Non-canonical pichia pastoris gene editing system and application thereof

Abstract

The present application relates to the technical field of gene editing, and specifically provides a non-typical Pichia pastoris gene editing system and application thereof.The present application provides a non-typical Pichia pastoris gene editing plasmid, comprising a prokaryotic replication origin, a resistance screening marker gene, a Cas9 gene expression cassette, a gRNA gene expression cassette and an autonomous replication sequence; the Cas9 gene expression cassette comprises, from upstream to downstream, a Cas9 gene promoter, a Cas9 gene and a terminator; the gRNA gene expression cassette comprises, from upstream to downstream, an RNA pol III promoter, a tRNA Leu guide sequence, a scaffold RNA sequence and a polyT.The gene editing plasmid of the present application can edit the Pichia pastoris genome, has high editing efficiency and simple operation method, and provides a good gene editing system for Pichia pastoris gene modification research.

Description

Technical Field

[0001] This invention relates to the field of gene editing technology, and in particular to an atypical Pichia pastoris gene editing system and its applications. Background Technology

[0002] Gene editing systems are essential for gene and protein function research. The emergence of technologies such as ZFN, TALEN, and CRISPR / Cas9 has made gene editing simpler and more efficient.

[0003] Currently, there is some understanding of CRISPR / Cas9 gene editing technology. The CRISPR / Cas9 system applied to eukaryotic cells mainly consists of two parts: single-guide RNA (sgRNA) and Cas9 protein. sgRNA replaces the tracrRNA:crRNA complex transcribed from the CRISPR spacer sequence, guiding the Cas9 protein to bind to the protospacer adjacent motif (PAM) region of the target gene site. This triggers the activity of the RuvC and HNH nuclease functional sites on the Cas9 protein, cleaving the DNA strand to produce a DSB. In the design of the sgRNA sequence, the 3' end contains a scaffold RNA sequence, which can be transcribed to form a stem-loop double-stranded RNA for binding the Cas9 protein, while the 5' end contains a guide sequence consistent with the target gene sequence. By modifying the guide sequence on the sgRNA, the Cas9 protein can be guided to target and cleave any DNA double strand containing the PAM sequence, thereby achieving gene editing.

[0004] Pichia kudriavzevii (PK yeast) is a Pichia species that has attracted much attention in recent years due to its strong resistance to adverse conditions and rapid growth. Compared with traditional yeasts, our understanding of the gene editing system of PK yeast is still very limited, and its gene editing system is not yet mature. Summary of the Invention

[0005] This invention provides an atypical Pichia pastoris gene editing system and its application, which addresses the deficiency of existing PK yeast genome editing systems and enables rapid and effective editing of PK yeast.

[0006] In a first aspect, the present invention provides a Cas9 gene suitable for editing in PK yeast, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] Existing Cas9 genes cannot edit PK yeast because most existing Cas9 genes are designed for model strains such as Saccharomyces cerevisiae and Pichia pastoris. PK yeast, as a non-model strain, has significantly different codon frequencies compared to these strains. Therefore, host codon optimization of Cas9 is necessary to ensure that its codons are high-frequency codons found in PK yeast, thereby better enabling it to perform its function.

[0008] Secondly, the present invention provides a primer pair, the sequence of which is shown in SEQ ID NO.14-15, for amplifying the Cas9 gene as shown in SEQ ID NO.1.

[0009] Thirdly, the present invention provides a PK yeast gene editing plasmid, the PK yeast gene editing plasmid comprising: a prokaryotic origin of replication, an resistance selection marker gene, a Cas9 gene expression cassette, a gRNA gene expression cassette, and an autonomous replication sequence; the Cas9 gene expression cassette contains a nucleotide sequence as shown in SEQ ID NO.1.

[0010] In the PK yeast gene editing plasmid provided by the present invention, the Cas9 gene expression cassette includes, from upstream to downstream, a Cas9 gene promoter, a Cas9 gene, and a terminator.

[0011] The nucleotide sequence of the Cas9 gene promoter is shown in SEQ ID NO.2; the nucleotide sequence of the Cas9 gene terminator is shown in SEQ ID NO.3.

[0012] Before finally selecting the aforementioned promoter and terminator, multiple combinations of promoter and terminator experiments were conducted: pHTX1 promoter, Gal1,10 promoter and DAS1 terminator, CYC1 terminator, and AOX terminator. The results showed that none of these combinations could express the Cas9 gene and gRNA, failing to effectively edit the yeast genome. Through experimentation, the aforementioned promoter and terminator, specifically those from the PK yeast genome, were ultimately chosen as the preferred choice.

[0013] In the PK yeast gene editing plasmid provided by this invention, the gRNA gene expression cassette includes, from upstream to downstream, an RNApol III promoter and a tRNA gene expression cassette. Leu Guide sequence, scaffold RNA sequence, and polyT.

[0014] In the PK yeast gene editing plasmid provided by this invention, the gRNA gene expression cassette is amplified by the primer pair shown in SEQ ID NO.6-7;

[0015] Preferably, the nucleotide sequence of the gRNA gene expression cassette is shown in SEQ ID NO.3.

[0016] In the PK yeast gene editing plasmid provided by this invention, the autonomous replication sequence is the ARS sequence of Saccharomyces cerevisiae; the ARS sequence is amplified by the primer pair shown in SEQ ID NO.12-13;

[0017] Preferably, the nucleotide sequence of the ARS sequence is as shown in SEQ ID NO.4.

[0018] The ARS sequence is an AT-rich origin of replication in eukaryotes, enabling plasmids to replicate independently in eukaryotic cells. All ARS sequences contain a conserved sequence; therefore, the ARS sequence of the model strain *Saccharomyces cerevisiae* was chosen.

[0019] In the PK yeast gene editing plasmid provided by this invention, the selection marker genes are ampicillin resistance genes and bleomycin resistance genes.

[0020] In the PK yeast gene editing plasmid provided by this invention, the prokaryotic replication origin is the replication origin of pBR322, and the pBR322 plasmid is one of the classic gene cloning vectors.

[0021] Fourthly, this invention provides a method for constructing the above-mentioned PK yeast gene editing plasmid.

[0022] The Cas9 gene promoter and Cas9 gene terminator were obtained by PCR amplification using PK yeast genomic DNA as a template; the gRNA gene expression cassette and autonomous replication sequence were obtained by PCR amplification using synthetic plasmid as a template.

[0023] The sequences shown in SEQ ID NO.1-5 were ligated into the vector pHTX1-Cas9-gRN A-GUT-AOXTT to construct the gene editing plasmid pk-Cas9, which was then transformed into Escherichia coli DH5α to obtain the recombinant plasmid pk-Cas9.

[0024] Fifthly, the present invention seeks protection for the application of the Cas9 gene or the PK yeast gene editing system described above in the efficient editing of PK yeast.

[0025] In the application provided by this invention, competent Pichia pastoris kudrica is mixed with pk-Cas9 plasmid and donor DNA, incubated on ice, and then electroporated. Immediately after electroporation, the mixture is added to sorbitol and YPD medium for resuscitation to obtain edited PK yeast.

[0026] The beneficial effects of this invention are as follows:

[0027] The gene editing system of this invention can edit the genome of PK yeast with high efficiency and simple operation. The gene editing plasmid of this invention provides an excellent gene editing system for PK yeast gene modification research. Attached Figure Description

[0028] Figure 1 This is a map of the pk-Cas9 plasmid.

[0029] Figure 2 To identify ura3 gene knockout by PCR, 1 is the DNA molecular weight standard, 2 is the ura3 gene knockout band, and 3 is the wild-type non-knockout ura3 band.

[0030] Figure 3 To validate the results of ura3 gene knockout for phenotypic purposes.

[0031] Figure 4 This is a diagram of the PCR identification results from Example 2.

[0032] Figure 5 This is a map of the pHTX1-Cas9-gRNA-GUT-AOXTT plasmid.

[0033] Figure 6 The image shows the PCR identification results from Comparative Example 1.

[0034] Figure 7 The image shows the PCR identification results from Comparative Example 2. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention are described clearly and completely below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0036] All reagents and consumables used in this embodiment are commercially available.

[0037] Example 1: Construction of Pichia kudriezwiyne gene-editing plasmid

[0038] 1. Cas9 codon optimization

[0039] The Cas9 protein codons were optimized based on the Codon Adaptation Index (CAI) of PK yeast, as follows:

[0040] Twenty open reading frames (ORFs) were randomly selected from the *Pichia pastoris* genome. The frequency of codon usage in these 20 ORFs was statistically analyzed to calculate the optimal codons in *Pichia pastoris*. Based on the *CAI* of *Pichia pastoris*, rare codons encoding the *Cas9* protein were replaced with the optimal codons of *Pichia pastoris*, and the bases were manually adjusted. The optimized *Cas9* codons, as shown in Sequence 1 of the sequence listing, were synthesized by Shanghai Sangon Biotech Co., Ltd.

[0041] 2. Constructing the pk-Cas9 plasmid

[0042] Genomic DNA was extracted from Pichia pastoris LC375240, and the extraction method was in accordance with "Molecular Cloning: A Laboratory Manual". The gRNA gene expression cassette sequence and ARS sequence were synthesized by Shanghai Sangon Biotech Co., Ltd.

[0043] The nucleotide sequences of primers P1 and P2, P3 and P4, P5 and P6, and P7 and P8 are designed as follows:

[0044] Primer P1: 5'-AGTCCAAAAGGCTCCACC-3' (SEQ ID NO.6);

[0045] Primer P2: 5'-AATTCTTTTGTTGTATTCTTAAAAAAAAGCACCGACTCGGTG-3' (SEQ ID NO.7);

[0046] Primer P3: 5'-CCGAGTCGGTGCTTTTTTTTAAGAATACAACAAAAGAATTTG-3' (SEQ ID NO. 8);

[0047] Primer P4: 5'-AGTTAGCAATAAGGGTATATC-3' (SEQ ID NO.9);

[0048] Primer P5: 5'-GTGAGCTGATAGTTTCTTC-3' (SEQ ID NO.10);

[0049] Primer P6: 5'-GGTAGTATTTGTTGGCGATCTTTACGTTTTCTTGTCGTGTG-3 (SEQ ID NO. 11)';

[0050] Primer P7: 5'-ACACGACAAGAAAACGTAAAGATCGCCAACAAATACTACC-3' (SEQ ID NO.12);

[0051] Primer P8: 5'-GATCGCTTGCCTGTAACTTAC-3' (SEQ ID NO.13);

[0052] Primer P9: 5'-TAAAACTACAACACGAAACGATCCAAAAAAGAAAAGAAAAGTTG-3' (SEQ ID NO. 14);

[0053] Primer P10: 5'-AAACTGTAAAGACTTCCCGTTTAAACTTTCCTTTTCTTTTTTGGATC-3' (SEQ IDNO.15);

[0054] Using PK yeast genomic DNA as a template, fragments A and B were amplified by PCR. Fragment A is an 800bp promoter, and fragment B is a 299bp terminator. Using the synthesized plasmid as a template, fragments C and D were amplified by PCR. Fragment C is a 577bp gRNA gene expression cassette, and fragment D is a 374bp Saccharomyces cerevisiae ARS sequence. The pk-Cas9 plasmid map is shown below. Figure 1 As shown.

[0055] More specifically, the PCR reaction system and reaction conditions for obtaining each fragment are as follows:

[0056] PCR reaction system (fragment A): 2×KOD Buffer 25μL, primers P3 and P4 1.5μL each (10μmol / L), DNA template 1μL (90ng / μL), dNTPs (2mM each) 10μL, KOD polymerase (TOYOBO, 1U / μL) 1μL, and finally add water to 50μL.

[0057] Reaction conditions (fragment A): 94℃ heat denaturation for 3 min; 98℃ denaturation for 10 s, 60℃ annealing for 30 s, 68℃ extension for 50 s, for a total of 35 cycles; 68℃ final extension for 5 min, and holding at 4℃.

[0058] PCR reaction system (fragment B): 2×KOD Buffer 25μL, primers P5 and P6 1.5μL each (10μmol / L), DNA template 1μL (90ng / μL), dNTPs (2mM each) 10μL, KOD polymerase (TOYOBO, 1U / μL) 1μL, and finally add water to 50μL.

[0059] Reaction conditions (fragment B): 94℃ heat denaturation for 3 min; 98℃ denaturation for 10 s, 60℃ annealing for 30 s, 68℃ extension for 20 s, for a total of 35 cycles; final extension at 68℃ for 5 min, followed by holding at 4℃.

[0060] PCR reaction system (fragment C): 2×KOD Buffer 25μL, primers P1 and P2 1.5μL each (10μmol / L), plasmid template 1μL (160ng / μL), dNTPs (2mM each) 10μL, KOD polymerase (TOYOBO, 1U / μL) 1μL, and finally add water to 50μL.

[0061] Reaction conditions (fragment C): 94℃ heat denaturation for 3 min; 98℃ denaturation for 10 s, 60℃ annealing for 30 s, 68℃ extension for 35 s, for a total of 35 cycles; 68℃ final extension for 5 min, and holding at 4℃.

[0062] PCR reaction system (fragment D): 2×KOD Buffer 25μL, primers P7 and P8 1.5μL each (10μmol / L), plasmid template 1μL (160ng / μL), dNTPs (2mM each) 10μL, KOD polymerase (TOYOBO, 1U / μL) 1μL, and finally add water to 50μL.

[0063] Reaction conditions (fragment D): 94℃ heat denaturation for 3 min; 98℃ denaturation for 10 s, 60℃ annealing for 30 s, 68℃ extension for 23 s, for a total of 35 cycles; final extension at 68℃ for 5 min, followed by holding at 4℃.

[0064] PCR reaction system (Cas9 fragment): 2×KOD Buffer 25μL, primers P9 and P10 1.5μL each (10μmol / L), plasmid template 1μL (160ng / μL), dNTPs (2mM each) 10μL, KOD polymerase (TOYOBO, 1U / μL) 1μL, and finally add water to 50μL.

[0065] Reaction conditions (Cas9 fragment): 94℃ heat denaturation for 3 min; 98℃ denaturation for 10 s, 60℃ annealing for 30 s, 68℃ extension for 4 min 10 s, for a total of 35 cycles; final extension at 68℃ for 5 min, followed by holding at 4℃.

[0066] PCR products (fragment A, fragment B, fragment C, fragment D, and Cas9 fragment) were recovered using a DNA gel extraction kit, following the instructions provided.

[0067] The optimized Cas9 fragment was ligated into a vector along with fragments A, B, C, and D to construct the gene editing plasmid pk-Cas9. This plasmid was then transformed into E. coli DH5α. The recombinant plasmid pk-Cas9 was sequenced, and the sequencing results showed that the nucleotide sequences of Cas9 fragments, fragments A, B, C, and D are as shown in sequences 1, 2, 3, 4, and 5 in the sequence listing.

[0068] 3. Knockout of the ura3 gene in Pichia pastoris kudriazwieldii

[0069] pk-Cas9 was co-transformed with donor DNA into Pichia pastoris LC375240. The donor DNA used was constructed using the following method:

[0070] Using PK yeast genomic DNA as a template, the upstream and downstream 1000bp fragments of the ura3 gene were amplified by PCR. Then, these two fragments were overlapped by PCR to finally form a 2000bp donor DNA.

[0071] Primer P11: 5'-GCCACAAATTAGGTTGAGGA-3' (SEQ ID NO.16);

[0072] Primer P12: 5'-ATCAAATCTTTGTGTAAGAACCTTGACAAA

[0073] CAAACTACTTTA-3' (SEQ ID NO. 17);

[0074] Primer P13: 5'-AAGTAGTTTGTTTGTCAAGGTTCTTACACAA

[0075] AGATTTGATAC-3' (SEQ ID NO. 18);

[0076] Primer P14: 5'-GCATGGTTTTTAAGCAGAAG-3' (SEQ ID NO.19).

[0077] PCR reaction system (ura3 upstream fragment): 2×KOD Buffer 25μL, primers P11 and P12 1.5μL each (10μmol / L), DNA template 1μL (90ng / μL), dNTPs (2mM each) 10μL, KOD polymerase (TOYOBO, 1U / μL) 1μL, and finally add water to 50μL.

[0078] PCR reaction system (ura3 downstream fragment): 2×KOD Buffer 25μL, primers P13 and P14 1.5μL each (10μmol / L), DNA template 1μL (90ng / μL), dNTPs (2mM each) 10μL, KOD polymerase (TOYOBO, 1U / μL) 1μL, and finally add water to 50μL.

[0079] Reaction conditions (Ura3 upstream and downstream segments): 94℃ heat denaturation for 3 min; 98℃ denaturation for 10 s, 60℃ annealing for 30 s, 68℃ extension for 1 min, for a total of 35 cycles; final extension at 68℃ for 5 min, followed by holding at 4℃.

[0080] PCR reaction system (donor DNA fragment): 2×KOD Buffer 25μL, primers P11 and P14 1.5μL each (10μmol / L), ura3 upstream fragment 1μL, ura3 downstream fragment 1μL, dNTPs (2mM each) 10μL, KOD polymerase (TOYOBO, 1U / μL) 1μL, and finally add water to 50μL.

[0081] Reaction conditions (donor DNA fragment): denaturation at 94℃ for 3 min; denaturation at 98℃ for 10 s, annealing at 60℃ for 30 s, extension at 68℃ for 2 min, for a total of 35 cycles; final extension at 68℃ for 5 min, followed by incubation at 4℃.

[0082] The knockout method is as follows:

[0083] Single colonies of *Pichia pastoris* LC375240 were picked and cultured overnight at 30°C and 200 rpm in 5 mL of YPD medium (20 g / L tryptone, 10 g / L yeast extract, 10 g / L glucose). The overnight culture was then inoculated at a 1% inoculum into 50 mL of YPD medium and cultured at 30°C and 200 rpm for expansion.

[0084] When the OD600 of the yeast culture to be expanded reaches 1.3–1.5, the culture is placed in a 50 mL centrifuge tube, incubated at 5000 g, 4 °C, for 5 min, and the supernatant is discarded. The precipitated cells are washed twice with 50 mL of sterile ddH2O water. 20 mL of pretreatment solution (100 mM lithium acetate, 10 mM DTT, 0.6 M sorbitol, 10 mM Tris-HCl, pH 7.5) is added, and the culture is incubated at 30 °C, 100 rpm for 30 min. The supernatant is then discarded by centrifugation. The cells are washed twice with 20 mL of sorbitol (1 mol / L). Finally, the cells are resuspended in 200 μL (1 mol / L) sorbitol.

[0085] Mix 80 μL of competent yeast cells with 500 ng pk-Cas9 plasmid and 1 μg of donor DNA, add to a pre-cooled 2 mm electroporation cuvette, incubate on ice for 5 min, and then electroporate (2.5 kV, 5 ms, 25 μF). Immediately after electroporation, add 1 mL sorbitol and 1 mL YPD, and incubate at 30 °C for 2 h. Finally, take 200 μL and spread it on a selection plate (Minimal SD Base 26.7 g / L, uracil 100 μg / mL, 5FOA 1 mg / mL, zeocin 100 μg / mL), and incubate at 30 °C for 2–3 days. After transformants grow on the plate, perform PCR verification and phenotypic verification, using wild-type Pichia pastoris as a control.

[0086] For the amount of competent yeast cells, plasmid, and donor DNA, refer to the general yeast electroporation transformation procedure. The total volume of pk-Cas9 plasmid and donor DNA should not exceed 1 / 10 of the competent cell volume. For electroporation and recovery conditions, refer to the general yeast electroporation and recovery procedure.

[0087] Figure 2 To identify ura3 gene knockout by PCR, 1 is the DNA molecular weight standard, 2 is the ura3 gene knockout band, and 3 is the wild-type non-knockout ura3 band.

[0088] Phenotypic validation of the ura3 gene knockout results are as follows: Figure 3 As shown, Figure 3 The left image shows the basic culture medium without uracil, and the right image shows the culture medium with uracil. This shows that PK yeast ura3 auxotroph does not grow in the basic culture medium without uracil, while PK yeast ura3 auxotroph grows when uracil is added to the basic culture medium.

[0089] The above experimental results show that the ura3 gene in Pichia kudriazweiss was knocked out, and that the pk-Cas9 plasmid can edit the yeast genome.

[0090] Example 2: The MSDS gene fragment was introduced into Pichia pastoris kudria.

[0091] This embodiment uses the pk-Cas9 plasmid obtained in Example 1 to replace the msds gene fragment at the position of the och1 gene in the PK yeast genome, thus achieving gene substitution. The steps are as follows:

[0092] Donor DNA was constructed by overlapping PCR of 1000 bp upstream and downstream of the och1 gene with the msds gene (och1 up-msds-och1 down). A suitable target site was selected in the och1 gene, and a 20 bp target sequence was constructed into the pk-Cas9 plasmid. The specific procedure is similar to that for knocking out the ura3 gene.

[0093] PCR identification results are shown below Figure 4 A 2000bp band indicates successful gene replacement, while a 1718bp band indicates unsuccessful gene replacement. Figure 4 The results showed that the gene was successfully replaced in 9 out of 14 replicates.

[0094] Comparative Example 1: Effects of other gene editing systems on PK yeast

[0095] When using the Pichia pastoris gene editing system (pHTX1-Cas9-gRNA-GUT-AOXTT plasmid and donor DNA) to perform gene editing on PK yeast, it was found that the pHTX1-Cas9-gRNA-GUT-AOXTT plasmid was... Figure 5 .

[0096] This editing system cannot effectively edit the genome of PK yeast.

[0097] PCR identification diagram (see image) Figure 6 The band at 1500 bp indicates that the ura3 gene was not knocked out, while the band at 711 bp indicates that the ura3 gene was knocked out. The PCR results show that gene editing of PK yeast using the Pichia pastoris gene editing system failed to produce an ura3-deficient strain. The appearance of the double bands at 1500 bp and 711 bp may be due to the influence of the donor DNA, as PCR tests after multiple passages showed that the 711 bp band disappeared, leaving only the 1500 bp band that was not knocked out.

[0098] Comparative Example 2: pk-Cas9 plasmids obtained by optimizing different Cas9 codons

[0099] Without host codon optimization of Cas9, the ura3-deficient strain could not be obtained. During the codon optimization process, it was found that the codon usage frequency of Pichia pastoris was significantly different from that of PK yeast.

[0100] Furthermore, in step 1 of Example 1, randomly selecting different open reading frames yielded different Cas9 codon results. During Cas9 codon optimization, other sequences were used to ligate the optimized Cas9 fragment with fragments A, B, C, and D into the vector. When constructing the gene-editing plasmid pk-Cas9, a low ura3 gene knockout success rate was observed. PCR identification results are shown below. Figure 7 The band at 1500 bp indicates that the ura3 gene was not knocked out, while the band at 711 bp indicates that the ura3 gene was knocked out.

[0101] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A PK yeast gene-editing plasmid, characterized in that, The PK yeast gene editing plasmid includes: a prokaryotic origin of replication, an resistance selection marker gene, a Cas9 gene expression cassette, a gRNA gene expression cassette, and an autonomous replication sequence; The Cas9 gene expression cassette comprises, from upstream to downstream, a Cas9 gene promoter, a Cas9 gene, and a terminator; the nucleotide sequence of the Cas9 gene is shown in SEQ ID NO.1; the nucleotide sequence of the Cas9 gene promoter is shown in SEQ ID NO.2; and the nucleotide sequence of the terminator is shown in SEQ ID NO.

3. The gRNA gene expression cassette, from upstream to downstream, includes the RNA polⅢ promoter, tRNA, and other components sequentially. Leu The gRNA gene expression cassette contains a guide sequence, a scaffold RNA sequence, and a polyT sequence; the gRNA gene expression cassette was amplified using the primer pairs shown in SEQ ID NO. 6-7. The autonomously replicating sequence is the ARS sequence of Saccharomyces cerevisiae; the ARS sequence was amplified by the primer pair shown in SEQ ID NO.12-13; The resistance selection marker genes are ampicillin resistance genes and bleomycin resistance genes; The prokaryotic replication start point is the replication start point of pBR322.

2. The PK yeast gene-editing plasmid according to claim 1, characterized in that, The nucleotide sequence of the gRNA gene expression cassette is shown in SEQ ID NO.

4.

3. The PK yeast gene-editing plasmid according to claim 1, characterized in that, The nucleotide sequence of the ARS sequence is shown in SEQ ID NO.

5.

4. The method for constructing the PK yeast gene-editing plasmid according to any one of claims 1-3, characterized in that, The Cas9 gene promoter and Cas9 gene terminator were obtained by PCR amplification using PK yeast genomic DNA as a template; the gRNA gene expression cassette and autonomous replication sequence were obtained by PCR amplification using synthetic plasmid as a template. The sequences shown in SEQ ID NO.1-5 were ligated into a vector to construct the gene editing plasmid pk-Cas9, which was then transformed into Escherichia coli DH5α to obtain the PK yeast gene editing plasmid.

5. The application of the PK yeast gene editing plasmid according to any one of claims 1-3 in the efficient editing of PK yeast.

6. The application according to claim 5, characterized in that, The competent Pichia pastoris kudrica was mixed with the PK yeast gene editing plasmid and donor DNA, and after being placed in an ice bath, it was transformed by electroporation. Immediately after electroporation, it was added to sorbitol and YPD medium for resuscitation to obtain the edited PK yeast.

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