Method for improving yeast CRISPR-Cas gene editing efficiency

Abstract

The invention discloses a method for improving yeast CRISPR-Cas gene editing efficiency, and belongs to the technical field of gene engineering. Firstly, SPC plasmids are transferred into host bacteria, the SPC plasmids contain gRNA1 expression cassettes targeting target genes 1, 5'ends of the gRNA1 expression cassettes are connected with Checkpoint promoters, the gRNA1 expression cassettes serving as host cells are transferred into pCasT plasmids, the plasmids and gRNA2 targeting yeast genome target genes 2 achieve DNA double-strand breakage through Cas protein, the Checkpoint promoters of the SPC plasmids are activated, transcription of the gRNA1 is started, and gene editing of the target genes 1 is achieved. On the contrary, the Cas protein and the gRNA1 expression cassette do not realize gene editing on the target gene 1, the unedited strain dies by absorbing the externally added toxic substances, and the successfully edited strain survives due to the lack of the functions. According to the method, the gene editing efficiency of a single target site can be improved, the gene knockout, large-fragment DNA insertion and recombination efficiency can be improved, and the high-throughput genome editing efficiency is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and in particular relates to a method for improving the efficiency of yeast CRISPR-Cas gene editing. Background Art

[0002] As the global energy crisis and climate issues become increasingly serious, my country has proposed the dual carbon goals of "carbon peak and carbon neutrality" to reduce carbon emissions and promote sustainable development. Against this background, biomanufacturing, as an important means to achieve a low-carbon economy, is gradually becoming the focus of competition and development direction among countries. Biomanufacturing can not only effectively reduce dependence on fossil energy, but also provide strong support for the production and application of green energy through biotechnology. With the rapid growth of global demand for bio-based products, microbial cell factories - the core "chips" of the bioindustry - have received unprecedented attention and challenges. The strains of these cell factories not only need to have strong production capacity, but also must be able to adapt to various complex environmental conditions. Gene editing technology provides more possibilities for improving the metabolic efficiency of strains and developing new biological products. As the first model eukaryote to complete whole genome sequencing, Saccharomyces cerevisiae is widely used in the production of food, biochemicals and bioenergy due to its clear genetic background, easy genetic modification and excellent fermentation performance. It is a key chassis cell in the industrial field.

[0003] Since DiCarlo et al. (DiCarlo JE, Norville JE, Mali P, et al. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Research, 2013, 41(7): 4336-4343.) first used CRISPR-Cas9 technology to knock out a single gene in Saccharomyces cerevisiae using a shorter homologous sequence in 2013, the CRISPR-Cas system has gradually become the core strategy for gene editing in Saccharomyces cerevisiae. In 2015, Bao et al. (Z Bao, H Xiao, J Liang, et al., Homology-integrated CRISPR-Cas (HI-CRISPR) system for one-step multigene disruption in Saccharomyces cerevisiae. ACS Synthetic Biology, 2015. 4 (5): 585-94.) introduced the CRISPR-Cas9 system into Saccharomyces cerevisiae and established the HI-CRISPR (Homology-integrated CRISPR) method by optimizing culture conditions and using Cas9 protein variants. The knockout efficiency was 27%-80% for three genes at the same time, and it was increased to 100% after prolonging the culture time. In 2019, researchers (Y Zhang, J Wang, Z Wang, et al., AgRNA-tRNA array for CRISPR-Cas9 based rapid multiplexed genome editing in Saccharomyces cerevisiae. Nature Communications, 2019. 10(1): 1053.) developed a sgRNA-tRNA array system GTR-CRISPR based on CRISPR-Cas9. Through two rounds of iterative editing, they achieved simultaneous knockout of eight genes with an efficiency of 87%, significantly enhancing the efficiency of gene editing.

[0004] In recent years, the improvement of Saccharomyces cerevisiae has shifted from small-scale, specific metabolic pathway modification to extensive gene editing and regulation at the whole genome level. However, the network regulation of microbial cell factories is highly complex. The construction of cell factories usually involves a lot of design, construction and testing of expression elements. Therefore, the development of efficient high-throughput CRISPR genome editing technology is a very challenging task. This process places strict requirements on high-throughput CRISPR genome editing technology, especially in terms of high-throughput DNA synthesis and library construction, efficient genome editing, library screening, and genotype-phenotype association tracking. Lian et al. (J Lian, C Schultz, M Cao, et al., Multi-functional genome-wide CRISPR system for high throughput genotype-phenotype mapping. Nature Communications, 2019.10(1):5794.) developed a multifunctional whole genome evolution technology (Multi-functional genome-wide CRISPR system, MAGIC) based on the CRISPR-AID system and constructed a diverse yeast genome library. Different library plasmids were designed for gene knockout, transcriptional activation and transcriptional repression according to the yeast genome, and genome evolution was achieved through high-throughput screening. Liu et al. (R Liu, L Liang, A Choudhury, et al., Multiplex navigation of global regulatory networks (MINR) in yeast for improved ethanol tolerance and production. Metabolic Engineering, 2019. 51: 50-58.) developed a traceable high-throughput CRISPR genome editing method in Saccharomyces cerevisiae, and targeted the transcriptional regulatory network of Saccharomyces cerevisiae to construct a global metabolic network multiple regulatory technology (MINR). Through oligonucleotide chip technology and developed mutation library computer design tools, high-throughput synthesis of rational or semi-rational design of gene editing units and high-throughput genome editing were achieved. Although the above technology overcomes the limitations of many traditional strain improvement technologies, realizes high-throughput genome editing, and greatly improves the ability to reprogram biological systems. However, its editing efficiency is relatively low and its traceability efficiency is relatively poor, which limits the efficient development of Saccharomyces cerevisiae cell factories.

[0005] In order to enhance the efficiency of CRISPR gene editing, researchers have adopted a variety of strategies for optimization, including exploring new CRISPR gene editing systems, improving the structure and expression of gene editing enzymes, and optimizing the design structure of gRNA and donor DNA. Despite this, there is still no single method or strategy that can ensure efficient editing of all different genes and sites. Therefore, it is crucial to develop a CRISPR-Cas gene editing technology to improve the gene editing efficiency of yeast. Summary of the invention

[0006] When using the CRISPR-Cas gene editing system for gene modification in yeast, there is a problem of different gene editing efficiencies for different targeted gene sites under different conditions. The purpose of the present invention is to provide a method for improving the efficiency of yeast CRISPR-Cas gene editing. By constructing a "gene editing purification system" based on CRISPR-Cas gene editing technology combined with a Checkpoint promoter that can respond to the yeast DNA damage repair mechanism, unmodified cells can be removed during the gene editing process, so that the surviving cells are all genetically modified cells, thereby improving the gene editing efficiency using the CRISPR-Cas gene editing system.

[0007] The present invention provides the following technical solutions:

[0008] The present invention provides a method for improving the efficiency of CRISPR-Cas gene editing in cells, comprising the following steps:

[0009] (1) The SP_C plasmid is transferred into a host cell, and a recombinant strain containing a Checkpoint promoter that responds to DNA double-strand breaks is obtained by screening through an auxotrophic screening marker; wherein the SP_C plasmid contains a gRNA1 expression cassette targeting target gene 1, and a Checkpoint promoter that can respond to the cell DNA damage mechanism is inserted into its 5' end, and the Checkpoint promoter is activated by the Cas protein shearing the DNA double strand of the target gene 2;

[0010] (2) transferring the pCas_T plasmid into the recombinant strain obtained in step (1), wherein the pCas_T plasmid is inserted with a Cas protein and a gRNA2 expression cassette (containing a homologous repair template) targeting a target gene 2 of the cell genome;

[0011] (3) The Cas protein expressed by the pCas_T plasmid binds to the gRNA2 expression cassette to cut the DNA double strand of the target gene 2, activates the Checkpoint promoter in the SP_C plasmid, transcribes and synthesizes the gRNA1 expression cassette to bind to the Cas protein, and performs gene editing on the target gene 1. The strains that have only undergone gene editing are screened using a plate containing the screening substance.

[0012] Based on the above technical scheme, further, when the Cas protein expressed by the pCas_T plasmid combines with the gRNA2 expression cassette targeting the cell genome target gene 2, the cell genome target gene 2 is sheared, resulting in a DNA double-strand break, activating the Checkpoint promoter in the SP_C plasmid in response to the DNA double-strand break signal, and starting the transcription and synthesis of the gRNA1 expression cassette targeting the cell genome target gene 1. The transcribed gRNA1 expression cassette combines with the Cas protein to perform gene editing on the target gene 1. Since the target gene 1 has been modified, the gene-edited strain can grow normally on the plate containing the screening substance; if the Cas protein expressed by the pCas_T plasmid and the gRNA1 expression cassette transcribed by the SP_C plasmid do not modify the target targeting gene 1, the strain cannot grow on the plate containing the screening substance, resulting in the death of the strain, thereby removing the unedited strain.

[0013] Based on the above technical solution, further, the Checkpoint promoter described in step (1) includes but is not limited to CRT1, HUG1, RNR1, RNR2, RNR3, and RNR4.

[0014] Based on the above technical solution, further, the target gene 1 described in step (1) includes but is not limited to the CAN1 gene.

[0015] Based on the above technical solution, further, the nucleic acid sequence of the gRNA1 expression cassette described in step (1) is shown as SEQ ID NO.1 or SEQ ID NO.2.

[0016] Based on the above technical solution, further, the host cell described in step (1) includes but is not limited to Saccharomyces cerevisiae.

[0017] Based on the above technical solution, further, the screening markers in the SP_C plasmid described in step (1) and the pCas_T plasmid described in step (2) include but are not limited to HIS3, LEU2, URA3 and TRP1.

[0018] Based on the above technical solution, further, the screening markers in the SP_C plasmid described in step (1) and the pCas_T plasmid described in step (2) are different.

[0019] Based on the above technical solution, further, the vector backbone of the SP_C plasmid described in step (1) and the pCas_T plasmid described in step (2) includes but is not limited to pCRCT.

[0020] Based on the above technical solution, further, the transformation methods in step (1) and step (2) include but are not limited to chemical transformation and electroporation transformation.

[0021] Based on the above technical solution, further, the Cas protein in step (2) includes but is not limited to Cas9, AsCas12a, FnCas12a, LbCas12a and MAD7.

[0022] Based on the above technical solution, further, the nucleotide sequence of the gRNA2 expression box in step (2) is shown as SEQ ID NO.3 or SEQ ID NO.4.

[0023] Based on the above technical solution, further, the target gene 2 described in step (2) includes but is not limited to the ADE2 gene.

[0024] Based on the above technical solution, further, the screening substances in step (3) include but are not limited to canavanine (L-canavanine), 5-fluoroorotic acid (5-FOA), and 2-amino-5-fluorobenzoic acid (5-FAA).

[0025] The beneficial effects of the present invention compared with the prior art are as follows:

[0026] The method of the present invention can not only significantly improve the gene editing efficiency of CRISPR-Cas9 and CRISPR-Cas12a at a single gene site, including gene mutation, knockout, and insertion and recombination of large DNA fragments, but also greatly enhance the efficiency of high-throughput gene editing, accelerate the speed of yeast cell factory construction and transformation, and remove untransformed cells during the gene editing process, so that the surviving cells are all genetically modified cells, which is of great significance to the development and improvement of gene editing technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention, the drawings related to the embodiments are briefly introduced below.

[0028] Figure 1 Flow chart of the gene editing method of Example 2 of the present invention.

[0029] Figure 2 Flow chart of the gene editing method of Example 3 of the present invention.

[0030] Figure 3 This is a comparison chart of the gene editing efficiency of the experimental group and the control group in Example 2 of the present invention.

[0031] Figure 4This is a comparison chart of the gene editing efficiency of the experimental group and the control group of Example 3 of the present invention when different proportions of non-targeted designed plasmids are added, wherein A is Checkpoint promoter CRT1, B is Checkpoint promoter HUG1, C is Checkpoint promoter RNR1, and D is Checkpoint promoter RNR2.

[0032] Figure 5 This is a comparison diagram of colony color changes in the experimental group and the control group of Example 3 of the present invention, wherein A is the Checkpoint promoter RNR2, and B is the Checkpoint promoter CRT1.

[0033] Specific implementation method

[0034] The present invention is described in detail below with reference to the embodiments, but the embodiments of the present invention are not limited thereto. Obviously, the embodiments described below are only partial embodiments of the present invention. For those skilled in the art, other similar embodiments obtained without creative labor all fall within the protection scope of the present invention.

[0035] Example 1

[0036] SP_C plasmid construction process: Use primers In-Cas9-CRT1-F / R (see Table 1) to PCR amplify the genome of Saccharomyces cerevisiae BY4741 to obtain the sequences of CRT1p, HUG1p, RNR1p, RNR2p, RNR3p, and RNR4p (hereinafter collectively referred to as Checkpoint promoter). The gRNA1 expression cassette (containing a homologous repair template) targeting target gene 1 (such as CAN1 gene) was synthesized by total gene synthesis. Using pCRCT (addgene Plasmid #60621) as a plasmid, BB-Checkpoint-F / R primers in Table 1 were used for PCR amplification to obtain the BB-Checkpoint vector fragment (pCRCT vector backbone). After DNA purification, the Checkpoint promoter, gRNA1 expression cassette and BB-Checkpoint vector fragment were sequentially connected using the Gibson seamless cloning method to construct the SP_C plasmid. If the Cas12a protein is used, the reverse primer of each In-Cas9 in Table 1 is replaced with the reverse primer of each In-Cas12a in Table 1.

[0037] Table 1 Primer sequences used in the construction of SP_C plasmid

[0038]

[0039]

[0040] Nucleotide sequence of the gRNA1 expression cassette (containing the homologous repair template) targeting target gene 1 (the sequence can be variable according to different targets)

[0041] If the pCas_T plasmid contains the Streptococcus pyogenes Cas9 (SpCas9) protein gene or the Cas9 protein gene with NGG as the PAM sequence, the sequence of the gRNA1 expression cassette (including the homologous repair template) can be:

[0042]

[0043] The wavy line sequence is the homology arm required for Gibson seamless cloning, which can be replaced according to the different promoter sequences to be connected. The underlined sequence is the homology repair template (5'-3'), and the sequence marked in gray is the targeting sequence.

[0044] If the pCas_T plasmid contains AsCas12a, LbCas12a, MAD7 protein based or Cas12a protein with TTTN as PAM sequence, the sequence of the gRNA1 expression cassette (including the homologous repair template) can be:

[0045]

[0046]

[0047] The wavy line sequence is the homology arm required for Gibson seamless cloning, which can be replaced according to the different promoter sequences to be connected. The underlined sequence is the homology repair template (5'-3'), and the sequence marked in gray is the targeting sequence.

[0048] Construction process of pCas_T plasmid: RNA2 expression cassette (homologous repair template) targeting yeast genome target gene 2 (such as ADE2 gene) was synthesized by gene total synthesis. Using pCRCT plasmid as template, BB-pCRCT-F / R primers in Table 2 were used for PCR amplification to obtain BB-pCRCT vector fragment, and HIS3-F / R or LEU2-F / R primers in Table 2 were used for PCR amplification of plasmid pRS423 or pRS425 to obtain HIS3 or LEU2 insert fragments. After PCR product purification, Gibson assembly seamless cloning was performed to sequentially connect the gRNA2 expression cassette, BB-pCRCT vector fragment (containing Cas protein) and HIS3 or LEU2 fragment to construct pCas_T plasmid.

[0049] Construction of pCas_N plasmid: The expression cassette of non-targeted gRNA_N was synthesized by total gene synthesis. Using the pCas_T plasmid as a template, PCR amplification was performed using the bb-Cas9-Non-F / R primers in Table 2 to obtain the vector fragment bb_Cas_N. After purification of the PCR product, the vector fragment bb_Cas_N was connected to the gCas_N fragment (for non-targeted editing, if Cas9 protein is used, the expression cassette of gCas_N uses the gRNA2 expression cassette in the form of Cas12a; on the contrary, if Cas12a protein is used, the expression cassette of gCas_N uses the gRNA2 expression cassette in the form of Cas9) by the Gibson assembly seamless cloning connection method to construct the pCas_N plasmid.

[0050] If the plasmid of AsCas12a is used, the plasmid of pCas_N is used as a template, and the BB-As-F / R primers in Table 2 are used to amplify the vector fragment of pCRCT-As (containing the gRNA2 expression cassette and the pCRCT vector fragment). Using plasmid pY010 (addgene Plasmid #69982) as a template, the protein of AsCa12a is amplified using primers AsCpf1-F / R. After PCR product purification, Gibson assembly seamless cloning is performed to sequentially connect the AsCas12a protein with the pCRCT-As vector fragment to construct the pCas_T plasmid.

[0051] Table 2 Primer sequences used in the construction of pCas plasmid

[0052]

[0053]

[0054] Nucleotide sequence of the gRNA2 expression cassette (containing a homologous repair template) targeting target gene 2 in the yeast genome (the sequence can be variable according to different targets)

[0055] If the pCas_T plasmid contains the Streptococcus pyogenes Cas9 (SpCas9) protein gene or the Cas9 protein gene with NGG as the PAM sequence, the sequence of the gRNA2 expression cassette (containing the homologous repair template) can be:

[0056] The underlined sequence is the homology repair template (5'-3'), and the sequence marked in gray is the targeting sequence.

[0057] If the pCas_T plasmid contains AsCas12a, LbCas12a, MAD7 protein gene or Cas12a protein gene with TTTN as PAM sequence, the sequence of gRNA2 expression cassette (containing homologous repair template) can be:

[0058]

[0059] The underlined sequence is the homology repair template (5'-3'), and the sequence marked in gray is the targeting sequence.

[0060] Example 2

[0061] See also Figure 1 As shown, Figure 1Flow chart of the method for improving the efficiency of yeast CRISPR-Cas gene editing in Example 2 of the present invention, the host strain used in this embodiment is Saccharomyces cerevisiae BY4741, and the specific implementation steps are as follows: First, the SP_C plasmid (Checkpoint promoters are CRT1, HUG1, RNR1, RNR2, respectively) is introduced into the Saccharomyces cerevisiae cell BY4741 by electroporation transformation or chemical transformation, and the YC yeast strain is obtained by screening through SC-Ura medium (Bao Biological, Dalian, China). Yeast competent cells are then prepared using the YC strain as the host cell, and the pCas_T plasmid is introduced into the YC yeast competent cell by electroporation transformation or chemical transformation. The Cas protein expressed by the pCas_T plasmid and the gRNA2 targeting the yeast genome target gene 2 (such as ADE2 gene, The isolation of adenylosuccinate synthetase mutants in yeast by selection for constitutive behavior in pigmented strains. Genetics 61 (2): 377-89) work together to shear DNA and break the DNA double strand. In this process, DNA double-strand breaks activate the Checkpoint promoter on the SP_C plasmid, transcribe and synthesize gRNA1 targeting target gene 1 (such as CAN1 gene, Mutagenicity of methylated N-nitrosopiperidines in Saccharomycescerevisiae. Mutat Res 57(2):155-61), and work together with the Cas protein expressed by the pCas_T plasmid to cleave the CAN1 gene. At the same time, the homologous repair template in the gRNA1 expression box is used for DNA repair, in which two "TAATAA" termination codons are inserted into the homologous repair template, so that the plasma membrane arginine permease encoded by the CAN1 gene is inactivated, and the successfully edited strain can survive in a medium containing canavanine. If the Cas protein expressed by the pCas_T plasmid and the gRNA1 on the SP_C plasmid do not modify the target gene, the CAN1 gene of the strain continues to be expressed, and the cells can absorb canavanine through the plasma membrane arginine permease. Canavanine is toxic to cells and prevents them from growing normally.

[0062] Compared with the control group (without canavanine selection), the editing efficiency of the ADE2 gene of the yeast strain grown in the medium supplemented with canavanine was close to 100%, and the gene editing efficiency was as follows Figure 3 In summary, this embodiment can significantly improve the efficiency of gene editing of yeast strains.

[0063] Checkpoint promoters that respond to DNA double-strand breaks in yeast include but are not limited to CRT1, HUG1, RNR1, RNR2, RNR3, and RNR4, which can all serve as promoters that respond to DNA double-strand break signals in the SP_C plasmid.

[0064] Example 3

[0065] See also Figure 2 As shown, Figure 2 Flow chart of the method for improving the efficiency of yeast CRISPR-Cas gene editing of Example 3 of the present invention, the host strain used in this embodiment is Saccharomyces cerevisiae BY4741; the specific implementation steps are as follows: first, the SP_C plasmid (including Checkpoint promoters CRT1, HUG1, RNR1, RNR2) is introduced into Saccharomyces cerevisiae cells by electroporation transformation or chemical transformation, and the YC yeast strain is obtained by SC-Ura medium screening. Then use it as a host cell to make yeast competent, and then mix the pCas_N plasmid and the pCas_T plasmid in different proportions (1: 10, 1: 50, 1: 100, 1: 1000) to test the resistance of the system to unnecessary interference introduced during the gene editing process. Subsequently, it is introduced into yeast cells by electroporation transformation or chemical transformation. The pCas_T plasmid is introduced into the YC yeast competent cell by electroporation transformation or chemical transformation. The Cas protein expressed by the pCas_T plasmid and the gRNA2 targeting the yeast genome target gene 2 (ADE2 gene) work together to shear the yeast genomic DNA. In this process, the DNA double-strand break will activate the Checkpoint promoter on the SP_C plasmid, transcribe and synthesize the gRNA1 targeting the target gene 1 (CAN1 gene), and work together with the Cas protein expressed by the pCas_T plasmid to cleave the gene. At the same time, the homologous repair template in the gRNA1 expression box is used for DNA repair. Two "TAATAA" termination codons are inserted into the homologous repair template, which inactivates the plasma membrane arginine permease encoded by the CAN1 gene, and the host cells can survive in the medium containing canavanine. If the Cas protein expressed by the pCas_T plasmid and the gRNA1 on the SP_C plasmid do not transform the target gene, the CAN1 gene of the yeast strain continues to express, and the cells can absorb canavanine through the plasma membrane arginine permease. Canavanine is toxic to the cells and prevents them from growing normally.

[0066] Gene editing efficiency Figure 4As shown, at a ratio of 1:10, the gene editing efficiency of ADE2 in the control group (without canavanine screening) remained low in all tests (Checkpoint promoters were CRT1, HUG1, RNR1, and RNR2, respectively), ranging from 5.0% to 16.6%. In contrast, the experimental group significantly improved the editing efficiency, up to 76.6%. As the proportion of pCas_T plasmids gradually increased, the editing efficiency continued to increase, reaching 94.2% to 99.2%. Comparison of colony color changes in the experimental and control groups before and after screening Figure 5 In summary, even if a certain amount of non-targeted designed plasmid is added, this embodiment can still maintain a stable and high gene editing efficiency.

[0067] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A method for improving the efficiency of CRISPR-Cas gene editing in cells, characterized in that: The steps include: (1) The SP_C plasmid is transferred into the host cell. The SP_C plasmid contains a gRNA1 expression cassette targeting the target gene 1, and a Checkpoint promoter capable of responding to the cell DNA damage mechanism is inserted into the 5' end. The Checkpoint promoter is activated by the Cas protein shearing the DNA double strand of the target gene 2; (2) transferring the pCas_T plasmid into the recombinant strain obtained in step (1), wherein the pCas_T plasmid is inserted with a Cas protein and a gRNA2 expression cassette targeting a target gene 2 of the cell genome; (3) The Cas protein expressed by the pCas_T plasmid binds to the gRNA2 expression cassette to cut the DNA double strand of the target gene 2, activate the Checkpoint promoter in the SP_C plasmid, and the transcribed gRNA1 expression cassette binds to the Cas protein to perform gene editing on the target gene 1. The strains that have only undergone gene editing are screened using a plate containing the screening substance.

2. The method according to claim 1, characterized in that The checkpoint promoters described in step (1) include but are not limited to CRT1, HUG1, RNR1, RNR2, RNR3, and RNR4; the target gene 1 includes but is not limited to the CAN1 gene.

3. The method according to claim 1, characterized in that The nucleic acid sequence of the gRNA1 expression cassette described in step (1) is shown in SEQ ID NO.1 or SEQ ID NO.2; the host cell includes but is not limited to Saccharomyces cerevisiae.

4. The method according to claim 1, characterized in that: The screening markers in the SP_C plasmid described in step (1) and the pCas_T plasmid described in step (2) include but are not limited to HIS3, LEU2, URA3 and TRP1, and the screening markers in the SP_C plasmid and the pCas_T plasmid are different.

5. The method according to claim 1, characterized in that The vector backbones of the SP_C plasmid described in step (1) and the pCas_T plasmid described in step (2) include but are not limited to pCRCT.

6. The method according to claim 1, characterized in that The transformation methods in step (1) and step (2) include but are not limited to chemical transformation and electroporation transformation.

7. The method according to claim 1, characterized in that The Cas proteins in step (2) include but are not limited to Cas9, AsCas12a, FnCas12a, LbCas12a and MAD7.

8. The method according to claim 1, characterized in that The nucleotide sequence of the gRNA2 expression cassette in step (2) is shown in SEQ ID NO.3 or SEQ ID NO.

4.

9. The method according to claim 1, characterized in that: The target gene 2 mentioned in step (2) includes but is not limited to the ADE2 gene.

10. The method according to claim 1, characterized in that The screening substances in step (3) include, but are not limited to, canavanine (L-canavanine), 5-fluoroorotic acid (5-FOA), and 2-amino-5-fluorobenzoic acid (5-FAA).

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