Method for synthesizing random gRNA library by in vitro transcription and application

Abstract

The application provides a method for synthesizing a random gRNA library by in vitro transcription and application. The method comprises the following steps: synthesizing a single-stranded DNA primer library containing 20 degenerate bases by chemical synthesis, obtaining a double-stranded gDNA library by specific primer PCR amplification, and obtaining a random gRNA library by in vitro transcription mediated by T7 RNA polymerase. The random gRNA library is used for genome directed evolution of a beta-carotene producing Saccharomyces cerevisiae strain, a Cas9-NG expression frame is introduced, the gRNA library is electrotransformed, a non-fidelity repair mechanism is used to introduce diversified mutations, and finally a mutant strain with significantly improved beta-carotene yield is obtained. The application has the advantages of simple process, low cost, high gRNA diversity, wide host range and the like, can effectively improve the modification efficiency of a microbial strain, and is suitable for industrial microbial breeding.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a method and application for synthesizing random gRNA libraries through in vitro transcription. Background Technology

[0002] Microbial cell factories are the core carriers of modern biomanufacturing, playing an irreplaceable role in the efficient synthesis of high-value-added compounds such as natural products, amino acids, organic acids, vitamins, and functional sugars. However, the metabolic regulatory network of microorganisms is extremely complex, and rational metabolic engineering strategies are often difficult to predict and modify precisely. Therefore, irrational random genomic mutagenesis technology remains an indispensable tool for improving strain performance. Traditional physicochemical mutagenesis methods have limitations such as difficulty in controlling the direction of mutation, limited mutation types, susceptibility to cytotoxicity, and the need for specialized equipment.

[0003] In recent years, CRISPR-Cas-based genome mutation technology has developed rapidly, providing new tools for laboratory evolution. Among them, a technique called CRISPR-Cas-assisted random mutagenesis has shown promising application prospects. This technique involves constructing a gRNA plasmid library containing random degenerate base sequences, introducing it into cells, and expressing it to produce diverse gRNAs. These gRNAs guide Cas proteins to randomly cleave the genome, thereby introducing a wide range of mutations using the cell's own non-fidelity repair mechanisms.

[0004] However, this technology still faces a key bottleneck in practical applications: obtaining random gRNA libraries relies on constructing large plasmid libraries. This construction process involves multiple complex steps, including PCR amplification, restriction enzyme digestion, ligation, transformation of E. coli, amplification culture, and plasmid extraction. This process is not only lengthy and time-consuming, but also expensive in terms of materials and reagents. More importantly, the copy number of plasmids within host cells is limited and heterogeneous, fundamentally limiting the types and quantities of effective gRNAs that can be accommodated in a single cell. Even with optimization, each cell can only express an average of about 130 different gRNAs, which is far from sufficient to cover the complex genomes of microorganisms and generate sufficiently rich mutational diversity. Furthermore, this plasmid-dependent strategy also faces challenges such as poor universality, low electroporation efficiency of large plasmid libraries, and the need for additional plasmid removal during subsequent iterative evolution, further increasing the complexity of the technology's application.

[0005] Therefore, how to simplify the synthesis process of random gRNA libraries, reduce their cost, and overcome the limitations of intracellular gRNA types and quantities, thereby developing a more efficient, convenient, and universal microbial genome mutation technology, is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method and application for synthesizing random gRNA libraries through in vitro transcription, thereby solving the technical problems of "the existing technology relying on plasmid library construction, resulting in complex operation, poor universality, and insufficient types and quantities of effective gRNAs in cells due to limitations in plasmid copy number".

[0007] To achieve the above objectives, the present invention is implemented using the following technical solution: In a first aspect, the present invention provides a method for synthesizing a random gRNA library by in vitro transcription. The method first constructs a single-stranded DNA primer library containing 20 consecutive degenerate bases, wherein the degenerate base regions correspond to the target sequences of gRNAs; using the primer library as a template, PCR amplification is performed using specific upstream and downstream primers to obtain a double-stranded gDNA library; then, using the double-stranded gDNA library as a template, in vitro transcription is mediated by T7 RNA polymerase to finally obtain a random gRNA library.

[0008] The nucleotide sequence of the single-stranded DNA primer library is shown in SEQ ID NO. 1. Each single-stranded DNA molecule in the library consists of a T7 promoter sequence, a 20-base gRNA targeting sequence, and an 82-base gRNA backbone sequence, from the 5′ end to the 3′ end. The 20-base targeting sequence is completely randomized, with each site independently containing four bases: A, T, C, and G. The proportions of these four bases are relatively balanced, resulting in high sequence diversity in the library.

[0009] SEQ ID NO.1: TAATACGACTCACTATAGGNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT.

[0010] Using the aforementioned random primer library as a template, PCR amplification with upstream primer gRNA-F and downstream primer gRNA-R yields a structurally complete double-stranded gDNA library suitable for in vitro transcription. Using this gDNA library as a template, T7 RNA polymerase is added to the in vitro reaction system. The enzyme specifically recognizes and binds to the T7 promoter region, initiating the transcription reaction and ultimately obtaining a complete random gRNA library.

[0011] The gRNA-F sequence is shown in SEQ ID NO.2: TAATACGACTCACTATAGG.

[0012] The gRNA-R sequence is shown in SEQ ID NO.3: AAAAAAGCACCGACTCGGTGCCAC.

[0013] The second aspect of this invention provides the application of the above-mentioned in vitro transcription synthesis random gRNA library, specifically, using the library to perform genome-directed evolution of β-carotene-producing Saccharomyces cerevisiae strains to obtain mutant strains with significantly enhanced β-carotene synthesis capabilities.

[0014] The β-carotene-producing Saccharomyces cerevisiae strain uses Saccharomyces cerevisiae BY4741 as the starting strain. Key gene expression frames essential for β-carotene synthesis are integrated into specific sites in its genome. The specific integration sites and gene combinations include: HOΔ::PTEF1-crtI, Gal80Δ::PHXT7-crtYB, Gal1-7Δ::PTEF1-tHMG1, and PHXT7-crtE, enabling the strain to stably synthesize and accumulate β-carotene.

[0015] In the aforementioned β-carotene-producing strains, an expression cassette containing the Cas9-NG protein coding sequence was further introduced, enabling the host cells to stably express the Cas9-NG protein. After introducing a random gRNA library obtained through in vitro transcription into the host cells via electroporation, the gRNA binds to the intracellularly expressed Cas9-NG protein, performing random double-stranded DNA cleavage of the yeast genome. The cells repair the break sites using their own non-fidelity DNA repair system, introducing diverse mutations such as base insertions, deletions, inversions, duplications, and chromosomal rearrangements during the repair process, thereby achieving highly efficient random mutations at the whole genome scale.

[0016] The Cas9-NG is a modified spCas9 protein mutant with amino acids mutated at positions 1111, 1135, 1218, 1219, 1322, 1335, and 1337 to arginine, valine, arginine, phenylalanine, arginine, valine, and arginine, respectively. Compared to wild-type Cas9, Cas9-NG can recognize a wider range of PAM sites, including NG, NAC, NTG, NTT, and NCG, significantly expanding the genome targeting range and improving mutation coverage.

[0017] Based on the mutant strains obtained from the above mutation system, positive mutant strains with significantly increased β-carotene production can be obtained through shake-flask fermentation and product detection screening, thus achieving rapid and efficient directed evolution of the strains.

[0018] Compared with the prior art, the beneficial effects achieved by the present invention are: (1) This invention directly synthesizes random gRNA libraries through in vitro transcription, without the need to construct plasmid libraries, which greatly simplifies the operation process and reduces the preparation cost. At the same time, it avoids the limitation of plasmid copy number on gRNA diversity and significantly improves the coverage and efficiency of random mutations in the genome.

[0019] (2) The present invention uses Cas9-NG protein as a nuclease tool, which can recognize a wider range of PAM sites, effectively expand the mutation target range, and introduce multiple mutation types with the help of cell non-fidelity repair, which is conducive to rapidly obtaining mutant strains with improved traits.

[0020] (3) The present invention applies the constructed random gRNA library to the directed evolution of high β-carotene-producing Saccharomyces cerevisiae strains, combined with rapid phenotypic screening, to achieve efficient breeding of strains. It is versatile, easy to operate, and has good application prospects in the field of industrial microbial modification. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the in vitro synthesis process of the random gRNA library of the present invention.

[0022] Figure 2 This is an electrophoresis diagram of a random gRNA library.

[0023] Figure 3 This is a schematic diagram illustrating the mechanism of action of random gRNA library-mediated random mutations across the entire genome in this invention.

[0024] Figure 4 This is a schematic diagram illustrating the phenotypic differences of the mutant strains of the present invention.

[0025] Figure 5 This is a graph showing the yield analysis of β-carotene during shake-flask fermentation of the mutant strain. Detailed Implementation

[0026] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0027] Example 1: In vitro transcription synthesis of random gRNA libraries This embodiment illustrates the complete preparation process and quality control methods for random gRNA libraries.

[0028] The primer library used in the experiment was chemically synthesized by a professional biotechnology company. The high-fidelity DNA polymerase, high-fidelity buffer, dNTPs, T7 in vitro transcription kit, RNA purification kit and other reagents were all commercially available products. The ultra-micro spectrophotometer, PCR instrument, constant temperature metal bath, agarose electrophoresis instrument and other equipment were all routine molecular biology equipment.

[0029] The nucleotide sequence of the gRNA in the random primer library is shown in SEQ ID NO.1: TAATACGACTCACTATAGGNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT.

[0030] The gRNA-F nucleotide sequence is shown in SEQ ID NO.2: TAATACGACTCACTATAGG.

[0031] The gRNA-R nucleotide sequence is shown in SEQ ID NO.3: AAAAAAGCACCGACTCGGTGCCAC.

[0032] Since the initial random primer library contains very little template, it cannot be directly used for subsequent in vitro transcription. Therefore, it is necessary to obtain a sufficient amount of double-stranded DNA template through PCR amplification first. To obtain a sufficient amount of structurally homogeneous double-stranded DNA template, this embodiment first performs amplification of the random double-stranded DNA library. The total volume of the reaction system is 50 μL, including 10 μL of high-fidelity buffer, 1 μL of 10 mmol / L dNTPs, 2.5 μL of 10 μmol / L upstream primer gRNA-F, 2.5 μL of 10 μmol / L downstream primer gRNA-R, 1 μL of 10 ng / μL random primer library template, 0.5 μL of high-fidelity DNA polymerase, and the remaining volume is made up with deionized water. The PCR reaction program was set to pre-denaturation at 98℃ for 3 min, followed by 35 cycles of amplification reaction. Each cycle included denaturation at 98℃ for 10 s, annealing at 58℃ for 15 s, extension at 72℃ for 15 s, final extension at 72℃ for 5 min after the cycle, and finally storage at 4℃.

[0033] After PCR amplification, the product needs to be structurally confirmed and purified to provide a suitable template for subsequent in vitro transcription.

[0034] Combination Figure 1As shown in the synthesis process, the chemically synthesized single-stranded DNA random primer library in this embodiment contains, from the 5′ end to the 3′ end, a T7 RNA polymerase binding region, a random targeting region of 20 degenerate bases, and an 82-base gRNA backbone region. Using the above single-stranded DNA random primer library as a template, PCR amplification is performed using upstream primer gRNA-F and downstream primer gRNA-R to obtain a double-stranded DNA library that covers the complete expression element and contains 20 random base sequences, which is named gDNA-20N.

[0035] To verify the amplification effect, the PCR products were detected by 1.5% agarose gel electrophoresis. The results showed that the target band was single and clear, with no obvious impurities or primer dimers, indicating good amplification results. Subsequently, gDNA-20N was purified using a DNA purification kit to remove enzymes, salt ions, and non-specific fragments, finally obtaining purified gDNA-20N suitable for subsequent in vitro transcription.

[0036] Using purified gDNA-20N as a template, in vitro transcription was performed using T7 RNA polymerase to obtain a random gRNA library. After in vitro transcription, residual DNA template was removed by DNase I digestion, and then RNA was purified to obtain a high-purity random gRNA library.

[0037] To ensure that the prepared random gRNA library meets the requirements of subsequent experiments, its concentration, purity, and integrity need to be tested. The random gRNA library was quality-tested using an ultra-micro spectrophotometer. The results showed that the purified random gRNA library concentration reached 1715 ng / μL, and the OD260 / OD280 ratio was 2.1, indicating high product purity and low protein and organic solvent residues, meeting the requirements for in vivo cell experiments.

[0038] like Figure 2 As shown, the random gRNA library bands are single, clear, and without obvious impurities or degradation tails. The band positions are between 100bp and 250bp, which is in complete agreement with the theoretical length of the gRNA designed in this invention, which is about 120bp. This further verifies that the synthesized random gRNA library has a complete structure, high purity, and no degradation, which meets the requirements for subsequent microbial cell electroporation and whole-genome random mutation experiments.

[0039] Example 2; Construction of β-carotene-producing Saccharomyces cerevisiae strain C0 The core objective of this embodiment is to construct a basic strain that can be used for subsequent whole-genome random mutation experiments, namely, a Saccharomyces cerevisiae strain C0 that stably expresses the Cas9-NG protein and can synthesize β-carotene.

[0040] As can be seen from Example 1 above, the present invention has successfully prepared a random gRNA library. For the random gRNA library to function, it must rely on a host cell that can express the Cas9-NG protein. At the same time, in order to quickly screen for high-yield mutant strains, the host cell must also have the ability to stably synthesize β-carotene. Therefore, this example specifically carried out the construction of strain C0 to provide a qualified experimental vector for subsequent mutation experiments.

[0041] The starting strain used in the experiment was Saccharomyces cerevisiae BY4741, and the plasmid used was the pCas9-NG vector, which can stably express the Cas9-NG protein.

[0042] The construction process mainly consists of two steps: first, enabling the starter strain to synthesize β-carotene, and then introducing it into the Cas9-NG protein expression vector. The specific steps are as follows: First, key gene expression frames for the β-carotene synthesis pathway were integrated into specific sites in the *Saccharomyces cerevisiae* BY4741 genome. These integration sites and gene combinations included: HOΔ::PTEF1-crtI, Gal80Δ::PHXT7-crtYB, Gal1-7Δ::PTEF1-tHMG1, and PHXT7-crtE. These four genes are essential for β-carotene synthesis; integrating them into the host genome enabled the BY4741 strain to stably synthesize and accumulate β-carotene.

[0043] The Cas9-NG is a modified spCas9 mutant, with its amino acids at positions 1111, 1135, 1218, 1219, 1322, 1335, and 1337 mutated to arginine, valine, arginine, phenylalanine, arginine, valine, and arginine, respectively. It can recognize broad-spectrum PAM sites such as NG, NAC, NTG, NTT, and NCG.

[0044] To ensure that the selected strains fully met the experimental requirements, colony PCR and sequencing were performed on the colonies grown on the plates to confirm that the Cas9-NG protein gene sequence was correct, without mutations, and capable of normal expression of the functional protein. After the above series of screening and verification, a Saccharomyces cerevisiae strain C0 that stably expresses the Cas9-NG protein and can stably synthesize β-carotene was obtained.

[0045] Example 3: Random gRNA library-mediated genome-wide random mutation and mutant screening The core objective of this embodiment is to introduce the random gRNA library prepared in Example 1 into strain C0 constructed in Example 2, induce random mutations throughout the strain's genome, and screen for mutants with phenotypic differences to provide candidate strains for subsequent yield verification.

[0046] (1) Preparation of competent cells of strain C0: First, pick a single colony of strain C0 and inoculate it into 5 mL of SD-Leu liquid medium. Incubate at 30℃ and 220 r / min until the OD600 value reaches 1.0. Then expand the culture to 50 mL and continue to culture until the OD600 value reaches 0.9. (2) Treatment of cells as competent cells: The bacterial culture was placed on ice for 30 min, centrifuged at 4℃ and 4000 r / min for 5 min to collect the cells, the supernatant was discarded, the cells were washed twice with sterile water, and then washed twice with 1 mol / L sorbitol. Finally, the cells were resuspended in 100 μL of 1 mol / L sorbitol solution to prepare fresh competent cells. (3) Introduce the random gRNA library into competent cells: Take 5 μg of the random gRNA library prepared in Example 1 and mix it gently with 100 μL of the above competent cells. Transfer the mixture into a pre-cooled 2 mm electroporation cuvette and place it on ice for 5 min. Set the electroporation conditions to 1.5 kV, 200 Ω, and 25 μF. Immediately after electroporation, add 1 mL of 1 mol / L sorbitol solution, mix gently by pipetting, and incubate at 30 °C for 2 h to obtain the recovered bacterial culture. (4) Screening and preliminary verification of mutants: The above-mentioned revived bacterial culture was serially diluted to 10. -3 Take 100 μL of the diluted solution and spread it evenly on an SD-Leu plate. Incubate at 30°C for 48 h to obtain mutant colonies.

[0047] Combination Figure 3 The diagram shown illustrates the principle and application process of random gRNA library-mediated random mutation of the entire microbial genome, fully demonstrating the entire process from gRNA library import to mutant strain screening.

[0048] like Figure 3 As shown on the left, in host cells expressing the Cas9-NG protein, a random gRNA library binds to Cas9-NG to form a complex, recognizing PAM sites across the entire genome and performing double-stranded DNA cleavage. Cells repair these breaks via their own non-fidelity non-homologous end joining pathway. This process requires no exogenous template and can spontaneously introduce diverse mutations such as insertions, deletions, inversions, duplications, and chromosomal rearrangements, thereby achieving random mutations across the entire genome and providing rich genetic diversity for strain improvement.

[0049] like Figure 3 As shown on the right, after transformation and mutation repair, cells were plated on screening plates. Due to differences in β-carotene accumulation, the colonies exhibited significant color differences, allowing for rapid initial screening of high-yielding candidate strains by visual inspection. Further shake-flask fermentation and quantitative analysis yielded superior mutant strains with significantly enhanced target products, completing the entire process from random mutation to targeted screening.

[0050] like Figure 4 As shown, mutant colonies exhibit distinct color variations due to differences in β-carotene accumulation: most colonies are light-colored, consistent with the starting strain C0, while colonies with significantly deepened orange-yellow color are high-yielding candidate mutants, which can be screened directly by the naked eye.

[0051] Example 4: Analysis of fermentation and β-carotene yield, stability and iterative evolution of mutant strains The core objective of this embodiment is to build upon the initial screening results of Example 3, confirm the β-carotene production enhancement effect of the positive mutant strain, verify the industrial application potential of the mutant strain, and demonstrate that the technology of this invention can achieve multiple rounds of iterative evolution to further improve the performance of the strain.

[0052] As can be seen from Example 3, a batch of mutant colonies with significantly deepened orange-yellow color were obtained from SD-Leu plates through initial screening by the naked eye. However, visual observation can only preliminarily determine the phenotypic differences and cannot accurately quantify the yield increase. At the same time, in industrial applications, the strains need to have stable production performance and should not show a decrease in yield after subculturing. In addition, in order to verify the reproducibility and efficiency of the present invention, multiple rounds of iterative evolution experiments need to be carried out to verify the technical effect of the present invention and support the value service of industrial applications.

[0053] (1) Five mutant colonies with significantly deepened orange-yellow color were selected from the SD-Leu plate of Example 3 and numbered CS1-1, CS1-2, CS1-3, CS1-4 and CS1-5 respectively. These mutant colonies were inoculated into 5mLYPD test tubes and cultured at 30℃ and 220r / min for 24h to complete seed activation. (2) After the seeds are activated, the seed liquid is transferred to a 250 mL shake flask containing 50 mL of LYPD medium at an inoculation rate of 1%, and cultured at 30 °C and 220 r / min for 72 h to complete shake flask fermentation. (3) After fermentation, take 1 mL of fermentation broth, centrifuge at 8000 r / min for 5 min to collect cells, discard the supernatant, then add 3 mL of acetone solution, vortex for 10 min to mix thoroughly, and then extract at 55℃ in the dark for 15 min. After extraction, centrifuge again at 8000 r / min for 10 min, take the supernatant, and measure the OD450 value on a spectrophotometer. To ensure accurate quantitative results, this invention uses commercially available β-carotene standards to plot a standard curve and simultaneously measures the dry weight of the bacterial cells, ultimately calculating the β-carotene yield of each mutant strain.

[0054] like Figure 5As shown, all the mutant strains obtained from the initial screening had higher β-carotene production than the starting strain C0. The CS1-2 strain showed the most significant increase, with a relative yield 1.35 times that of the starting strain. The experimental error bars were small, indicating that the experimental data were stable and reliable. Specifically, the measured results showed that the β-carotene production of the starting strain C0 was 10.4 mg / gCDW, while the mutant strain CS1-2 produced 14.04 mg / gCDW. This fully demonstrates that the random gRNA library-mediated whole-genome mutation technology provided in this invention can effectively improve the β-carotene synthesis capacity of strains, with a significant improvement effect.

[0055] The mutant strain CS1-2 with the highest yield was passaged five times on YPD plates without selection pressure. Single colonies were picked from each generation, and the yield of β-carotene was determined according to the shake-flask fermentation and quantitative detection methods described above.

[0056] The results showed that after five consecutive generations, the relative deviation of β-carotene production in the mutant strain CS1-2 was less than ±3%, and there was no obvious downward trend. This indicates that the mutant strain has excellent genetic stability and will not undergo significant reversion mutations during the generation process, thus possessing potential value for industrial-scale application.

[0057] To further increase β-carotene production, this invention uses the high-yielding strain CS1-2 obtained in the first round of evolution as a new starting strain. Following the method in Example 3, the random gRNA library prepared in Example 1 is electroporated. The process of mutation, initial screening, and fermentation verification is repeated to quickly complete the second round of iterative evolution, and finally the mutant strain CS2-6 is obtained.

[0058] Shake-flask fermentation tests showed that the β-carotene production of the mutant strain CS2-6 reached 1.62 times that of the starting strain C0, with the evolutionary effect continuously accumulating. Moreover, no plasmid elimination step was required throughout the process, and a complete evolution cycle could be completed in just about 3 days. This fully demonstrates that the iterative evolution process of the present invention is efficient and convenient, and can quickly obtain ultra-high-yielding strains, further highlighting the practicality and superiority of the present invention.

[0059] In summary, this embodiment confirmed the high-yield characteristics of the initially screened mutant through quantitative yield analysis, verifying the improvement effect of the technology of the present invention; verified the industrial application potential of the mutant through genetic stability verification; and demonstrated the reproducibility and efficiency of the technology of the present invention through multiple rounds of iterative evolution verification.

[0060] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No markings in the claims should be construed as limiting the scope of the claims.

Claims

1. A method for synthesizing a random gRNA library via in vitro transcription, characterized in that, Includes the following steps: (1) A chemically synthesized single-stranded DNA random primer library, wherein the primer library contains, from the 5′ end to the 3′ end, a T7 RNA polymerase binding region, a random targeting region of 20 consecutive degenerate bases, and a gRNA backbone region; (2) Using the single-stranded DNA random primer library as a template, PCR amplification was performed using specific upstream and downstream primers to obtain a double-stranded DNA library; (3) The double-stranded DNA library is purified to obtain a purified double-stranded gDNA library; (4) Using the purified double-stranded gDNA library as a template, in vitro transcription was performed using T7 RNA polymerase to obtain a random gRNA library.

2. The method according to claim 1, characterized in that, The nucleotide sequence of the single-stranded DNA random primer library is shown in SEQ ID NO.

1.

3. The method according to claim 1, characterized in that, The nucleotide sequence of the specific upstream primer is shown in SEQ ID NO.

2.

4. The method according to claim 1, characterized in that, The nucleotide sequence of the specific downstream primer is shown in SEQ ID NO.

3.

5. The use of a random gRNA library as described in any one of claims 1-4 in the preparation of a high-β-carotene-producing Saccharomyces cerevisiae mutant strain.

6. The application according to claim 5, characterized in that, The β-carotene-containing Saccharomyces cerevisiae mutant strain was obtained by introducing the random gRNA library into host cells expressing Cas9-NG protein via electroporation, mediating double-stranded DNA cleavage of the genome, and then repairing the cell's own non-fidelity DNA.

7. The application according to claim 6, characterized in that, The Cas9-NG is a modified spCas9 protein mutant, in which amino acids at positions 1111, 1135, 1218, 1219, 1322, 1335, and 1337 of the spCas9 protein are mutated to arginine, valine, arginine, phenylalanine, arginine, valine, and arginine, respectively; the PAM sites that the Cas9-NG can recognize include NG, NAC, NTG, NTT, and NCG.

8. The application according to claim 6, characterized in that, The mutations mediated by the random gRNA library include base insertions, deletions, inversions, duplications, and chromosomal rearrangements.

9. The application according to claim 6, characterized in that, The host cell was Saccharomyces cerevisiae BY4741.

10. The application according to claim 6, characterized in that, The host cell contains the pCas9-NG vector.

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