Transformation method of liverwort for producing therapeutic protein, gene editing vector and gene editing transformant

Abstract

The invention belongs to the technical field of gene engineering, and relates to a transformation method of liverwort for producing therapeutic protein, which is characterized in that xylose transferase genes and fucose transferase genes in the liverwort are mutated, so that the liverwort loses xylose transferase activity and fucose transferase activity. According to the invention, a liverwort plant lacking xylosyltransferase and fucose transferase activity is constructed to obtain a new variety of liverwort capable of producing therapeutic protein, plant specific glycosylation is eliminated, adverse effects on product quality, safety and effectiveness are avoided, and the method has important significance on production of recombinant therapeutic protein in liverwort.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and relates to a method for transforming Marchantia polymorpha for producing therapeutic proteins, a gene editing vector and a gene editing transformant. Background Art

[0002] Marchantia polymorpha L. is also known as a cloud, Bagulong, rice seaweed, pus scab grass, ground thrush, ground duckweed, lichen, longan grass, etc. It is a spore plant of the genus Marchantia in the family Mosses. The plant body is light green or dark green, and the fronds are flat and ribbon-shaped. Marchantia belongs to the common group of Mosses plants, widely distributed all over the world, and is also an emerging model plant.

[0003] The production of recombinant therapeutic proteins is a rapidly developing field in the pharmaceutical industry. So far, most therapeutic proteins are produced in mammalian cell culture, namely Chinese hamster ovary cells (CHO cells). Although stable and safe recombinant proteins can be obtained, their cost is high, which greatly limits the large-scale production of therapeutic protein drugs. Although the expression of antibodies using expression systems such as yeast and bacteria is low-cost, there are significant differences in post-translational glycosylation modification from mammals, making it difficult to be used clinically. Plants have similar N-glycosylation pathways to mammals and can be grown on a large scale at a low cost. Therefore, the use of plants to produce therapeutic proteins has become a new research direction. Summary of the invention

[0004] In order to produce a homozygous Marchantia polymorpha plant lacking β-1,2-xylose transferase and α-1,3 fucosyl transferase activities, the present invention uses gene editing technology to target and knock out the xylose transferase and fucosyl transferase genes in Marchantia polymorpha, and the modified Marchantia polymorpha plant can be used to produce therapeutic proteins that do not contain plant-specific xylose and fucosyl glycosyl groups. The present invention provides a method for obtaining a Marchantia polymorpha plant lacking xylose transferase and fucosyl transferase activities, which is of great significance for the production of recombinant therapeutic proteins in Marchantia polymorpha.

[0005] The present invention adopts the following technical solution:

[0006] A method for modifying Marchantia polymorpha for producing therapeutic proteins comprises mutating a xylose transferase gene and a fucosyl transferase gene in the Marchantia polymorpha so that the Marchantia polymorpha loses the xylose transferase activity and the fucosyl transferase activity.

[0007] Most therapeutic proteins are composed of glycoproteins, so the differences in protein glycosylation between humans and plants must be considered and plant-specific glycosylation must be eliminated to avoid adverse effects on product quality, safety and effectiveness. Therefore, the plant-specific xylose transferase and fucosyl transferase activities are eliminated in this scheme to achieve the purpose of producing therapeutic proteins. Marchantia polyphylla is particularly suitable for modifying glycosylation pathways as a chassis to produce therapeutic glycoproteins because of its simple genetic background, small genome and haploidy. Therefore, glycosylation modification of Marchantia polyphylla so that it does not contain plant-specific xylose and fucose glycosyls is of great significance for using plants to produce therapeutic proteins suitable for humans.

[0008] Preferably, the mutation of the xylose transferase gene is located at the sequence listed in SEQ ID NO: 1, and the mutation of the fucosyl transferase gene is located at the sequence listed in SEQ ID NO: 2.

[0009] Preferably, Cas9 endonuclease is used to induce mutations in the xylose transferase gene and the fucosyl transferase gene in Marchantia polymorpha.

[0010] As a preference, the method specifically comprises the following steps:

[0011] S1. Design the target RNA fragments corresponding to the xylose transferase gene and the fucosyl transferase gene respectively;

[0012] S2. The target RNA fragment, the Marchantia polymorpha U6 promoter and the shortguide RNA standard element E1 from the GoldenBraid kit are connected to the pDGB3-α1 vector in the GoldenBraid kit to construct an α1-guide expression vector;

[0013] S3. The plant Cas9 endonuclease gene sequence optimized by Marchantia polymorpha codon was connected to the pDGB3-α2 vector in the GoldenBraid kit together with the CaMV35S promoter and CaMV35S terminator to construct an α2-Cas9 expression vector;

[0014] S4. Connect the α1-guide expression vector and the α2-Cas9 expression vector to the pDGB3-Ω1 vector in the GoldenBraid kit to construct an Ω1-guide-Cas9 plant gene editing vector;

[0015] S5. The hygromycin HYG gene nucleotide sequence was connected to the pDGB3-Ω2 vector in the GoldenBraid kit together with the CaMV35S promoter and the CaMV35S terminator to construct an Ω2-HYG expression vector;

[0016] S6. Connect the Ω1-guide-Cas9 plant gene editing vector and the Ω2-HYG expression vector to the pDGB3-α1 vector in the GoldenBraid kit to construct the α1-guide-Cas9-HYG plant gene editing vector;

[0017] S7. transferring the α1-guide-Cas9-HYG plant gene editing vector into the Agrobacterium GV3101 strain to obtain an Agrobacterium engineering strain containing the gene editing vector;

[0018] S8. The engineered Agrobacterium strain constructed above is transformed into Marchantia polymorpha, and resistant seedlings are obtained by hygromycin screening. Plants that are positive in PCR detection are transgenic Marchantia polymorpha seedlings.

[0019] Preferably, the transgenic Marchantia ternata plant detected by PCR refers to a transgenic Marchantia ternata plant obtained by designing and synthesizing detection primers of the Cas9 encoding gene sequence and performing DNA amplification. The positive strain in which the target band is observed under ultraviolet light is the transgenic Marchantia ternata plant.

[0020] The present invention also provides a gene editing vector of Marchantia polyphylla for producing therapeutic proteins, the vector comprising a gene sequence encoding a Cas9 endonuclease and a targeting sequence for editing β-1,2-xylose transferase and α-1,3-fucosyltransferase, the targeting sequence being shown in SEQ ID NO: 3-10.

[0021] The present invention also provides a gene-edited transformant of Marchantia polyphylla for producing therapeutic proteins, the transformant comprising a gene sequence encoding Cas9 endonuclease and a targeting sequence for editing β-1,2-xylose transferase and α-1,3 fucosyl transferase. Preferably, the host strain of the transformant is Agrobacterium tumefaciens GV3101.

[0022] By implementing the above technical solution, the present invention has the following advantages:

[0023] The present invention constructs a Marchantia polymorpha plant lacking xylose transferase and fucosyl transferase activities to obtain a new variety of Marchantia polymorpha capable of producing therapeutic proteins, eliminates plant-specific glycosylation, and avoids adverse effects on product quality, safety and effectiveness, which is of great significance for the production of recombinant therapeutic proteins in Marchantia polymorpha. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0025] Figure 1The target site of the β-1,2-xylosyltransferase gene is bound by the Cas9 endonuclease. The figure shows a partial DNA sequence of the xylosyltransferase gene, starting from the start codon (ATG). The underlined sequence represents the target site of the Cas9 endonuclease that recognizes the xylosyltransferase gene.

[0026] Figure 2 The α-1,3 fucosyltransferase gene is the target site for Cas9 endonuclease binding. The figure shows a partial DNA sequence of the fucosyltransferase gene, starting from the start codon (ATG). The underlined sequence represents the target site of the Cas9 endonuclease that recognizes the fucosyltransferase gene.

[0027] Figure 3 This is an example of a Cas9 endonuclease-induced mutation in the β-1,2-xylosyltransferase gene. The numbers on the left indicate transgenic positive lines, red indicates mutations or insertions, and - indicates deletions.

[0028] Figure 4 This is an example of a Cas9 endonuclease-induced mutation in the α-1,3 fucosyltransferase gene. The numbers on the left indicate transgenic positive lines, red indicates mutations or insertions, and - indicates deletions.

[0029] Figure 5 The presence of plant complex glycosylation in total protein was detected by western blot experiment to verify the absence of xylose transferase and fucosyl transferase at the protein level. The lane of wild-type Marchantia polyphylla (WT) had multiple bands, representing the presence of plant complex glycosylation xylose and fucose. XFT ) lane, there is no band, indicating that there is no detectable plant complex glycosylation, indicating that xylose transferase and fucosyl transferase are missing in the gene-edited mutant Marchantia polyphylla. DETAILED DESCRIPTION

[0030] The technical solutions in the embodiments of the present invention are described clearly and completely below through the accompanying drawings. The described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] The experimental methods in the following examples where specific conditions are not specified are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer; the materials, reagents, instruments, etc. used are commercially available unless otherwise specified.

[0032] The GoldenBraid kit used in the following examples, including PUPD2 vector, pDGB3-α1, pDGB3-Br, pDGB3-Br, pDGB3-Ω2 vector, etc., can be purchased publicly on the market. For details on vector usage and connection methods, please refer to the GoldenBraid kit manual and product website https: / / gbcloning.upv.es.

[0033] Example 1

[0034] Construction of plant gene editing vector containing Marchantia polymorpha U6 promoter, xylose transferase and fucosyl transferase gene targeting sequences and Cas9 endonuclease coding sequence

[0035] 1. Use the online tool https: / / gbcloning.upv.es / do / crispr / to design the corresponding target RNA fragments in the coding regions of the xylose transferase and fucosyl transferase genes, which were synthesized by Sangon Biotech in the form of a pair of primers. Then gradient annealing was performed to obtain the complete target RNA fragments.

[0036] 2. Connect the U6 promoter from Marchantia polyphylla into the PUPD2 vector in the GoldenBraid kit;

[0037] 3. Connect the target RNA fragment, Marchantia polymorpha U6 promoter and the shortguide RNA standard element E1 from the GoldenBraid kit to the pDGB3-α1 vector in the GoldenBraid kit to construct an α1-guide expression vector;

[0038] 4. Connect the plant Cas9 endonuclease gene sequence optimized by Marchantia polymorpha codons together with the CaMV35S promoter and CaMV35S terminator to the pDGB3-α2 vector in the GoldenBraid kit to construct the α2-Cas9 expression vector;

[0039] 5. Connect the α1-guide expression vector and the α2-Cas9 expression vector to the pDGB3-Ω1 vector in the GoldenBraid kit to construct the Ω1-guide-Cas9 plant gene editing vector;

[0040] 6. Connect the hygromycin HYG gene nucleotide sequence together with the CaMV35S promoter and CaMV35S terminator to the pDGB3-Ω2 vector in the GoldenBraid kit to construct the Ω2-HYG expression vector;

[0041] 7. Connect the Ω1-guide-Cas9 plant gene editing vector and the Ω2-HYG expression vector to the pDGB3-α1 vector in the GoldenBraid kit to construct the α1-guide-Cas9-HYG plant gene editing vector.

[0042] Example 2

[0043] Genetic transformation of Marchantia polymorpha with Agrobacterium tumefaciens-mediated α1-guide-Cas9-HYG plant gene editing vector to obtain transgenic Marchantia polymorpha plants

[0044] 1. Obtaining the engineered Agrobacterium tumefaciens containing the α1-guide-Cas9-HYG plant gene editing vector

[0045] The plant gene editing vector containing α1-guide-Cas9-HYG in Example 1 was transferred into Agrobacterium tumefaciens GV3101 by freeze-thaw method, and PCR verification was performed. The results showed that the plant expression vector containing the gene had been successfully constructed into the Agrobacterium tumefaciens strain.

[0046] 2. Agrobacterium tumefaciens-mediated transformation of Marchantia polymorpha with α1-guide-Cas9-HYG vector

[0047] 2.1. Pre-culture of Marchantia polyphylla embryos

[0048] Liquid pre-culture: Pour about 20 mL of 1 / 2 B5 liquid culture medium into a conical flask, take the embryo of Marchantia polymorpha and place it in the liquid culture medium for static pre-culture, culture at 21°C and 16h / 8h (light / dark) light for 6 days, and it can be used for Marchantia polymorpha transformation.

[0049] 2.2. Co-cultivation of Agrobacterium and embryos

[0050] The pre-cultured embryos of Marchantia polyphylla were added to the activated MES-Mgcl2 suspension of Agrobacterium tumefaciens engineered bacteria containing the α1-guide-Cas9-HYG plant gene editing vector, so that the embryos were fully in contact with the bacterial solution, and allowed to infect for 10 minutes under a vacuum environment. Excess bacterial solution was aspirated, washed 3 times with sterile water, and the washed embryos were evenly spread on 1 / 2B5 solid transformation medium and cultured in the dark at 21°C for 2 days. The embryos added with the MES-Mgcl2 suspension of Agrobacterium tumefaciens without the target gene were used as controls.

[0051] 2.3. Screening of resistant regenerated plants

[0052] Wash the co-cultured embryos on 1 / 2B5 solid medium with sterile water several times to wash off the Agrobacterium propagated during dark culture until the washing liquid is clear. Then add 5mL of screening solution (50μg / mL hygromycin + 2000μg / mL cephalosporin), shake slightly to evenly distribute the screening solution in the culture dish, suck away 4mL of screening solution and leave 1mL, let it dry naturally, and place it at 21°C and 16h / 8h light conditions to continue culturing for 2-3 weeks. Move the well-growing resistant regenerated embryos to the screening medium (1 / 2B5 + 5μg / mL hygromycin + 100μg / mL cephalosporin) for culture, so as to obtain hygromycin-resistant regenerated Marchantia plants.

[0053] 3. PCR detection of transgenic Marchantia polyphylla plants

[0054] According to the gene sequence of Cas9 endonuclease, forward detection primer (Cas9-F: 5'-AGGCTCAGGTTTCAGGAC-3') and reverse detection primer (Cas9-R: 5'-GAGTAAGCCACGGTAGGA-3') were designed to detect positive overexpression plants. The results showed that specific DNA fragments could be amplified using the designed PCR specific detection primers. However, no fragments were amplified when non-transformed Marchantia ovata genomic DNA was used as a template.

[0055] In this example, the plant expression vector was transformed into Agrobacterium tumefaciens GV3101 to obtain an Agrobacterium tumefaciens strain containing an α1-guide-Cas9-HYG plant gene editing vector for transforming Marchantia polyphylla, and the constructed Agrobacterium tumefaciens strain was used to transform Marchantia polyphylla to obtain a transgenic Marchantia polyphylla plant that was positive by PCR detection. The acquisition of the transgenic Marchantia polyphylla plant provides materials for Sanger sequencing of mutants.

[0056] Example 3

[0057] Identification of gene mutations of xylosyltransferase and fucosyltransferase in transgenic Marchantia polymorpha by Sanger sequencing 1. Extraction of Marchantia polymorpha genomic DNA

[0058] Place the Marchantia polyphylla tissue in a 2 mL centrifuge tube, add 2 sterilized steel beads, freeze with liquid nitrogen, and grind with a grinder; add 1000 μL 2x CTAB extraction buffer, vortex mix, and place in a 65°C water bath for 30 min; after cooling to room temperature, add 900 μL chloroform and mix by inversion; centrifuge at 12000 rpm for 10 min, transfer the upper aqueous phase to a new 2 mL centrifuge tube, add 750 μL isopropanol and mix by inversion, and precipitate at -20°C for more than 30 min; centrifuge at 12000 rpm for 10 min, discard the supernatant, add 75% ethanol to wash twice; dry at room temperature, and add water to dissolve the DNA.

[0059] 2. PCR amplification of xylose transferase and fucosyl transferase genes

[0060] According to the sequence of the xylose transferase gene, a forward primer (XylT-F: 5'-AAACCGATTGATTGATGG-3') and a reverse primer (XylT-R: 5'-GAAGCTCCTGTTCGTAATT-3') were designed to amplify the xylose transferase gene from the transgenic Marchantia polymorpha that was positive in PCR detection;

[0061] According to the sequence of the fucosyltransferase gene, a forward primer (Fuc-F: 5'-ATACGCATTGTGATTAGAAG-3') and a reverse primer (Fuc-R: 5'-CAACGAATAAGGGAGAAG-3') were designed to amplify the fucosyltransferase gene from the transgenic Marchantia polymorpha that were positive in PCR detection;

[0062] 3. Sanger sequencing to identify gene mutations

[0063] The PCR amplified products were subjected to Sanger sequencing by Shanghai Sangon Biotechnology Co., Ltd., and then the sequencing results were compared with the xylose transferase and fucosyl transferase gene sequences of wild-type Marchantia polymorpha to screen out transgenic Marchantia polymorpha plants with double mutations of the two enzymes.

[0064] Example 4

[0065] Detection of complex glycosylation in transgenic Marchantha polymorpha by western blot

[0066] 1. Extraction of total protein from plant tissues

[0067] Take 0.1g of transgenic Marchantia polyphylla leaves with mutations in both glycosylases, grind into powder in liquid nitrogen, add an equal volume of pre-cooled lysis buffer (PBS+0.1% Triton X100+cocktail+pefabloc), quickly vortex mix, insert on ice for 20min, centrifuge at 12000rpm at 4℃ for 10min, take the supernatant into a new 1.5mL centrifuge tube, repeat the centrifugation several times, and take the supernatant, which is the total protein extract.

[0068] 2. SDS-PAGE gel electrophoresis

[0069] Take 60 μL of total protein extract and add it to 20 μL of 4× loading buffer, mix well, boil in 100℃ water bath for 10 min, centrifuge at 12000 rpm for 2 min. Take 20 μL of boiled protein extract and add it to the well of denatured protein precast gel (Yisheng Biotechnology Shanghai Co., Ltd.), keep constant voltage at 100V, and perform SDS-PAGE electrophoresis for 1h.

[0070] 3. Transfer (wet transfer)

[0071] Prepare two 3M filter papers (10cm×7cm) and a PVDF membrane (9cm×6cm), and place the PVDF membrane in methanol to wake up for 30s. In 1×Transfer buffee, clamp the film in the order of sponge-3M filter paper-protein glue-PVDF membrane-3M filter paper-sponge, put it in the transfer tank, and transfer the membrane for 50min at a constant current of 200mA.

[0072] 4. Closure

[0073] Soak the transferred membrane in 1×PBS for 5 minutes, discard the PBS, add blocking solution (5% skim milk powder + PBST) for 1 hour, and wash three times with 1×PBST (1L PBS + 1mL Tween20), each time for 5 minutes.

[0074] 5. Primary Antibody Incubation

[0075] Anti-HRP monoclonal antibody (Thermo Fisher) was added to 4 mL 3% BSA solution (3% BSA + PBST) at a ratio of 1:2000, and the antibody solution was poured into the blocked and washed membrane and incubated at 4°C overnight.

[0076] 6. Secondary Antibody Incubation

[0077] The membrane was washed three times with 1×PBST for 10 min each time, rabbit secondary antibody was added to 5% skim milk powder at a ratio of 1:10,000, and incubated at room temperature for 2 h. The membrane was washed three times with 1×PBST for 10 min each time.

[0078] 7. Development

[0079] The washed membrane was placed in a black developing box, developer was added dropwise, and the membrane was developed for 1 min in the dark. The membrane was then developed and photographed using the Tanon 4600SF fully automatic chemiluminescence image analysis system.

[0080] In this example, a western blot experiment was used to detect the level of complex glycosylation of proteins in gene-edited mutant Marchantia polymorpha plants. A large number of complex glycosylated protein bands could be detected in the wild-type Marchantia polymorpha, while no complex glycosylation was detected in the Marchantia polymorpha with double mutations in the glycosylases, indicating that the activity of xylose transferase and fucosyl transferase was absent in the modified mutant Marchantia polymorpha. The mutant Marchantia polymorpha does not contain plant-specific xylose and fucose glycosylation and can be used to produce humanized therapeutic proteins.

[0081] The gene-edited liverwort plant of the present invention does not contain xylosyl and fucosylation, and a Target RNA fragment is designed according to the gene sequence of the xylose transferase and fucosylation of the liverwort, and the Target RNA is connected with the U6 promoter, shortguide RNA element and Cas9 gene of the liverwort to construct a plant gene editing vector; the gene editing vector is transferred into Agrobacterium, and the Agrobacterium is transferred into the liverwort, and antibiotic screening is performed to obtain a transformed cell containing the gene editing vector, and a transgenic plant is regenerated, and then the genomic DNA of the transgenic plant is extracted, and the mutation of the two glycosyltransferase genes is identified by Sanger sequencing. The two gene double mutant liverwort plants obtained in the present invention do not contain detectable xylosyl transferase and fucosylation activities. The present invention provides a method for obtaining a liverwort plant that does not contain xylosyl and fucosylation, and the plant can be used to produce a humanized therapeutic protein that does not contain plant-specific glycosylation.

[0082] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for modifying Marchantia polymorpha for producing therapeutic proteins, characterized in that: The xylose transferase gene and the fucosyl transferase gene in Marchantia polyphylla were mutated to make Marchantia polyphylla lose the xylose transferase activity and the fucosyl transferase activity.

2. The method for transforming Marchantia polymorpha for producing therapeutic proteins according to claim 1, characterized in that: The mutation of the xylose transferase gene is located at the sequence listed in SEQ ID NO: 1, and the mutation of the fucosyl transferase gene is located at the sequence listed in SEQ ID NO:

2.

3. A method for transforming Marchantia polymorpha for producing therapeutic proteins according to claim 1 or 2, characterized in that: Cas9 endonuclease was used to induce mutations in the xylosyltransferase and fucosyltransferase genes in Marchantia polymorpha.

4. The method for transforming Marchantia polymorpha for producing therapeutic proteins according to claim 3, characterized in that: The specific steps include: S1. Design the target RNA fragments corresponding to the xylose transferase gene and the fucosyl transferase gene respectively; S2. Connect the target RNA fragment, U6 promoter and short guide RNA standard element E1 from the GoldenBraid kit to the pDGB3-α1 vector in the GoldenBraid kit to construct an α1-guide expression vector; S3. The plant Cas9 endonuclease gene sequence optimized by Marchantia polymorpha codon was connected to the pDGB3-α2 vector in the GoldenBraid kit together with the CaMV35S promoter and CaMV35S terminator to construct an α2-Cas9 expression vector; S4. Connect the α1-guide expression vector and the α2-Cas9 expression vector to the pDGB3-Ω1 vector in the GoldenBraid kit to construct an Ω1-guide-Cas9 plant gene editing vector; S5. The hygromycin HYG gene nucleotide sequence was connected to the pDGB3-Ω2 vector in the GoldenBraid kit together with the CaMV35S promoter and the CaMV35S terminator to construct an Ω2-HYG expression vector; S6. Connect the Ω1-guide-Cas9 plant gene editing vector and the Ω2-HYG expression vector to the pDGB3-α1 vector in the GoldenBraid kit to construct the α1-guide-Cas9-HYG plant gene editing vector; S7. transferring the α1-guide-Cas9-HYG plant gene editing vector into Agrobacterium to obtain an Agrobacterium engineering strain containing the gene editing vector; S8. The constructed Agrobacterium engineering strain is transformed into Marchantia polymorpha, and resistant seedlings are obtained by hygromycin screening. Plants that are positive in PCR detection are transgenic Marchantia polymorpha plants.

5. The method for transforming Marchantia polymorpha for producing therapeutic proteins according to claim 4, characterized in that: The U6 promoter is a U6 promoter derived from Marchantia polyphylla.

6. The method for transforming Marchantia polymorpha for producing therapeutic proteins according to claim 4, characterized in that: The transgenic Marchantia ternata plant detected by PCR refers to a transgenic Marchantia ternata plant obtained by designing and synthesizing detection primers of the Cas9 encoding gene sequence and performing DNA amplification. The positive strain in which the target band is observed under ultraviolet light is the transgenic Marchantia ternata plant.

7. A gene editing vector of Marchantia polymorpha for producing therapeutic proteins, characterized in that: The gene editing vector includes a gene sequence encoding a Cas9 endonuclease and a targeting sequence for editing β-1,2-xylose transferase and α-1,3-fucosyl transferase.

8. The gene editing vector of Marchantia polymorpha for producing therapeutic proteins according to claim 7, characterized in that: The targeting sequences are shown in SEQ ID NOs: 3-10.

9. A gene-edited transformant of Marchantia polymorpha for producing therapeutic proteins, characterized in that: The gene editing transformant comprises a gene sequence encoding a Cas9 endonuclease and a targeting sequence for editing β-1,2-xylose transferase and α-1,3-fucosyl transferase.

10. The gene-edited transformant of Marchantia polymorpha for producing therapeutic proteins according to claim 9, characterized in that: The host strain of the transformant is Agrobacterium tumefaciens GV3101.

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