Skin organoid construction method of human embryonic stem cells based on high-expression recombinant human COL17A1 protein and application of skin organoid construction method

Abstract

The invention provides a skin organoid construction method of human embryonic stem cells based on high-expression recombinant human COL17A1 protein and application of the skin organoid construction method, and relates to the technical field of molecular biology, and the skin organoid construction method comprises the following steps: integrating nucleic acid molecules for coding COL17A1 protein to a KRT5 site of genome DNA of the human embryonic stem cells to obtain recombinant human embryonic stem cells; the nucleic acid molecule for coding the COL17A1 protein contains the gene for coding the COL17A1 protein, and compared with the traditional'random integration 'or'universal safe port site' (AAVS1), the yield of the recombinant human COL17A1 protein is remarkably improved by integrating at the KRT5 site, and the stable and efficient expression of the recombinant human COL17A1 protein is realized. The technical problem that in the prior art, the expression quantity of recombinant human COL17A1 protein is low when mammalian cells such as HEK-293T and CHO are used for expressing the recombinant human COL17A1 protein is solved.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to a method for constructing skin organoids based on human embryonic stem cells with high expression of recombinant human COL17A1 protein and its application. Background Technology

[0002] COL17A1 (collagen type XVII α1 chain) belongs to the XVII collagen family and is an important transmembrane protein in the basement membrane region. As a core component of hemidesmosomes, COL17A1 exists in the basement membrane region of the epidermis and dermis, playing a crucial role in maintaining skin structural stability, cell adhesion, signal transduction, and tissue regeneration. It also regulates the stem cell microenvironment, promotes stem cell proliferation, regulates keratinocyte migration, and promotes wound healing. In recent years, it has been widely used in the treatment of skin diseases, gene therapy, regenerative medicine, and anti-aging.

[0003] COL17A1 is present in extremely low amounts in humans and animals, making extraction difficult and yielding very low quantities, which is insufficient to meet application demands. Currently, its main sources rely on recombinant expression in *E. coli*, *Pichia pastoris*, or mammalian cells (such as HEK-293T and CHO). However, recombinant expression systems still face numerous technical challenges. While recombinant expression of COL17A1 using *E. coli* or *Pichia pastoris* is inexpensive and yields high quantities, the expressed COL17A1 lacks glycosylation, easily forms inclusion bodies, and may contain endotoxins. Recombinant human COL17A1 protein expressed in mammalian cells such as HEK-293T and CHO is close to natural glycosylation and has higher biological activity, making it suitable for therapeutic research. However, its expression level is low (usually <1 g / L), and purification steps such as affinity chromatography and ion exchange are required, significantly increasing production costs.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] One of the objectives of this invention is to provide a method for constructing recombinant human embryonic stem cells that highly express human COL17A1 protein, in order to solve the technical problem of low expression levels of recombinant human COL17A1 protein when using mammalian cells such as HEK-293T and CHO in the prior art.

[0006] The second objective of this invention is to provide a kit for preparing recombinant human embryonic stem cells that highly express human COL17A1 protein.

[0007] A third objective of this invention is to provide the application of recombinant human embryonic stem cells constructed by the above-described method or prepared using the above-described kit in the preparation of skin organoids.

[0008] The fourth objective of this invention is to provide a skin organoid that highly expresses human COL17A1 protein.

[0009] The fifth objective of this invention is to provide the application of the above-mentioned skin organoids in the production of human COL17A1 protein.

[0010] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: In a first aspect, the present invention provides a method for constructing recombinant human embryonic stem cells that highly express human COL17A1 protein, the method comprising integrating a nucleic acid molecule encoding COL17A1 protein into the KRT5 site of the genomic DNA of human embryonic stem cells to obtain recombinant human embryonic stem cells; The nucleic acid molecule encoding the COL17A1 protein contains a gene encoding the COL17A1 protein.

[0011] Furthermore, this includes co-introducing a homologous recombinant plasmid expressing COL17A1, a plasmid expressing sgRNA, and a plasmid expressing Cas9 or Cas12 into human embryonic stem cells. The sgRNA is a specific sgRNA targeting the KRT5 site, and the sequence of the sgRNA is shown in SEQ ID NO.3.

[0012] Furthermore, the homologous recombination plasmid includes a nucleic acid molecule encoding the COL17A1 protein, and has an upstream homologous arm upstream of the nucleic acid molecule encoding the COL17A1 protein and a downstream homologous arm downstream of the nucleic acid molecule encoding the COL17A1 protein; the upstream and downstream homologous arms are used to integrate the nucleic acid molecule encoding the COL17A1 protein into the KRT5 site of the genomic DNA of human embryonic stem cells; The nucleotide sequence of the upstream homologous arm is shown in SEQ ID NO.4, and the nucleotide sequence of the downstream homologous arm is shown in SEQ ID NO.5.

[0013] Furthermore, the plasmid expressing sgRNA and the plasmid expressing Cas9 or Cas12 are the same plasmid.

[0014] Furthermore, the nucleic acid molecule encoding the COL17A1 protein also contains a selection marker gene; Preferably, the screening marker gene includes a puromycin resistance gene; Preferably, the nucleotide sequence of the COL17A1 protein is shown in SEQ ID NO.1; Preferably, the gene encoding the COL17A1 protein is shown in SEQ ID NO.2.

[0015] Secondly, the present invention provides a kit for preparing recombinant human embryonic stem cells that highly express human COL17A1 protein, the kit comprising the above-mentioned homologous recombinant plasmid expressing COL17A1 and plasmid expressing sgRNA.

[0016] Furthermore, the kit also includes plasmids expressing Cas9 or Cas12; Preferably, the plasmid expressing sgRNA and the plasmid expressing Cas9 or Cas12 are the same plasmid.

[0017] Thirdly, the present invention provides the application of recombinant human embryonic stem cells constructed by the above-described method or prepared using the above-described kit in the preparation of skin organoids.

[0018] Fourthly, the present invention provides a skin organoid that highly expresses human COL17A1 protein, obtained by differentiation from genetically engineered human embryonic stem cells constructed by the above method or genetically engineered human embryonic stem cells constructed using the above kit.

[0019] Fifthly, the present invention provides the application of the above-mentioned skin organoids in the production of human COL17A1 protein.

[0020] The present invention provides a method for constructing recombinant human embryonic stem cells with high expression of human COL17A1 protein. Utilizing CRISPR / Cas gene editing technology, the gene encoding human COL17A1 protein is precisely integrated into a specific site—the KRT5 site—in the genome of human embryonic stem cells (hESCs). Compared to traditional "random integration" or "universal safe harbor site" (AAVS1), integration at the KRT5 site significantly increases the yield of recombinant human COL17A1 protein, achieving stable and efficient expression of the recombinant human COL17A1 protein. This solves the technical problem of low expression levels of recombinant human COL17A1 protein when using mammalian cells such as HEK-293T and CHO in existing technologies. Attached Figure Description To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0021] Figure 1 This is a schematic diagram of the Donor structure, a homologous recombinant plasmid expressing COL17A1 protein at the AAVS1 gene site provided in Example 1 of the present invention. Figure 2This is a schematic diagram of the structure of the homologous recombinant plasmid Donor, which expresses COL17A1 protein at the KRT5 gene site, as provided in Example 1 of the present invention. Figure 3 This is a schematic diagram of the structure of the sgRNA sequence cloned into the pLentiV2-Cas9-U6-sgRNA plasmid provided in Example 2 of the present invention; Figure 4 This is a statistical chart of COL17A1 protein expression levels in recombinant cells expressing COL17A1 protein at different gene loci, provided in Example 2 of the present invention. Figure 5 This is a statistical diagram of COL17A1 protein expression levels in skin organoids expressing COL17A1 protein at different gene loci, as provided in Example 3 of the present invention. Figure 6 The results of the effect of different gene loci expressing COL17A1 protein on fibroblast adhesion provided in Example 5 of the present invention; Figure 7 The results of the effect of different gene loci expressing COL17A1 protein on fibroblast proliferation provided in Example 6 of the present invention; Figure 8 The results of the effect of different gene loci expressing COL17A1 protein on fibroblast migration provided in Example 7 of the present invention. Detailed Implementation

[0022] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0023] Generally, the nomenclature and techniques used in cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization, together with those described herein, are those well-known and commonly used in the art. Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well-known in the art and described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature, laboratory procedures, and techniques used in analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry, together with those described herein, are those well-known and commonly used in the art.

[0024] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] The present invention provides a method for constructing recombinant human embryonic stem cells that highly express human COL17A1 protein, the method comprising integrating a nucleic acid molecule encoding COL17A1 protein into the KRT5 site of the genomic DNA of human embryonic stem cells to obtain recombinant human embryonic stem cells; The nucleic acid molecule encoding the COL17A1 protein contains a gene encoding the COL17A1 protein.

[0026] Using CRISPR / Cas gene editing technology, the gene encoding the human COL17A1 protein was precisely integrated into a specific site—the KRT5 site—in the genome of human embryonic stem cells (hESCs). Compared to traditional "random integration" or "universal safe harbor site" (AAVS1), integration at the KRT5 site significantly increased the yield of recombinant human COL17A1 protein, achieving stable and efficient expression of the recombinant human COL17A1 protein. This solves the technical problem of low expression levels of recombinant human COL17A1 protein in existing mammalian cells such as HEK-293T and CHO.

[0027] In some specific embodiments, the method includes co-introducing a homologous recombinant plasmid expressing COL17A1, a plasmid expressing sgRNA, and a plasmid expressing Cas9 or Cas12 into human embryonic stem cells; wherein the sgRNA is a specific sgRNA targeting the KRT5 site, and the sequence of the sgRNA is shown in SEQ ID NO.3.

[0028] Cas9 or Cas12 and sgRNA can be expressed in the same plasmid. In some specific embodiments, the plasmid expressing sgRNA and the plasmid expressing Cas9 or Cas12 are the same plasmid.

[0029] In some specific embodiments, the homologous recombinant plasmid includes a nucleic acid molecule encoding the COL17A1 protein, and has an upstream homologous arm upstream of the nucleic acid molecule encoding the COL17A1 protein and a downstream homologous arm downstream of the nucleic acid molecule encoding the COL17A1 protein; the upstream and downstream homologous arms are used to integrate the nucleic acid molecule encoding the COL17A1 protein into the KRT5 site of the genomic DNA of human embryonic stem cells; the nucleotide sequence of the upstream homologous arm is shown in SEQ ID NO.4, and the nucleotide sequence of the downstream homologous arm is shown in SEQ ID NO.5.

[0030] In some specific embodiments, the nucleic acid molecule encoding the COL17A1 protein also contains a selection marker gene to identify and enrich successfully edited cells.

[0031] The selection marker genes can be antibiotic resistance markers, metabolic markers, or fluorescent protein reporter genes. Antibiotic resistance markers include neomycin resistance genes, hygromycin B resistance genes, blast fungicide resistance genes, puromycin resistance genes, or bleomycin resistance genes. Metabolic markers include dihydrofolate reductase (DHFR) systems, glutamine synthase (GS) systems, or TK... - / GCV system (negative selection). Fluorescent protein reporter genes include EGFP / eGFP, mCherry / RPF, or Luciferase. In some specific embodiments, the selection marker gene is a puromycin resistance gene.

[0032] In some specific embodiments, the nucleotide sequence of the COL17A1 protein is shown in SEQ ID NO.1; in some specific embodiments, the gene encoding the COL17A1 protein is shown in SEQ ID NO.2.

[0033] According to another aspect of the present invention, a kit for preparing recombinant human embryonic stem cells that highly express human COL17A1 protein is also provided, the kit comprising the above-mentioned homologous recombinant plasmid expressing COL17A1 and plasmid expressing sgRNA.

[0034] In some specific embodiments, the kit further includes a plasmid expressing Cas9 or Cas12; in some specific embodiments, the plasmid expressing sgRNA and the plasmid expressing Cas9 or Cas12 are the same plasmid.

[0035] According to another aspect of the present invention, the application of recombinant human embryonic stem cells constructed by the above-described method or prepared using the above-described kit in the preparation of skin organoids is also provided.

[0036] Among them, skin organoids include epidermis, dermis and / or skin appendage structures, wherein the skin appendage structures include at least one of hair follicles, sebaceous glands or sweat glands.

[0037] According to another aspect of the present invention, a skin organoid highly expressing human COL17A1 protein is also provided, which is obtained by differentiation of genetically engineered human embryonic stem cells constructed by the above method or genetically engineered human embryonic stem cells constructed using the above kit.

[0038] According to another aspect of the invention, the use of the above-described skin organoids in the production of human COL17A1 protein is also provided.

[0039] Skin organoids can be used in bioreactors to maintain and continuously secrete target proteins over long periods, producing highly efficient and stable recombinant human COL17A1 protein, which is more efficient and stable than traditional batch culture. Designing the expression and secretion of COL17A1 protein into the culture supernatant simplifies the harvesting and purification process, eliminating the need for cell disruption and significantly reducing the difficulty and cost of downstream purification. Furthermore, the produced recombinant human COL17A1 protein promotes cell proliferation, adhesion, and migration, exhibiting biological activity.

[0040] The present invention will be further illustrated below by way of examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0041] The amino acid sequence of the COL17A1 protein is shown in SEQ ID NO.1. Specifically, COL17A1 expresses a signal peptide secreted into the supernatant at its anterior end and a 6xhix tag before the stop codon at its posterior end. The gene encoding COL17A1 used in the following examples is shown in SEQ ID NO.2.

[0042] Example 1: Construction of the Donor vector, a homologous recombinant plasmid expressing COL17A1 protein at different gene loci. 550bp homologous arms were designed on both sides of the AAVS1 or KRT5 cleavage site. These homologous arms were cloned into the Donor vector. Then, the expression sequences encoding the COL17A1 gene and the puro gene were cloned into the Donor vector containing the homologous arms, resulting in homologous recombinant plasmids Donor vectors expressing the COL17A1 protein, named pAAVS1-COL17A1-Donor and pKRT5-COL17A1-Donor, respectively. Their structural diagrams are shown below. Figure 1 and Figure 2As shown, the expression sequences encoding the COL17A1 and puro genes are located between the two homologous arms of the insertion site. The specific sequences of the Donor vector, a homologous recombinant plasmid expressing the COL17A1 protein, include: the left homologous arm sequence of the target gene, the sequence of the gene encoding COL17A1 and puro, and the right homologous arm sequence of the target gene. The spacer sequence of the sgRNA targeting the KRT5 cleavage site (KRT5-sgRNA) and its homologous arm sequences, and the spacer sequence of the sgRNA targeting the AAVS1 cleavage site (AAVS1-sgRNA) and its homologous arm sequences are shown in SEQ ID NO.3 to SEQ ID NO.8, respectively.

[0043] Example 2 Construction of recombinant H1ES cells expressing COL17A1 protein at different gene loci The AAVS1-sgRNA and KRT5-sgRNA sequences used for gene editing were cloned into the pLentiV2-Cas9-U6-sgRNA plasmid expression vector, respectively, to obtain the pLentiV2-Cas9-U6-AAVS1-sgRNA and pLentiV2-Cas9-U6-KRT5-sgRNA expression plasmids, with the structures shown below. Figure 3 As shown. pAAVS1-COL17A1-Donor and pLentiV2-Cas9-U6-AAVS1-sgRNA were mixed at a plasmid molar ratio of 1:1, incubated with Lipofectamine™ 3000 transfection reagent, and transfected into H1ES cells. After clonal selection, H1ES monoclonal cell lines stably expressing COL17A1 protein at the AAVS1 gene locus were obtained, designated as H1-AAVS1-COL17A1 cell lines. Similarly, pKRT5-COL17A1-Donor and pLentiV2-Cas9-U6-KRT5-sgRNA were mixed at a plasmid molar ratio of 1:1, incubated with Lipofectamine™ 3000 transfection reagent, and transfected into H1ES cells. After clonal selection, H1ES monoclonal cell lines stably expressing COL17A1 protein at the KRT5 gene locus were obtained, designated as H1-KRT5-COL17A1 cell lines.

[0044] Culture supernatant from H1-AAVS1-COL17A1 and H1-KRT5-COL17A1 cells was collected after 72 hours, and the expression level of COL17A1 protein was compared using Western blot. Results are as follows: Figure 4As shown, the total protein loading amount was consistent in each well. Both the H1-AAVS1-COL17A1 and H1-KRT5-COL17A1 cell lines successfully expressed COL17A1 protein, but the expression level of COL17A1 protein in the H1-KRT5-COL17A1 cell line was significantly higher than that in the H1-AAVS1-COL17A1 cell line.

[0045] Example 3: Comparison of protein expression levels of skin organoids expressing COL17A1 protein at different gene loci Two cell lines, H1-AAVS1-COL17A1 and H1-KRT5-COL17A1, were differentiated into 3D skin organoids using a method disclosed in Chinese Patent Application No. 202111322147.9, based on existing laboratory differentiation techniques. The culture supernatant from mature skin organoids was collected after 72 hours, and the expression level of COL17A1 protein was compared using Western blot. The results are as follows... Figure 5 As shown, when the total protein loading amount per well was consistent, the expression level of COL17A1 protein in Organoid-KRT5-COL17A1 skin organoids was significantly higher than that in Organoid-AAVS1-COL17A1 skin organoids.

[0046] Example 4: Production and Preparation of Human COL17A1 Protein for High-Efficiency Secretion by Skin Organoids Skin organoids differentiated from H1-AAVS1-COL17A1 and H1-KRT5-COL17A1 cell lines were seeded into a bioreactor and cultured with feed for 10 days. The supernatant of the skin organoid culture medium was collected, and the COL17A1 protein corresponding to the elution peak was eluted and collected using a nickel ion affinity chromatography column to obtain recombinant human COL17A1 protein.

[0047] The BCA method was used to quantitatively detect the COL17A1 protein yield of recombinant human skin organoids. The COL17A1 yield of H1-AAVS1-COL17A1 was 0.23 g / L, and that of H1-KRT5-COL17A1 was 2.5 g / L. Although the gene encoding COL17A1 integrated at the AAVS1 site could be expressed, the expression level was low and could not achieve the goal of producing COL17A1 protein.

[0048] Example 5: The role of COL17A1 protein in promoting fibroblast adhesion The H1-AAVS1-COL17A1 and H1-KRT5-COL17A1 proteins obtained in Example 4 were dissolved in PBS to prepare a 1 mg / ml protein solution. For the cell adhesion assay in 96-well plates, 100 μL of COL17A1 protein solution was added to each well of the experimental group, and PBS solution was used for the negative group. The plates were coated overnight at 4°C, then incubated with 1% denatured BSA for 1 hour. The plates were then washed three times with serum-free medium to remove excess gel. Fibroblasts were digested, and 5000 cells were seeded in each well. A blank control (no cells, only medium) was added. The plates were incubated at 37°C for 1 hour. After 1 hour, the medium was removed from each well, and the cells were gently washed three times with PBS to remove non-adhered cells. 100 μL of medium containing 10% CCK-8 was added, and the plates were incubated at 37°C for another 2 hours. The absorbance at 450 nm was measured using a microplate reader, and the cell adhesion rate was calculated. Results are as follows: Figure 6 As shown, the cell adhesion rate of COL17A1 protein-coated well plates was significantly higher than that of PBS-coated well plates, and the COL17A1 protein derived from H1-KRT5-COL17A1 had a more significant cell adhesion-promoting effect than that derived from H1-AAVS1-COL17A1.

[0049] Example 6: Effect of COL17A1 protein on fibroblast proliferation The H1-AAVS1-COL17A1 and H1-KRT5-COL17A1 proteins obtained in Example 4 were diluted to appropriate concentrations using DMEM basal medium: 10 μg / ml, 5 μg / ml, 2.5 μg / ml, 1.25 μg / ml, 0.625 μg / ml, 0.3125 μg / ml, and 0 μg / ml. The sample group consisted of the above-mentioned diluted COL17A1, while the blank control group used DMEM basal medium. Each group had three replicates. One day prior to incubation, fibroblasts were seeded into 96-well plates at 2000 cells per well, with no cells seeded in the outer wells. PBS was added to prevent the inner medium from drying out. Cells were incubated overnight at 37°C. Each cell group was then incubated with a series of diluted COL17A1 protein concentrations and DMEM basal medium, and cultured for another 48 hours. After 48 hours, the culture medium in each well was removed, the cells were washed three times with PBS, and culture medium containing 10% CCK-8 was added. The cells were then incubated at 37°C for another 2 hours, and the absorbance at 450 nm was measured using a microplate reader. Results are as follows: Figure 7 As shown, with the increase of COL17A1 protein concentration, the proliferation activity of fibroblasts gradually increased, and compared with H1-AAVS1-COL17A1, COL17A1 protein derived from H1-KRT5-COL17A1 showed a more significant cell proliferation promoting effect.

[0050] Example 7: The role of COL17A1 protein in promoting fibroblast migration In a 12-well plate, use a marker to draw evenly spaced horizontal lines on the back of the plate using a ruler. Digest and count the fibroblasts in the logarithmic growth phase, and seed 3 × 10⁶ cells per well. 5 After 24 hours of cell adhesion, the confluence reached 100%. Using a 200µl pipette tip, the cells were scratched vertically along the bottom line of the plate. After scratching, the cells were rinsed three times with PBS. In the experimental group, each well was replaced with 1ml of DMEM basal medium containing 1mg / mL H1-AAVS1-COL17A1 and H1-KRT5-COL17A1 proteins prepared in Example 4. In the negative control group, each well was replaced with DMEM basal medium without COL17A1 protein. Cells were cultured at 37°C, and the scratched areas were observed and photographed at 0h and 24h. Results are as follows: Figure 8 As shown, fibroblasts cultured with H1-KRT5-COL17A1 protein showed a significant reduction in the area of ​​the central scratch after 24 hours, and their wound healing rate was greater than that of fibroblasts in the DMEM group and the H1-AAVS1-COL17A1 group. This demonstrates that the H1-KRT5-COL17A1 protein prepared in this invention significantly promotes fibroblast migration. 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 or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for constructing recombinant human embryonic stem cells that highly express human COL17A1 protein, characterized in that, The method includes integrating a nucleic acid molecule encoding the COL17A1 protein into the KRT5 site of the genomic DNA of human embryonic stem cells to obtain recombinant human embryonic stem cells; The nucleic acid molecule encoding the COL17A1 protein contains a gene encoding the COL17A1 protein.

2. The method according to claim 1, characterized in that, This includes introducing a homologous recombinant plasmid expressing COL17A1, a plasmid expressing sgRNA, and a plasmid expressing Cas9 or Cas12 into human embryonic stem cells. The sgRNA is a specific sgRNA targeting the KRT5 site, and the sequence of the sgRNA is shown in SEQ ID NO.

3.

3. The method according to claim 2, characterized in that, The homologous recombination plasmid includes a nucleic acid molecule encoding the COL17A1 protein, and has an upstream homologous arm upstream of the nucleic acid molecule encoding the COL17A1 protein and a downstream homologous arm downstream of the nucleic acid molecule encoding the COL17A1 protein; the upstream and downstream homologous arms are used to integrate the nucleic acid molecule encoding the COL17A1 protein into the KRT5 site of the genomic DNA of human embryonic stem cells; The nucleotide sequence of the upstream homologous arm is shown in SEQ ID NO.4, and the nucleotide sequence of the downstream homologous arm is shown in SEQ ID NO.

5.

4. The method according to claim 2, characterized in that, The plasmid expressing sgRNA and the plasmid expressing Cas9 or Cas12 are the same plasmid.

5. The method according to any one of claims 1 to 4, characterized in that, The nucleic acid molecule encoding the COL17A1 protein also contains a selection marker gene; Preferably, the screening marker gene includes a puromycin resistance gene; Preferably, the nucleotide sequence of the COL17A1 protein is shown in SEQ ID NO.1; Preferably, the gene encoding the COL17A1 protein is shown in SEQ ID NO.

2.

6. A kit for preparing recombinant human embryonic stem cells with high expression of human COL17A1 protein, characterized in that, The kit includes the homologous recombinant plasmid expressing COL17A1 as described in any one of claims 2 to 4 and the plasmid expressing sgRNA.

7. The reagent kit according to claim 6, characterized in that, The kit also includes plasmids expressing Cas9 or Cas12; Preferably, the plasmid expressing sgRNA and the plasmid expressing Cas9 or Cas12 are the same plasmid.

8. The use of recombinant human embryonic stem cells constructed by the method of any one of claims 1 to 5, or recombinant human embryonic stem cells prepared using the kit of claim 6 or 7, in the preparation of skin organoids.

9. A skin organoid highly expressing human COL17A1 protein, characterized in that, The genetically engineered human embryonic stem cells constructed by the method described in any one of claims 1 to 5, or genetically engineered human embryonic stem cells constructed using the kit described in claim 6 or 7, are obtained by differentiation.

10. The use of the skin organoid of claim 9 in the production of human COL17A1 protein.

Images