cas9 continuous expression to cells, improving the survival rate of strains; combining with auxotrophic marker (HIS3) and resistance screening (5-FOA) to significantly improve the screening efficiency of positive clones.

[0014] (3) Wide application scenarios: used for industrial strain improvement, can quickly construct mutant library of high-yield metabolic products such as ethyl; accelerate the adaptive evolution of Saccharomyces cerevisiae to extreme environment (high osmotic pressure, high ethanol concentration); provide efficient mutation tool for analyzing yeast gene function. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 Gene module results of BY4741aΔatg1 strain construction.

[0016] Figure 2 pRS423-gRNA(URA3-1)-Cas9 vector structure diagram.

[0017] Figure 3 CRISPT / Cas9 editing induction and editing effect evaluation.

[0018] Figure 4 Evaluation of cell survival rate and editing effect of wild-type strain with exogenous addition of methionine.

[0019] Figure 5 Evaluation of cell survival rate and editing effect of autophagy-deficient strain Δatg11 and Met addition. DETAILED DESCRIPTION

[0020] In order to facilitate the understanding of the present application, the present application will be described more fully below. However, the present application can be realized in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present application more thorough and comprehensive.

[0021] Example 1 Construction of autophagy-deficient strain (Δatg11) Recombinant fragment preparation: KanMX tag (primers: KanMX-F / R) was amplified with pUG6 plasmid as template, and 50bp upstream homologous arm+KanMX+50bp downstream homologous arm recombinant fragment was amplified with KanMX as template and primers: ATG11-up-F / R; Transformation and screening: the recombinant fragment was transformed into BY4741a competent cells, and coated on YPD plate containing G418 (200 mg / L), and cultured at 30°C for 48h, and single colonies were picked for PCR verification (primers: ATG11-verify-F / R), and it was confirmed that ATG11 the gene was successfully knocked outFigure 1 ).

[0022] Construction and transformation of CRISPR / Cas9 system (1) Vector backbone: pRS423 plasmid with multiple copies (2µ-based) was used, which carries HIS3 auxotrophic marker to ensure stable replication of the vector in yeast and positive transformant screening; (2) Cas9 expression cassette: Cas9 protein N-terminal fusion SV40 nuclear localization signal (NLS), driven by the GAL1 promoter induced by galactose, and terminated by CYC1 terminator; (3) gRNA expression cassette: guide RNA (gRNA) is driven by SNR52 promoter (yeast endogenous high-efficiency promoter) and terminated by SNR52 terminator to ensure efficient transcription and targeting specificity of gRNA.

[0023] (4) Transform pRS423-gRNA-Cas9 vector into wild type and Δatg11 strains, and screen positive transformants in SD-His auxotrophic medium.

[0024] According to the instructions of the yeast transformation kit (Zymo Research), 200 ng of the above editing plasmid and empty plasmid pRS423 were transformed into the host strain BY4741a, respectively, and positive transformants were screened on SD-His auxotrophic plates. The correct construction of the vector was confirmed by enzyme digestion and sequencing (FIG. 1). Figure 2 ).

[0025] CRISPR editing and mutation screening The positive transformants were inoculated into SD-His liquid medium containing 20 g / L galactose and divided into four groups: wild type group: adding 150 mg / L Met (methionine); control group (wild type): no Met added; Δatg11 group: no Met added; Δatg11 group: adding Met; 30°C shaking culture for 36 h, inducing Cas9 and gRNA expression, realizing target site cutting and NHEJ repair; the induced bacterial liquid was gradiently diluted and plated on SD+Ura+5-FOA plates (containing 5-FOA 1.5 g / L), and cultured at 30°C for 48 h. The number of surviving bacteria (i.e. mutant strains) was counted; the surviving colonies were picked and genomic DNA was extracted, PCR amplified with URA3-vf-F / R primers and sequenced to confirm the presence of Indel mutations at the target site (FIG. 2). Figure 3 ).

[0026] Mutation efficiency statistics The mutant strain is screened through SD+Ura+5-FOA screening medium (5-FOA can kill cells with normal URA3 function, and only mutant strains survive), and sequencing primers (URA3-vf-F, URA3-vf-R) are used for sequencing verification.

[0027] The results show that: the cell survival rate of the control group wild type is 2.11±0.32%, and the mutation efficiency is 21.5±0.17‰; the cell survival rate of the wild type+Met group is 1.25±0.17%, and the mutation efficiency is 45.84±0.48‰; the cell survival rate of the Δatg11 group is 0.76±0.03%, and the mutation efficiency is 33.40±0.54‰; the cell survival rate of the Δatg11+Met group is 00.80±0.05%, and the mutation efficiency is 55.47±0.63‰. Figures 4-5 .

[0028] The results fully prove that the method can effectively improve the CRISPR editing mutation efficiency of Saccharomyces cerevisiae, and provides an efficient tool for industrial strain improvement.

Claims

1. A method for improving the gene editing efficiency of Saccharomyces cerevisiae, characterized in that, Includes the following steps: (1) Knock out key autophagy genes in Saccharomyces cerevisiae using homologous recombination; (2) Design the target site sequence of Saccharomyces cerevisiae DNA and synthesize gRNA; (3) Construct the pRS423-gRNA-Cas9 vector; transfect the pRS423-gRNA-Cas9 vector into Saccharomyces cerevisiae cells or autophagy-deficient strains, and screen for positive transformants; (4) Under galactose culture conditions of 10-30 g / L, cell editing of wild-type or autophagy-deficient strains was induced by exogenous addition of 100-200 mg / L methionine or by direct culture to induce cell editing of autophagy-deficient strains; (5) Screen mutant strains by screening culture medium, sequence them, and verify the mutation effect.

2. The method for improving gene editing efficiency in Saccharomyces cerevisiae according to claim 1, characterized in that, In step (1), the key autophagy gene is ATG11 Homologous recombination fragments are ATG11 The upstream 500 bp homologous arm, KanMX tag and ATG11 The downstream 500 bp homologous arm.

3. The method for improving the gene editing efficiency of Saccharomyces cerevisiae according to claim 1, characterized in that, The pRS423-gRNA-Cas9 vector includes gRNA and CRISPR / Cas9 protein genes; Its backbone plasmid is a multi-copy pRS423 carrying the HIS3 auxotrophic marker; the nuclease Cas9 gene and Cpf1 gene are respectively fused with the SV40 nucleic acid localization signal at the N-terminus, and their expression is controlled by the galactose-inducible GAL1 promoter and CYC1 terminator; the gRNA expression cassette is controlled by the SNR52 promoter and SNR52 terminator.

4. The method for improving the gene editing efficiency of Saccharomyces cerevisiae according to claim 1, characterized in that, In step (2), the targeting gene of the brewer's yeast is URA3 The gRNA sequence is shown in SEQ ID NO:

1.

5. The method for improving gene editing efficiency in Saccharomyces cerevisiae according to claim 1, characterized in that, In step (3), the culture medium used for screening is SD-His auxotrophic medium.

6. The method for improving gene editing efficiency in Saccharomyces cerevisiae according to claim 1, characterized in that, The screening medium in step (5) is SD+Ura+5-FOA medium.

7. The method for improving gene editing efficiency in Saccharomyces cerevisiae according to claim 1, characterized in that, The sequencing primer sequences mentioned in step (5) are URA3-vf-F and URA3-vf-R; their nucleotide sequences are shown in SEQ ID NO: 2-3, respectively.

8. The method for improving gene editing efficiency in Saccharomyces cerevisiae according to claim 1, characterized in that, The brewer's yeast cells mentioned are the BY4741a type strain.

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