Composition for gene editing and application thereof in gene editing

Abstract

The invention discloses a composition for gene editing and application thereof in gene editing, and belongs to the technical field of genetic engineering. The invention provides an efficient traceless gene editing method for pichia pastoris. According to the technical scheme provided by the invention, traceless knockin or knockout of the pichia pastoris gene can be realized only by constructing the donor fragment, and Ura3 can be repeatedly used as a selection marker, so that knockin or knockout of a plurality of genes is realized, and the pain point that no selection marker is available in multiple rounds of gene editing in pichia pastoris is solved.

Description

Technical Field

[0001] This application belongs to the field of genetic engineering technology, specifically relating to compositions for gene editing and their application in gene editing. Background Technology

[0002] Pichia pastoris ( Pichia pastoris Pichia pastoris is widely used in modern industry. As a methyltrophic microorganism, it can grow using methanol as its sole carbon source. Due to its advantages such as rapid growth, relatively simple gene manipulation, numerous strong promoters, high protein expression efficiency, ability to perform post-translational modifications, low secretion of extraneous proteins, and ease of high-density fermentation, it has been widely used to express various heterologous proteins. However, compared to protein production, the engineered biosynthesis of compounds typically requires the integration of multiple heterologous genes and the optimization of multiple metabolic pathways.

[0003] The URA3 gene is a coding gene on yeast chromosome V, and its product, orotidine 5-phosphate decarboxylase, is involved in the biosynthesis of uracil nucleotides. The absence of this enzyme prevents yeast from synthesizing uracil autonomously, resulting in an auxotrophic phenotype. This gene is widely used as a selection marker in gene editing technology, enabling efficient screening of transformed strains through the 5-fluoroorotidine (5-FOA) toxicity screening system. Researchers have used its upstream and downstream sequences to construct homologous recombination arms, developing a multi-species applicable genetic operating system. Targeted gene knockout can be achieved by PCR amplification of the URA3 upstream and downstream sequences and their connection to selection markers (such as the G418 resistance gene) to form a knockout cassette.

[0004] Currently used gene editing technologies rely on the simultaneous recombination of the target gene and a selection marker. Each round of editing consumes a selection marker, but Pichia pastoris only has 3-4 efficient markers available, leading to marker depletion during multi-gene sequential editing. To enable multi-round gene editing, several marker-free editing systems have been established in Pichia pastoris. For example, systems based on Flp / FRT and Cre / loxP recombinases have been developed. However, these systems still leave redundant sequences such as recombinase recognition sites (FRT / loxP) after genome manipulation. These residual sequences may interfere with host genome stability, thus increasing the risk of unintended gene recombination. In recent years, the CRISPR-Cas9 system has also been used in Pichia pastoris genome modification. However, the main repair mechanism in Pichia pastoris is non-homologous end joining (NHEJ). The Cas9-mediated gRNA cleavage of the genome depends on both the sgRNA cleavage efficiency and homologous recombination (HR), resulting in lower integration efficiency compared to double crossover integration with selection markers. Performing multiple rounds of gene editing requires repeated elimination of the edited plasmid, and without appropriate selection pressure, the edited plasmid is difficult to eliminate.

[0005] Therefore, it is particularly important to improve the homologous recombination (HR) efficiency of Pichia pastoris and to develop reusable screening markers to support multiple rounds of high-efficiency strain construction. Summary of the Invention

[0006] The technical problem this application aims to solve is how to address the issues of insufficient selection markers (limiting multiple gene editing), easy residues of selection markers or other redundant sequences in the genome, and low editing efficiency in existing technologies. To solve this technical problem, this application provides the following technical solution: This application provides compositions for gene editing, said compositions comprising donor DNA pair A or donor DNA pair B; The donor DNA pair A may consist of two double-stranded DNA fragments named donor DNA fragment A1 and donor DNA fragment A2. One single strand of donor DNA fragment A1 may contain, from upstream to downstream, an upstream homologous arm, a downstream homologous arm, and a portion of the Ura3 expression frame for homologous recombination with the cell to be edited. One single strand of donor DNA fragment A2 may contain, from upstream to downstream, another portion of the Ura3 expression frame and the downstream homologous arm. The 3' end of the single strand of donor DNA fragment A1 and the 5' end of the single strand of donor DNA fragment A2 contain a sequence-identical overlap portion located in the Ura3 expression frame. The donor DNA pair B may consist of two double-stranded DNA fragments named donor DNA fragment B1 and donor DNA fragment B2. One single strand of donor DNA fragment B1 may contain a portion of the Ura3 expression frame, the upstream homologous arm, and the downstream homologous arm from upstream to downstream. One single strand of donor DNA fragment B2 may contain the upstream homologous arm and another portion of the Ura3 expression frame from upstream to downstream. The 5' end of the single strand of donor DNA fragment B1 and the 3' end of the single strand of donor DNA fragment B2 have an overlapping portion with the same sequence, and the overlapping portion is located in the Ura3 expression frame.

[0007] In this application, the gene editing may be gene knockout or knock-in.

[0008] In this application, the length of the upstream homologous arm and / or the downstream homologous arm may be at least one of the following: 100 to 2000 bp, 200 to 1900 bp, 300 to 1800 bp, 400 to 1700 bp, 500 to 1600 bp, 600 to 1500 bp, 700 to 1400 bp, 800 to 1300 bp, 900 to 1200 bp, 900 to 1100 bp, 910 to 1090 bp, 920 to 1080 bp, 930 to 1070 bp, 940 to 1060 bp, 950 to 1050 bp, 960 to 1040 bp, 970 to 1030 bp, 980 to 1020 bp, 990 to 1010 bp, and 1000 bp.

[0009] In this application, the size of the overlapping portion described in A1) and / or the overlapping portion described in A2) may be at least one of the following: 100 to 2000 bp, 200 to 1900 bp, 300 to 1800 bp, 400 to 1700 bp, 500 to 1600 bp, 600 to 1500 bp, 700 to 1400 bp, 700 to 1300 bp, 700 to 1200 bp, 700 to 1100 bp, 710 to 1090 bp, 720 to 1080 bp, 730 to 1070 bp, 740 to 1060 bp, 750 to 1050 bp, 760 to 1040 bp, or 770 to 1030 bp.

[0010] In the above composition, the gene editing can be gene knockout, the sequence of the upstream homologous arm is the same as the upstream sequence of the target gene to be knocked out in the cell to be edited, and the sequence of the downstream homologous arm is the same as the downstream sequence of the target gene to be knocked out.

[0011] In the above composition, the gene editing can be gene knock-in, and the upstream homologous arm and the downstream homologous arm of the donor DNA fragment A1 and the donor DNA fragment B1 further contain a gene expression cassette to be knocked in; The sequence of the upstream homologous arm is the same as the upstream sequence of the insertion site, and the sequence of the downstream homologous arm is the same as the downstream sequence of the insertion site. The insertion site is the insertion location of the gene expression cassette to be knocked in.

[0012] In this application, the gene to be knocked in is the cell to be introduced into the cell to be edited.

[0013] In the above composition, the donor DNA fragment A1 and the donor DNA fragment A2 can undergo homologous recombination and integration into recombinant DNA donor fragment A in the cell to be edited, and the donor DNA fragment B1 and the donor DNA fragment B2 can undergo homologous recombination and integration into recombinant DNA donor fragment B in the cell to be edited. The recombinant DNA donor fragment A may be at least one of the following: One strand of the recombinant DNA donor fragment A contains, from upstream to downstream, the following elements: an upstream homologous arm and a downstream homologous arm for homologous recombination with the cell to be edited, the Ura3 expression cassette, and the downstream homologous arm; Aii) One strand of the recombinant DNA donor fragment A contains, from upstream to downstream, the following elements: an upstream homologous arm that undergoes homologous recombination with the cell to be edited, a gene knock-in expression cassette, a downstream homologous arm that undergoes homologous recombination with the cell to be edited, the Ura3 expression cassette, and the downstream homologous arm. The recombinant DNA donor fragment B may be at least one of the following: Bi) One strand of the recombinant DNA donor fragment B contains, from upstream to downstream, the following elements: an upstream homologous arm that undergoes homologous recombination with the cell to be edited, the Ura3 expression cassette, the upstream homologous arm, and a downstream homologous arm that undergoes homologous recombination with the cell to be edited; One strand of the recombinant DNA donor fragment B (Bii) contains, from upstream to downstream, the following elements: an upstream homologous arm that undergoes homologous recombination with the cell to be edited, the Ura3 expression cassette, the upstream homologous arm, the gene to be knocked in expression cassette, and a downstream homologous arm that undergoes homologous recombination with the cell to be edited.

[0014] In this application, the Ura3 gene is a coding gene on yeast chromosome V, and its product, orotidine 5-phosphate decarboxylase, participates in the biosynthesis of uracil nucleotides. This gene is widely used as a screening marker in gene editing technology, enabling efficient screening of transformed strains through the 5-fluoroorotidine (5-FOA) toxicity screening system.

[0015] In this application, the orotidine 5-phosphate decarboxylase encoded by the Ura3 gene is a protein containing 263 amino acid residues. The coding sequence of the Ura3 gene in this application is shown at positions 367 to 1158 of SEQ ID NO:1.

[0016] In some embodiments of this application, the nucleotide sequence of the Ura3 expression cassette is SEQ ID NO:1. Specifically, positions 1 to 366 of SEQ ID NO:1 are the promoter sequence, positions 367 to 1158 are the Ura3 coding sequence, and positions 1159 to 1366 are the stop sequence.

[0017] In some embodiments of the present invention, the donor DNA fragments A1 and A2 used for gene knockout undergo homologous recombination and integration into the cell to be edited to form the recombinant DNA donor fragment A (Ai).

[0018] The nucleotide sequence of the recombinant DNA donor fragment A shown in Ai is as shown in SEQ ID NO:3. In SEQ ID NO:3, positions 1 to 988 are the upstream homologous arms, positions 989 to 1967 are the downstream homologous arms, positions 1968 to 3333 are the Ura3 expression cassette, and positions 3334 to 4312 are the downstream homologous arms.

[0019] The nucleotide sequence of the DNA donor fragment A1 corresponding to Ai is positions 1 to 3114 of SEQ ID NO:3, and the nucleotide sequence of the DNA donor fragment A2 is positions 2343 to 4312 of SEQ ID NO:3. Positions 2343 to 3114 of SEQ ID NO:3 are the overlapping sequences of the donor DNA fragment A1 and the donor DNA fragment A2.

[0020] In some embodiments of this application, the donor DNA fragments A1 and A2 used for gene knock-in undergo homologous recombination and integration into the cell to be edited to form recombinant DNA donor fragment A as shown in Aii).

[0021] The nucleotide sequence of recombinant DNA donor fragment A shown in Aii) is as shown in SEQ ID NO:4. In SEQ ID NO:4, positions 1 to 1056 are the upstream sequence of the knock-in site Int6, positions 1057 to 4694 are the hLF gene expression cassette, positions 4695 to 5729 are the downstream sequence of the knock-in site Int6, positions 5730 to 7095 are the Ura3 expression cassette, and positions 7096 to 8130 are the downstream sequence of the knock-in site Int6.

[0022] The nucleotide sequence of donor DNA fragment A1 corresponding to Aii) is positions 1 to 6876 of SEQ ID NO:4, and the nucleotide sequence of donor DNA fragment A2 is positions 6105 to 8130 of SEQ ID NO:4, wherein positions 6105 to 6876 of SEQ ID NO:4 are the overlapping sequences of donor DNA fragment A1 and donor DNA fragment A2.

[0023] In this application, the composition for gene editing may be a composition for gene editing in microbial cells.

[0024] This application also provides the use of the above-described composition in gene editing, wherein the gene editing may be gene knockout.

[0025] This application also provides the application of the above-described composition in gene editing, wherein the gene editing may be gene knock-in.

[0026] In this application, the gene editing (gene knockout or gene knock-in) can be performed in microbial cells.

[0027] This application also provides a gene knockout method, wherein the knockout may include the following steps: S1) Introduce the donor DNA pair A described above into the cells to be edited, and screen for recombinant cells containing the recombinant DNA donor fragment A described above (Ai), or The donor DNA pair B described above was introduced into the cells to be edited, and recombinant cells containing the recombinant DNA donor fragment B described in Bi) were screened. S2) Screening for target cells that survive on a culture medium containing uracil and 5-FOA; The cells to be edited do not contain the Ura3 gene.

[0028] This application also provides a gene knock-in method, wherein the knock-in may include the following steps: S1') The donor DNA pair A described above is introduced into the cells to be edited, and recombinant cells containing the recombinant DNA donor fragment A described in Aii) above are screened, or The donor DNA pair B described above was introduced into the cells to be edited, and recombinant cells containing the recombinant DNA donor fragment B described in Bii) above were screened. S2') Screening for target cells that survive on a medium containing uracil and 5-FOA; The cells to be edited do not contain the Ura3 gene.

[0029] In this application, the screening in S1) or S1') can be performed by uracil screening: the target microorganism that has been successfully recombined can grow on a culture medium that does not contain uracil.

[0030] In this application, the screening in S1) or S1') can also be performed by PCR sequencing: clones that can obtain the target product by PCR amplification are positive clones.

[0031] In this application, the screening in S2) or S2') can be performed by screening with uracil and 5-FOA: target cells with Ura3 gene elimination can survive on a culture medium containing uracil and 5-FOA.

[0032] In this application, the screening in S2) or S2') can also be performed by PCR sequencing: clones that can obtain the target product by PCR amplification are positive clones.

[0033] In some embodiments of this application, the uracil-free culture medium is MD medium, with the following formula: YNB: 13.4 g / L; glucose: 10 g / L; agar: 20 g / L, and water as the solvent.

[0034] In some embodiments of this application, the culture medium containing uracil and 5-FOA is MDU+5-FOA medium, with the following formula: YNB: 13.4 g / L; glucose: 10 g / L; uracil: 100 mg / L; 5-FOA: 1 mg / mL; agar: 20 g / L; and water as the solvent.

[0035] In the above method, the number of operations of steps S1) to S2) and / or S1') to S2') is N, where N is an integer ≥1, and the difference between the N operations lies in the different target and / or introduction site.

[0036] In this application, the cell to be edited may be a microbial cell.

[0037] In this application, the microorganism may be bacteria or fungi.

[0038] In this application, the fungus may be yeast.

[0039] In this application, the yeast includes, but is not limited to: Pichia pastoris ( Pichia pastoris ), brewer's yeast ( Saccharomyces cerevisiae ), Candida genus, such as Candida utilis ( Candida utilis ), Kluyveromyces, for example Kluyveromyces lactis or Kluyveromyces marxianus For example, Hansenula genus. Hansenula polymorpha Fission yeast ( Schizosaccharomyces pombe ), such as the genus *Torulopsis*. Torulopsis versatilis And the genus Rhodotorula.

[0040] The above method also includes the step of knocking out the Ura3 gene in the cells to be edited.

[0041] In some embodiments of this application, the Ura3 gene in the cell to be edited is knocked out by homologous recombination. In some specific embodiments of this application, the nucleotide sequence of the Ura3 knockout donor fragment is SEQ ID NO:2.

[0042] The beneficial technical effects achieved by this application are as follows: The composition and gene editing method provided in this application can achieve traceless, multiple knockout or knock-in of genes in cells to be edited. Attached Figure Description

[0043] Figure 1 The diagram shows the positional relationship between primers Ura3-LR-F and Ura3-LR-R and their amplified upstream homologous arms, primers Ura3-RR-F and Ura3-RR-R and their amplified downstream homologous arms, and the Ura3 genome used in the PCR identification of Pichia pastoris Ura3-deficient strains.

[0044] Figure 2 This is a schematic diagram of the seamless knock-in of the gene when homologous arm 2 and homologous arm 3 are identical, and expression frame 2 is the Ura3 expression frame.

[0045] Figure 3 This is a schematic diagram of the seamless knock-in of the gene when homologous arm 1 and homologous arm 2 are identical and expression frame 1 is the Ura3 expression frame.

[0046] Figure 4 The following is a gene knock-in diagram using the X33ΔUra3 defective strain prepared in Example 1 as the host bacterium, hLF as the knock-in gene, and INT6 gene as the knock-in site, with the recombinant donor DNA structure described in "(1) A structure of a recombinant donor DNA fragment" as an example.

[0047] Figure 5 PCR validation of hLF knock-in at the Int6 site was performed on colonies. Figure A shows homozygous heterozygote identification; homozygotes did not amplify the wild-type band, while heterozygotes or wild-type clones amplified a 2861 bp band. Lanes 1, 2, 5, 6, 7, 9, 11, 12, 13, 15, and 16 were homozygous. Figures B and C show donor fragment integration identification. In Figure B, the positive clone amplified a 7242 bp band; lanes 1, 2, 3, 4, 5, 6, and 8 were positive clones. In Figure C, the positive clone amplified a 2805 bp band; lanes 1, 2, 3, 4, 5, 7, and 8 were positive clones. The marker was a Bomeide 1kb DNA Ladder, catalog number MD114.

[0048] Figure 6This is a schematic diagram of the recombinant DNA fragment and the gene knockout when homologous arm 2 and homologous arm 3 are identical.

[0049] Figure 7 This is a schematic diagram of recombinant DNA fragments and gene knockout when homologous arm 1 and homologous arm 2 are identical.

[0050] Figure 8 The following is a gene knockout diagram using the X33ΔUra3 defective strain prepared in Example 1 as the host bacterium, Pep4 as the knockout gene, and the recombinant donor DNA structure described in "(1) A structure of a recombinant donor DNA fragment" as an example.

[0051] Figure 9 PCR validation of Pep4 knockout colonies. In Figure A, lanes 1-8 show homozygous identification; homozygotes did not amplify the wild-type band, while heterozygotes or wild-type amplified a 1233bp band. Lane 6 is homozygous. Figure B shows donor fragment integration identification. The primers used for lanes 1-8 were Pep4-JD-F and Ura3-5-R1, with a theoretical size of 3267bp. Lanes 1, 2, 5, 6, 7, and 8 were the correct size. The primers used for lanes 9-16 were Ura3 promoter-F1 and Pep4-JD-R, with a theoretical size of 2505bp. Lanes 9, 11, 12, 13, 14, 15, and 16 were positive clones. The marker was a Bomeide 1kb DNA Ladder, catalog number MD114. Detailed Implementation

[0052] I. Terms used in this application: Examples of resources describing many of the molecular biology-related terms used in this article can be found in the following literature: Alberts et al., Molecular Biology of The Cell, 5th ed., Garland Science Publishing, Inc.: New York, 2007; Rieger et al., Glossary of Genetics: Classical and Molecular, 5th ed., Springer-Verlag: New York, 1991; King et al., A Dictionary of Genetics, 6th ed., Oxford University Press: New York, 2002; and Lewin, GenesIX, Oxford University Press: New York, 2007.

[0053] Any references cited in this article, including, for example, all patents, published patent applications and non-patent publications, are incorporated in their entirety by reference.

[0054] For ease of understanding of this disclosure, several terms and abbreviations used herein are defined as follows: In this application, "identity" refers to the similarity of amino acid or nucleotide sequences. The similarity of amino acid sequences (or nucleotide sequences) can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, and setting the Gap existence cost, Perresidue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing a search for the similarity of a pair of amino acid sequences, the similarity value (%) can be obtained.

[0055] Specifically, the consistency of 70% or more can be 75% or more. Specifically, the consistency of 75% or more can be 80% or more. Specifically, the consistency of 80% or more can be 85% or more. Specifically, the consistency of 85% or more can be 90% or more. Specifically, the consistency of 90% or more can be 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more. More specifically, the consistency of 70% or more can be at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% consistency.

[0056] When used in a list of two or more items, the term "and / or" means that any of the listed items can be used alone or in combination with any one or more of the listed items. For example, the expression "A and / or B" is intended to mean either or both of A and B, i.e., A alone, B alone, or a combination of A and B. The expression "A, B and / or C" means A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B and C.

[0057] As is commonly understood in the art, the term "promoter" generally refers to a DNA containing an RNA polymerase binding site, a transcription start site, and / or a TATA box that assists or promotes the transcription of transcribed DNA. Promoters can be artificially synthesized, modified, or derived from known or naturally occurring promoters. Promoters can also include chimeric promoters comprising combinations of two or more heterologous sequences. Therefore, the promoters of this application may include variants of promoter sequences that are compositionally similar but not identical to other promoter sequences provided herein.

[0058] The term "transcribed DNA" refers to DNA that can be transcribed into RNA molecules.

[0059] The term "operationally ligated" can refer to a functional connection between a promoter and transcribed DNA, enabling the promoter to function and initiate transcription of the transcribed DNA. The term "operationally ligated" can also refer to a functional connection between other regulatory elements and a target gene to regulate the transcription and / or expression of the target gene.

[0060] As used herein, an "expression cassette" means that it contains at least transcribed DNA operatively linked to one or more regulatory elements, typically at least a promoter and a 3'UTR (such as a terminator).

[0061] As used herein, the term "vector" refers to any construct that can be used for transformation purposes, i.e., to introduce heterologous DNA into a host cell. Examples include plasmids, granules, viruses, bacteriophages, or linear or circular DNA.

[0062] In this application, "editing" or "genome editing" means using targeted genome editing technology to produce a targeted mutation, deletion, inversion, or substitution of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 75, at least 100, at least 250, at least 500, at least 1000, at least 2500, at least 5000, or at least 10,000 nucleotides of endogenous genomic nucleic acid sequence to be edited.

[0063] In this application, “editing” or “genome editing” also covers the use of targeted genome editing technology to target and insert or integrate at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 75, at least 100, at least 250, at least 500, at least 750, at least 1000, at least 1500, at least 2000, at least 2500, at least 3000, at least 4000, at least 5000, or at least 10,000 nucleotides into the endogenous genome of the cell to be edited.

[0064] II. Implementation Examples The present application will now be described in further detail with reference to specific embodiments. The embodiments given are merely illustrative of the present application and are not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the present application in any way.

[0065] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0066] In the following examples, the YNB (amino-free yeast nitrogen source (containing ammonium sulfate, but not amino acids)) is a product of Boao Tuoda Company, with product number Y6050G; it is referred to as YNB in ​​the examples.

[0067] Unless otherwise specified, the quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0068] Example 1: Obtaining a Ura3-deficient yeast strain 1.1 Preparation method of Ura3-deficient yeast strain Using the recipient yeast genome as a template, primers Ura3-LR-F and Ura3-LR-R amplified the upstream homologous arm, and primers Ura3-RR-F and Ura3-RR-R amplified the downstream homologous arm; the fragments were then recovered from the gel. Using the two recovered homologous arms as templates, primers Ura3-LR-F and Ura3-RR-R amplified the Ura3 knockout donor fragment; the fragments were then recovered from the gel. The positional relationship between primers Ura3-LR-F and Ura3-LR-R and their amplified upstream homologous arm, primers Ura3-RR-F and Ura3-RR-R and their amplified downstream homologous arm, and URA3-JD-F and URA3-JD-R used in the PCR identification of the Pichia pastoris Ura3-deficient strain and the Ura3 genome is as follows: Figure 1 As shown.

[0069] 1.2 Obtaining the Pichia pastoris Ura3-deficient strain Taking Pichia pastoris X33 as an example, the specific explanation of method 1.1 is as follows: Using the X33 genome as a template, primers Ura3-LR-F and Ura3-LR-R amplified the upstream homologous arm, and primers Ura3-RR-F and Ura3-RR-R amplified the downstream homologous arm; the fragments were then recovered via gel electrophoresis. Using the two recovered homologous arms as templates, primers Ura3-LR-F and Ura3-RR-R amplified the Ura3 knockout donor fragment; the fragments were then recovered via gel electrophoresis. Sequencing results showed that the nucleotide sequence of the Ura3 knockout donor fragment was SEQ ID NO:2. Positions 1 to 1057 of SEQ ID NO:2 are the upstream homologous arm, and positions 1058 to 2086 are the downstream homologous arm. The obtained donor fragment was electroporated into X33 electroporation competent cells and plated on MDU+5-FOA plates. After single colonies grew, positive clones were identified using the following two methods. Clones that were positive in both methods were identified as Pichia pastoris Ura3-deficient strains and named X33ΔUra3-deficient strain.

[0070] Identification Method 1: Colony PCR identification was performed using primers URA3-JD-F and URA3-JD-R. The PCR amplification band of positive clones with successful Ura3 knockout was 2411bp, while the PCR amplification band of clones without knockout was 3203bp.

[0071] Identification Method 2: Single clones were cultured on MD and MDU+5-FOA plates respectively. Positive clones with successful Ura3 knockout could grow normally on MDU+5-FOA plates, but not on MD plates.

[0072] The formulation of MDU+5-FOA plates is as follows: YNB: 13.4 g / L; glucose: 10 g / L; uracil: 100 mg / L; 5-FOA: 1 mg / mL; agar: 20 g / L; and water as the solvent.

[0073] The formulation of MD plates is: YNB: 13.4 g / L; glucose: 10 g / L; agar: 20 g / L, with water as the solvent.

[0074]

[0075] Example 2: Scarless editing method for gene knock-in ΔUra3-deficient strains 2.1. Scarless editing method for gene knock-in ΔUra3-deficient strains Donor DNA fragment 1 and donor DNA fragment 2 were constructed, containing overlapping sequences. Donor DNA fragment 1 and donor DNA fragment 2 were transformed into a host strain (ΔUra3-deficient strain), resulting in a recombinant donor DNA fragment with a single-stranded DNA structure as follows: homologous arm 1 - expression frame 1 - homologous arm 2 - expression frame 2 - homologous arm 3. Here, expression frame 1 and expression frame 2 are either knock-in gene expression frames or Ura3 expression frames, respectively. Homologous arms 1, 2, and 3 satisfy the following conditions: homologous arm 1 is identical to homologous arm 2, or homologous arm 2 is identical to homologous arm 3; the expression frame between two identical homologous arms is the Ura3 expression frame, and the other expression frame is the knock-in gene expression frame.

[0076] (1) A structure of recombinant donor DNA fragment Homologous arm 2 is identical to homologous arm 3. When expression frame 2 is the Ura3 expression frame, the schematic diagram of the seamless gene knock-in is as follows: Figure 2 As shown in the figure, one single strand of donor DNA fragment 1 consists of, from upstream to downstream: the upstream sequence of the knock-in site (labeled UP-In), the knock-in gene expression cassette, the downstream sequence of the knock-in site (labeled DN-In), and a portion of the Ura3 expression cassette. One single strand of donor DNA fragment 2 consists of, from upstream to downstream: a portion of the Ura3 expression cassette and the downstream sequence of the knock-in site (labeled DN-In). There is approximately 700 bp overlap in the Ura3 expression cassettes of donor DNA fragments 1 and 2. During electroporation of donor DNA fragments 1 and 2 into host cells, the host's own DNA repair process allows donor DNA fragments 1 and 2 to combine into a complete donor fragment, resulting in a recombinant donor fragment integrated into the host genome. The structure of one single strand of DNA from the recombinant donor fragment is as follows: an upstream sequence of the knock-in site (marked as UP-In in the figure), a knock-in gene expression cassette, a downstream sequence of the knock-in site (marked as DN-In in the figure), an Ura3 expression cassette, and a downstream sequence of the knock-in site (marked as DN-In in the figure).

[0077] Screening for positive homozygotes containing recombinant donor fragments: The recombinant donor fragment undergoes homologous recombination with the host cell, integrating the donor DNA fragment into the knock-in site. Positive homozygotes are identified by PCR.

[0078] 5-FOA pressure screening: The selected positive homozygotes were further screened by MDU+5-FOA plates to finally obtain recombinant bacteria with Ura3 elimination, achieving scarless gene knock-in.

[0079] (2) Another structure of recombinant donor DNA fragments Homologous arm 1 is identical to homologous arm 2. When expression frame 1 is the Ura3 expression frame, the schematic diagram of the gene knock-in is as follows: Figure 3 As shown in the figure, one single strand of donor DNA fragment 1 consists of, from upstream to downstream: a portion of the Ura3 expression cassette, the upstream sequence of the knock-in site (labeled UP-In), the knock-in gene expression cassette, and the downstream sequence of the knock-in site (labeled DN-In). One single strand of donor DNA fragment 2 consists of, from upstream to downstream: the upstream sequence of the knock-in site (labeled UP-In) and a portion of the Ura3 expression cassette. There is approximately 700 bp overlap in the Ura3 expression cassettes of donor DNA fragments 1 and 2. During electroporation of donor DNA fragments 1 and 2 into host cells, the host's own DNA repair process allows donor DNA fragments 1 and 2 to combine into a complete donor fragment, resulting in a recombinant donor fragment integrated into the host genome. The structure of one DNA single strand of the recombinant donor fragment is as follows: upstream sequence of the knock-in site (marked as UP-In in the figure), Ura3 expression cassette, upstream sequence of the knock-in site (marked as UP-In in the figure), knock-in gene expression cassette, and downstream sequence of the knock-in site (marked as DN-In in the figure).

[0080] The screening of positive homozygotes containing recombinant donor fragments after electroporation of donor DNA fragment 1 and donor DNA fragment 2 and the 5-FOA pressure screening were conducted in accordance with the method in “(1) A structure of recombinant donor DNA fragment”, the only difference being the primer sequence.

[0081] 2.2. Knock hLF into the INT6 site of the X33ΔUra3 defective strain. Using the X33ΔUra3 defective strain prepared in Example 1 as the host bacterium, hLF as the knock-in gene, and the INT6 gene as the knock-in site, the method in 2.1 is specifically described below, taking the recombinant donor DNA structure described in "(1) A structure of a recombinant donor DNA fragment" as an example. The schematic diagram is shown below. Figure 4 As shown.

[0082] (1) Constructing hLF knock-in donors Because the donor contains two identical sequences, it is difficult to obtain a complete donor fragment using ordinary overlap PCR. Therefore, the donor fragment was constructed in two parts, with the two donors overlapping by approximately 700 bp at Ura3. After being transformed into yeast cells, the yeast's own DNA repair function was used to reassemble them into a complete donor. The specific construction is as follows: Using the X33 genome as a template, the upstream homologous arm was amplified using primers Int6-UP-F and Int6-UP-R, the downstream homologous arm was amplified using Int6-DN-F and Int6-DN-R, the Ura3 expression cassette (5-Ura3) was amplified using Ura3 promoter-F and Ura3-5-R1, and the Ura3 expression cassette (3-Ura3) was amplified using Ura3 promoter-F and Ura3tt-R. All fragments were recovered via gel electrophoresis. Using the pPICZαA-hLF plasmid as a template, the hLF expression cassette was amplified using primers pAOX1-F and AOX1tt-R, and recovered via gel electrophoresis. Using the recovered upstream homologous arm, hLF expression cassette, downstream homologous arm, and 5-Ura3 as templates, overlap was performed using primers Int6-UP-F and Ura3-5-R1 to obtain donor DNA fragment 1. Using the recovered downstream homologous arm and 3-Ura3 as templates, overlap PCR was performed with primers Ura3-3-F and Int6-DN-R to obtain donor DNA fragment 2.

[0083] The nucleotide sequence of donor DNA fragment 1 is positions 1 to 6876 of SEQ ID NO:4. Among them, positions 1 to 1056 of SEQ ID NO:4 are the upstream sequence of the knock-in site Int6 (marked as UP-Int6 in the figure), positions 1057 to 4694 are the hLF gene expression cassette (positions 1057 to 1995 are the promoter sequence, positions 2269 to 4344 are the coding sequence of the hLF gene, and positions 4448 to 4694 are the terminator sequence), positions 4695 to 5729 are the downstream sequence of the knock-in site Int6 (marked as DN-Int6 in the figure), and positions 5730 to 6876 are a partial sequence of the Ura3 expression cassette (positions 6105 to 6876 are overlapping sequences).

[0084] The nucleotide sequence of donor DNA fragment 2 is SEQ ID NO:4, positions 6105 to 8130. Positions 6105 to 7095 are part of the Ura3 expression cassette (positions 6105 to 6876 are overlapping sequences), and positions 7096 to 8130 are the downstream sequence of the knock-in site Int6 (marked as DN-Int6 in the figure).

[0085]

[0086] (2) Screening for positive clones The obtained donor DNA fragments 1 and 2 were electroporated into competent cells of the X33ΔUra3 defective strain and plated on MD plates. After single colonies grew, colony PCR was performed for identification. Since Pichia pastoris is diploid, homozygotes were first identified using primers Int6-JD-F and Int6-JD-R. Because the successfully integrated donor fragments contained two completely identical sequences that could not be amplified by PCR, those without wild-type bands were considered homozygous. Eight homozygotes were selected for further verification. Amplification was performed using Int6-JD-F and Ura3-5-R1, and Ura3 promoter-F and Int6-JD-R, respectively. Correct amplification of 7242 bp and 2805 bp bands, respectively, indicated that donor DNA fragments 1 and 2 were correctly integrated into the genome. Clones correctly amplified by both primer pairs were considered positive.

[0087] Figure 5 PCR validation of hLF knock-in at the Int6 site was performed on colonies. Figure A shows homozygous heterozygote identification; homozygotes did not amplify the wild-type band, while heterozygotes or wild-type clones amplified a 2861 bp band. Lanes 1, 2, 5, 6, 7, 9, 11, 12, 13, 15, and 16 were homozygous. Figures B and C show donor fragment integration identification. In Figure B, the positive clone amplified a 7242 bp band; lanes 1, 2, 3, 4, 5, 6, and 8 were positive clones. In Figure C, the positive clone amplified a 2805 bp band; lanes 1, 2, 3, 4, 5, 7, and 8 were positive clones. The marker was a Bomeide 1kb DNA Ladder, catalog number MD114.

[0088] The correctly integrated recombinant fragment is named the recombinant donor DNA fragment, and its nucleotide sequence is SEQ ID NO:4. Specifically, in SEQ ID NO:4, positions 1 to 1056 are the upstream sequence of the knock-in site Int6 (UP-Int6), positions 1057 to 4694 are the hLF gene expression cassette (positions 1057 to 1995 are the promoter sequence, positions 2269 to 4344 are the hLF gene coding sequence, and positions 4448 to 4694 are the terminator sequence), positions 4695 to 5729 are the downstream sequence of the knock-in site Int6 (DN-Int6), positions 5730 to 7095 are the sequence of the Ura3 expression cassette (positions 6105 to 6876 are the overlapping sequences of donor DNA fragment 1 and donor DNA fragment 2), and positions 7096 to 8130 are the downstream sequence of the knock-in site Int6 (DN-Int6).

[0089]

[0090] (3) Eliminate Ura3 The positive clones obtained in step 2 were incubated overnight with YPD via shaking. The bacterial culture was then diluted 10-fold and plated at a density of 100 μL onto an MDU+5-FOA plate. After single clones grew, they were spotted onto MD and MDU+5-FOA plates for verification. Clones that grew on MDU+5-FOA plates but not on MD plates successfully eliminated Ura3. A second verification was performed using primers Int6-JD-F and Int6-JD-R. If the primers amplified a target band of 6499 bp, then Ura3 elimination was successful.

[0091] The recombinant bacteria with successful Ura3 elimination achieved scarless gene knock-in. Furthermore, due to the successful Ura3 elimination, the obtained recombinant bacteria can be processed according to the procedures outlined in this application. Figure 3 , Figure 4 , Figure 6 and Figure 7 The method described in at least one of the above methods is used to perform multiple rounds of gene knock-in or knock-out.

[0092] Example 3: Gene Knockout 3.1. Scarless editing method for target gene knockout Donor DNA fragment 1 and donor DNA fragment 2 were constructed, containing overlapping sequences. Donor DNA fragment 1 and donor DNA fragment 2 were transformed into a host strain (ΔUra3-deficient strain), resulting in a recombinant donor DNA fragment in the host containing the following DNA elements: homologous arms 1, 2, and 3 from upstream to downstream, and an Ura3 expression cassette located between two identical homologous arms. Homologous arms 1, 2, and 3 satisfy the following conditions: homologous arm 2 is identical to homologous arm 1, or homologous arm 2 is identical to homologous arm 3; and the space between two identical homologous arms is the Ura3 expression cassette.

[0093] (1) A structure of recombinant donor DNA fragment A schematic diagram of recombinant DNA fragments and scarless gene knockout when homologous arm 2 and homologous arm 3 are identical is shown below. Figure 6As shown in the figure, one single strand of donor DNA fragment 1 consists of, from upstream to downstream, the upstream sequence of the target gene (labeled UP-T), the downstream sequence of the target gene (labeled DN-T), and a portion of the Ura3 expression frame. One single strand of donor DNA fragment 2 consists of, from upstream to downstream, the Ura3 expression frame and the downstream sequence of the target gene (labeled DN-T). There is approximately 700 bp overlap in the Ura3 expression frames of donor DNA fragments 1 and 2. During electroporation of donor DNA fragments 1 and 2 into host cells, the host's own DNA repair process allows donor DNA fragments 1 and 2 to combine into a complete donor fragment, resulting in a recombinant donor fragment integrated into the host genome. The structure of one single strand of the recombinant donor fragment is as follows: the upstream sequence of the target gene (labeled UP), the downstream sequence of the target gene (labeled DN-T), the Ura3 expression frame, and the downstream sequence of the target gene (labeled DN-T).

[0094] The screening of positive homozygotes containing recombinant donor fragments after electroporation of donor DNA fragment 1 and donor DNA fragment 2 and the 5-FOA pressure screening were conducted in accordance with the method in "(1) a structure of recombinant donor DNA fragment" in Example 2, the only difference being the primer sequence.

[0095] (2) Another structure of recombinant donor DNA fragments A schematic diagram of recombinant DNA fragments and scarless gene knockout when homologous arm 1 and homologous arm 2 are identical is shown below. Figure 7 As shown in the figure, one single strand of donor DNA fragment 1 consists of, from upstream to downstream: a portion of the Ura3 expression frame, the upstream sequence of the target gene (labeled UP-T in the figure), and the downstream sequence of the target gene (labeled DN-T in the figure). One single strand of donor DNA fragment 2 consists of, from upstream to downstream: the upstream sequence of the target gene (labeled UP-T in the figure) and a portion of the Ura3 expression frame. There is approximately 700 bp overlap in the Ura3 expression frames of donor DNA fragments 1 and 2. When donor DNA fragments 1 and 2 are electroporated into host cells, the host's own DNA repair process allows donor DNA fragments 1 and 2 to combine into a complete donor, resulting in a recombinant donor fragment integrated into the host genome. The structure of one single strand of the recombinant donor fragment is: the upstream sequence of the target gene (labeled UP-T in the figure), the Ura3 expression frame, the upstream sequence of the target gene (labeled UP-T in the figure), and the downstream sequence of the target gene (labeled DN-T in the figure).

[0096] The screening of positive homozygotes containing recombinant donor fragments after electroporation of donor DNA fragment 1 and donor DNA fragment 2 and the 5-FOA pressure screening were conducted in accordance with the method in "(1) a structure of recombinant donor DNA fragment" in Example 2, the only difference being the primer sequence.

[0097] 3.2 Knockout Example Using the X33ΔUra3-deficient strain prepared in Example 1 as the host bacterium, and Pep4 as the knockout gene, the method in 3.1 is specifically described below, taking the recombinant donor DNA structure described in "(1) A structure of a recombinant donor DNA fragment" as an example, where the schematic diagram is shown below. Figure 8 As shown.

[0098] (1) Constructing a knockout donor Using the X33 genome as a template, the upstream homologous arm was amplified using primers Pep4-LR-F and Pep4-LR-R, the downstream homologous arm was amplified using primers Pep4-RR-F and Pep4-RR-Ura3-R, and a portion of the Ura3 expression cassette (denoted as 5-Ura3) was amplified using primers Uep4-LR-F and Ura3-5-R1. After gel recovery, all primers were used together as templates, and overlapped with primers Pep4-LR-F and Ura3-5-R1 to obtain knockout donor fragment 1. Using the X33 genome as a template, a portion of the Ura3 expression cassette (denoted as 3-Ura3) was amplified using primers Ura3-3-F and Ura3tt-R1, and the downstream homologous arm was amplified using primers Pep4-RR-Ura3-F and Pep4-RR-R. After gel recovery, all primers were used together as templates, and overlapped with primers Ura3-3-F and Pep4-RR-R to obtain knockout donor fragment 2.

[0099] The nucleotide sequence of donor fragment 1 is positions 1 to 3114 of SEQ ID NO:3, where positions 1 to 988 are the upstream homologous arms of the pep4 gene, positions 989 to 1967 are the downstream homologous arms of the pep4 gene, and positions 1968 to 3114 are a portion of the Ura3 expression frame. The corresponding nucleotide sequence of DNA donor fragment 2 is positions 2343 to 4312 of SEQ ID NO:3, where positions 2343 to 3333 are a portion of the Ura3 expression frame, and positions 3334 to 4312 are the downstream homologous arms of the pep4 gene. Positions 2343 to 3114 of SEQ ID NO:3 represent the overlapping portion of donor fragment 1 and donor fragment 2.

[0100]

[0101] (2) Screening for positive clones The obtained donor DNA fragments 1 and 2 were electroporated into competent cells of the X33ΔUra3 defective strain and plated on MD plates. Single colonies were then identified by colony PCR. First, homozygotes were identified using primers Pep4-JP-F and Pep4-JP-R; homozygotes were those without wild-type bands amplified by PCR. Amplification was then performed using Pep4-JD-F and Ura3-5-R1 with Ura3 promoter-F1 and Pep4-JD-R, respectively. Correct amplification of 3267 bp and 2505 bp bands, respectively, indicated correct integration of donor DNA fragments 1 and 2 into the genome. Clones amplified with correct bands using all three primer pairs were considered positive clones.

[0102] Figure 9 PCR validation of Pep4 knockout colonies. In Figure A, lanes 1-8 show homozygous identification; homozygotes did not amplify the wild-type band, while heterozygotes or wild-type amplified a 1233bp band. Lane 6 is homozygous. Figure B shows donor fragment integration identification. The primers used for lanes 1-8 were Pep4-JD-F and Ura3-5-R1, with a theoretical size of 3267bp. Lanes 1, 2, 5, 6, 7, and 8 were the correct size. The primers used for lanes 9-16 were Ura3 promoter-F1 and Pep4-JD-R, with a theoretical size of 2505bp. Lanes 9, 11, 12, 13, 14, 15, and 16 were positive clones. The marker was a Bomeide 1kb DNA Ladder, catalog number MD114.

[0103] The correctly integrated recombinant fragment is named the recombinant donor DNA fragment, and its nucleotide sequence is SEQ ID NO:3. In SEQ ID NO:3, positions 1 to 988 are the upstream homologous arms of the pep4 gene, positions 989 to 1967 are the downstream homologous arms of the pep4 gene, positions 1968 to 4312 are the Ura3 expression frame (positions 2343 to 3114 are overlapping portions), and positions 3334 to 4312 are the downstream homologous arms of the pep4 gene.

[0104] (3) Eliminate Ura3 The positive clones obtained in step 2 were incubated overnight with YPD via shaking. The bacterial culture was then diluted 10-fold and plated in 100 μL onto MDU+5-FOA plates. After single clones grew, they were spotted onto MD and MDU+5-FOA plates for verification. Clones that grew on MDU+5-FOA plates but not on MD plates successfully eliminated Ura3. A second verification was performed using primers Pep4-JD-F and Pep4-JD-R. If the primers amplified a target band of 2280 bp, then Ura3 elimination was successful.

[0105] The successful elimination of Ura3 in the recombinant bacteria achieved scarless gene knockout. Furthermore, due to the successful elimination of Ura3, the obtained recombinant bacteria can be processed according to the procedures outlined in this application. Figure 3 , Figure 4 , Figure 6 and Figure 7 The method described in at least one of the above methods is used to perform multiple rounds of gene knock-in or knock-out.

[0106] The present application has been described in detail above. Those skilled in the art will recognize that the present application can be implemented in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments are given in this application, it should be understood that further modifications can be made to the present application. In summary, in accordance with the principles of this application, this application is intended to include any changes, uses, or improvements to the present application, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A composition for gene editing, characterized in that: The composition comprises donor DNA pair A or donor DNA pair B; The donor DNA pair A consists of two double-stranded DNA fragments named donor DNA fragment A1 and donor DNA fragment A2. One single strand of donor DNA fragment A1 contains, from upstream to downstream, an upstream homologous arm, a downstream homologous arm, and a portion of the Ura3 expression frame for homologous recombination with the cell to be edited. One single strand of donor DNA fragment A2 contains, from upstream to downstream, another portion of the Ura3 expression frame and the downstream homologous arm. The 3' end of the single strand of donor DNA fragment A1 and the 5' end of the single strand of donor DNA fragment A2 have an identical sequence overlap, which is located within the Ura3 expression frame. The donor DNA pair B consists of two double-stranded DNA fragments named donor DNA fragment B1 and donor DNA fragment B2. One single strand of donor DNA fragment B1 contains a portion of the Ura3 expression frame, the upstream homologous arm, and the downstream homologous arm from upstream to downstream. One single strand of donor DNA fragment B2 contains the upstream homologous arm and another portion of the Ura3 expression frame from upstream to downstream. The 5' end of the single strand of donor DNA fragment B1 and the 3' end of the single strand of donor DNA fragment B2 have an identical sequence overlap, which is located in the Ura3 expression frame.

2. The composition according to claim 1, characterized in that: The gene editing is gene knockout, and the sequence of the upstream homologous arm is the same as the upstream sequence of the target gene to be knocked out in the cell to be edited, and the sequence of the downstream homologous arm is the same as the downstream sequence of the target gene to be knocked out.

3. The composition according to claim 1, characterized in that: The gene editing is gene knock-in, and the upstream homologous arm and the downstream homologous arm of the donor DNA fragment A1 and the donor DNA fragment B1 also contain a gene expression frame to be knocked in; The sequence of the upstream homologous arm is the same as the upstream sequence of the insertion site, and the sequence of the downstream homologous arm is the same as the downstream sequence of the insertion site. The insertion site is the insertion location of the gene expression cassette to be knocked in.

4. The composition according to any one of claims 1 to 3, characterized in that: The donor DNA fragment A1 and the donor DNA fragment A2 can undergo homologous recombination and integration into recombinant DNA donor fragment A in the cell to be edited, and the donor DNA fragment B1 and the donor DNA fragment B2 can undergo homologous recombination and integration into recombinant DNA donor fragment B in the cell to be edited; The recombinant DNA donor fragment A is at least one of the following: One strand of the recombinant DNA donor fragment A contains, from upstream to downstream, the following elements: an upstream homologous arm and a downstream homologous arm for homologous recombination with the cell to be edited, the Ura3 expression cassette, and the downstream homologous arm; Aii) One strand of the recombinant DNA donor fragment A contains, from upstream to downstream, the following elements: an upstream homologous arm that undergoes homologous recombination with the cell to be edited, a gene knock-in expression cassette, a downstream homologous arm that undergoes homologous recombination with the cell to be edited, the Ura3 expression cassette, and the downstream homologous arm. The recombinant DNA donor fragment B is at least one of the following: Bi) One strand of the recombinant DNA donor fragment B contains, from upstream to downstream, the following elements: an upstream homologous arm that undergoes homologous recombination with the cell to be edited, the Ura3 expression cassette, the upstream homologous arm, and a downstream homologous arm that undergoes homologous recombination with the cell to be edited; Bii) One strand of the recombinant DNA donor fragment B contains, from upstream to downstream, the following elements: an upstream homologous arm for homologous recombination with the cell to be edited, the Ura3 expression cassette, the upstream homologous arm, the gene to be knocked in expression cassette, and a downstream homologous arm for homologous recombination with the cell to be edited.

5. An application, characterized in that: The application is at least one of the following: C1) The use of the composition according to claim 2 in gene knockout; C2) The use of the composition according to claim 3 in gene knock-in.

6. A gene knockout method, characterized in that: The knockout includes the following steps: S1) Introduce the donor DNA pair A as described in claim 1 or 2 into the cell to be edited, and screen for recombinant cells containing the recombinant DNA donor fragment A as described in claim 4 (Ai), or The donor DNA pair B as described in claim 1 or 2 is introduced into the cells to be edited, and recombinant cells containing the recombinant DNA donor fragment B as described in claim 4 are screened. S2) Screening for target cells that survive on a culture medium containing uracil and 5-FOA; The cells to be edited do not contain the Ura3 gene.

7. A gene knock-in method, characterized in that: The knock-in includes the following steps: S1') The donor DNA pair A as described in claim 3 is introduced into the cell to be edited, and recombinant cells containing the recombinant DNA donor fragment A as described in claim 4 (Aii) are screened, or The donor DNA pair B as described in claim 3 is introduced into the cells to be edited, and recombinant cells containing the recombinant DNA donor fragment B as described in claim 4 (Bii) are screened. S2') Screening for target cells that survive on a medium containing uracil and 5-FOA; The cells to be edited do not contain the Ura3 gene.

8. The method according to claim 6 or 7, characterized in that: The number of operations for steps S1) to S2) and / or steps S1') to S2') is N, where N is an integer ≥ 1.

9. The method according to claim 6 or 7, characterized in that: It also includes the step of knocking out the Ura3 gene in the cells to be edited.

10. The method according to claim 9, characterized in that: The cells to be edited are microbial cells.

Images