Human skin cell system containing fluorescent reporter for evaluating skin irritation

Abstract

The present invention relates to a human skin cell system containing a fluorescent reporter for evaluating skin irritation, wherein a CRISPR-Cas9 system was used to establish a human skin cell line in which a fluorescent marker is permanently endogenously expressed in a 3'-terminal region of the G3BP1 gene, which is a representative protein that undergoes the phase separation phenomenon, and phase separation, specifically the formation of stress granules, can be confirmed in real time after the human skin cells are treated with a test material for evaluating skin irritation, and thus the present invention can be effectively used for evaluating skin toxicity due to exposure to various harmful factors (medicines, chemicals, environmental harmful factors, and biological harmful factors).

Description

Human skin cell system with fluorescent reporter for skin irritation assessment

[0001] The present invention relates to a human skin cell system incorporating a fluorescent reporter for skin irritation evaluation that detects phase separation phenomena in real time.

[0002] This research was supported by the 'Development of a real-time lung toxicity assessment method for mixtures containing household chemical products using human lung organoids with fluorescent labels' project (Project No.: 1485019341, RS-2022-KE002021), a project of the Korea Environmental Industry & Technology Institute under (or affiliated with) the Ministry of Environment's household chemical product safety management technology development project.

[0003] Cosmetic safety assessments and skin irritation tests following exposure to hazardous chemicals have primarily used animal models; however, as regulations on the use of laboratory animals are strengthened, the development of alternative models to laboratory animals is actively underway.

[0004] Alternative animal testing (AAT) is a technology that replaces laboratory animals to evaluate the efficacy and toxicity of chemicals. It is considered a promising approach to overcome the inconsistencies between animal testing and clinical trials due to species differences. Furthermore, the technology holds significant future potential, with potential applications in regenerative therapeutics, personalized medicine, and disease modeling.

[0005] As interest in laboratory animal welfare grows, particularly in advanced economies, a growing movement is underway within related industries to discourage the use of laboratory animals. Beginning with the UK, at least 23 countries around the world have enacted laboratory animal protection laws. Europe has banned the sale of cosmetics tested on animals within the EU since 2013. In the US, state governments like New York and California are regulating the sale of cosmetics tested on animals.

[0006] Although human skin tissue models have recently become commercially available, the high cost of human skin tissue models makes it difficult to utilize them due to the high cost burden in maintaining various experimental conditions (concentration, number of repetitions, etc.) of basic experiments. In addition, after exposure to harmful factors, fixation is performed for tissue analysis and then staining is performed to observe cell death, etc., making it difficult to observe reversible responses and cell stimulation phenomena in response to repeated exposure.

[0007] Existing in vitro toxicity assessment methods include measuring cell viability and ROS following chemical exposure. However, no method has been reported for real-time monitoring of cytotoxicity following short-term exposure. Therefore, there is a need to identify new skin irritation markers following chemical exposure and develop new technologies to monitor them.

[0008] Meanwhile, Korean Patent No. 1455244 discloses a quantitative evaluation system and method for cytotoxicity using an AFM and a probe-type sensor, and Korean Patent No. 2466092 discloses a human liver chimeric non-human animal having a defective P450 oxidoreductase and a method for using the same. However, there is no disclosure at all regarding a human skin cell system with a fluorescent reporter for skin irritation evaluation that detects the phase separation phenomenon of the present invention in real time.

[0009] The present invention was derived from the above-mentioned needs, and provides a human skin cell system in which a fluorescent marker is introduced at the 3'-end (just before the stop codon) of the G3BP1 gene using the CRISPR-Cas9 system for skin irritation evaluation, and completes the present invention by confirming the phase separation phenomenon after treating the human skin cells with a test substance for skin irritation evaluation (a test substance causing toxicity).

[0010] To solve the above problem, the present invention provides a human skin cell line into which a fluorescent reporter for skin irritation evaluation is introduced, which detects a phase separation phenomenon in real time.

[0011] In addition, the present invention provides a composition for evaluating skin irritation comprising the human skin cell line.

[0012] In addition, the present invention provides a composition for screening skin irritant-inducing substances comprising the human skin cell line.

[0013] In addition, the present invention provides a method for producing a human skin cell line having a fluorescent reporter for skin irritation evaluation, comprising the step of introducing a fluorescent label into the 3'-end of a G3BP1 gene using a CRISPR-Cas9 system.

[0014] In addition, the present invention

[0015] 1) A step of treating the human skin cell line with a test substance for skin irritation evaluation; and

[0016] 2) A method for screening a skin irritant substance is provided, including a step of confirming a real-time phase separation phenomenon after the above step 1).

[0017] The present invention relates to a human skin cell system into which a fluorescent reporter for skin irritation evaluation has been introduced, and a human skin cell line that permanently and endogenously expresses a fluorescent marker at the 3'-terminal portion of the G3BP1 gene, which is one of the representative proteins constituting the phase separation phenomenon, was established using the CRISPR-Cas9 system, and after treating the human skin cells with a test substance for skin irritation evaluation, the phase separation phenomenon, specifically the formation of stress granules, can be confirmed in real time, and thus can be usefully utilized as a skin toxicity evaluation technology according to exposure to various harmful factors (medicines, chemicals, environmental harmful factors, and biological harmful factors).

[0018] Figure 1 is a schematic diagram of a CRISPR / Cas9-based gene insertion (knock-in) system for introducing a fluorescent marker into a phase separation marker gene (G3BP1).

[0019] Figure 2 shows the results of confirming the expression of fluorescent protein (EGFP) and G3BP1 protein in a human skin cell line (#10) into which a specific fluorescent marker for the phase separation marker gene (G3BP1) of the present invention has been introduced. Con. is a HaCat wild-type cell group.

[0020] Figure 3 shows the results of PCR analysis to confirm whether a phase separation marker-specific fluorescent marker has been introduced into a human skin cell line (#10) into which a phase separation marker gene (G3BP1)-specific fluorescent marker of the present invention has been introduced. M represents a size marker, and Con. represents a HaCat wild-type cell group.

[0021] Figure 4 shows the results of analyzing the expression of the phase separation marker gene (G3BP1) and the expression of the fluorescent marker according to NaAsO2 (Ars.) treatment in a human skin cell line (#10) into which the phase separation marker gene (G3BP1) specific fluorescent marker of the present invention has been introduced. WT is a HaCat wild-type cell group. Con. is a human skin cell line group into which the phase separation marker gene (G3BP1) specific fluorescent marker of the present invention has been introduced and which has not been treated with NaAsO2.

[0022] Figure 5 shows the results of real-time confirmation through fluorescence imaging analysis of the expression of the endogenous phase separation marker (G3BP1) and the expression of the fluorescent marker according to BPA (Bisphenol A) treatment in a human skin cell line (#10) into which the phase separation marker gene (G3BP1) specific fluorescent marker of the present invention has been introduced.

[0023] In order to achieve the purpose of the present invention, the present invention provides a human skin cell line into which a fluorescent reporter for skin irritation evaluation is introduced, which detects a phase separation phenomenon in real time.

[0024] The term 'phase separation phenomenon' of the present invention refers to a process in which macromolecules within a cell are separated into a dense phase and a relatively dilute phase by stimulation, and the phase separation phenomenon of the present invention is preferably characterized by the formation of stress granules.

[0025] In the present invention, skin irritation evaluation may be an evaluation of inflammation and irritation occurring in a living body without conducting animal testing, but is not limited thereto.

[0026] The fluorescent reporter for the skin irritation evaluation may be any protein that expresses fluorescence, and is preferably one selected from the group consisting of firefly luciferase, renilla luciferase, green fluorescent protein (GFP), modified green fluorescent protein, enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP), enhanced red fluorescent protein (ERFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), cyan fluorescent protein (CFP), enhanced cyan fluorescent protein (ECFP), and DsRed, but is not limited thereto.

[0027] In one embodiment of the present invention, the human skin cell line into which the fluorescent reporter for skin irritation evaluation is introduced may be one in which a fluorescent label is introduced into the G3BP1 gene using the CRISPR-Cas9 system, preferably one in which a fluorescent label is introduced just before the stop codon at the 3'-end of the G3BP1 gene using the CRISPR-Cas9 system, and more preferably one in which a fluorescent label is introduced into the 3'-end of the G3BP1 gene using the CRISPR-Cas9 system, such as firefly luciferase, renilla luciferase, green fluorescent protein (GFP), modified green fluorescent protein, enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP), enhanced red fluorescent protein (ERFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (EGFP), Any one fluorescent marker selected from the group consisting of, but not limited to, EBFP, yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), cyan fluorescent protein (CFP), enhanced cyan fluorescent protein (ECFP), and DsRed is introduced.

[0028] In one embodiment of the present invention, the human skin cell line into which the fluorescent reporter for skin irritation evaluation has been introduced is a human keratinocyte cell line, preferably a HaCaT cell line, but any human skin cell line that can be used for evaluating skin irritation may be used without limitation.

[0029] The 'skin irritation evaluation' of the present invention is to investigate the harm that a test substance (a pesticide, a medicine, a household chemical product, an industrial chemical, other environmental pollutants, or a biological hazard (bacteria and virus)) has on the skin, and to evaluate the degree of harm to a human skin cell line into which a fluorescent reporter for skin irritation evaluation, which detects the phase separation phenomenon of the present invention in real time, has been introduced.

[0030] In addition, the present invention provides a composition for evaluating skin irritation comprising the human skin cell line.

[0031] The composition for evaluating skin irritation above is characterized by measuring the degree of stress granule formation after treatment with a test substance using a fluorescent reporter, and the degree of stress granule formation can be confirmed using a microscope.

[0032] The above skin irritation evaluation is preferably, but not limited to, evaluating irritation caused by any one of pesticides; household chemical products or industrial chemicals; and biological hazards including bacteria or viruses.

[0033] The above household chemical product or industrial chemical is preferably, but not limited to, sodium arsenite (NaAsO2) or BPA (Bisphenol A).

[0034] In addition, the present invention provides a composition for screening skin irritant-inducing substances comprising the human skin cell line.

[0035] The above composition can be used to screen for substances that cause skin irritation by treating the test substance to determine whether it causes skin irritation and then checking the degree of stress granule formation.

[0036] The above skin irritant is preferably, but not limited to, one of pesticides; household chemical products or industrial chemicals; and biological hazards including bacteria or viruses.

[0037] In addition, the present invention provides a method for producing a human skin cell line having a fluorescent reporter for skin irritation evaluation, comprising the step of introducing a fluorescent label into the 3'-end of a G3BP1 gene using a CRISPR-Cas9 system.

[0038] The above manufacturing method preferably includes a step of transfecting skin cells with a CRISPR-Cas9-sgRNA vector including sgRNA; and a gene insertion (Knock-in) vector for introducing a fluorescent label into the G3BP1 gene; and introducing a fluorescent label into the 3'-end of the G3BP1 gene; and more preferably, it includes a step of introducing a fluorescent label into the 3'-end of the G3BP1 gene using a CRISPR-Cas9 system including sgRNA of SEQ ID NO: 1; but is not limited thereto.

[0039] In one embodiment of the present invention, a human skin cell line was established by transfecting skin cells with a CRISPR-Cas9-sgRNA vector for CRISPR-Cas9-based gene editing; and a gene insertion (Knock-in) vector for introducing a fluorescent marker into the phase separation marker (G3BP1) gene, thereby permanently endogenously expressing a fluorescent marker immediately before the 3'-terminal stop codon of the G3BP1 gene through CRISPR-Cas9-based gene insertion (Knock-in).

[0040] There is no particular limitation on the type of Cas9 vector as long as it is a vector that expresses the Cas9 protein, and the pX330 plasmid is preferred.

[0041] In order to produce a gene insertion (knock-in) vector for introducing a fluorescent label into the phase separation marker (G3BP1) gene, it is preferable to design a 5'HA (5' homology arm) of 500 to 600 bp and a 3'HA (3' homology arm) of 700 to 900 bp based on the stop codon sequence located in exon 12 of the G3BP1 gene, and to insert the EGFP gene therebetween, and more preferably, a 5'HA of 550 bp and a 3'HA of 800 bp based on the stop codon sequence located in exon 12 of the G3BP1 gene, and to insert the EGFP gene therebetween, but the present invention is not limited thereto.

[0042] In one embodiment of the present invention, a pDSRed vector may be used to produce a gene insertion (knock-in) vector for introducing a fluorescent marker into a phase separation marker (G3BP1) gene, but is not limited thereto.

[0043] In addition, the present invention

[0044] 1) A step of treating the human skin cell line with a test substance for skin irritation evaluation; and

[0045] 2) A method for screening a skin irritant substance is provided, including a step of confirming a real-time phase separation phenomenon after the above step 1).

[0046] The test substance for skin irritation evaluation in step 1) above refers to any substance that is expected to induce phase separation, specifically stress granule formation, when applied to cells.

[0047] The test substance for the above skin irritation evaluation is not limited to any substance for determining whether it is irritating to the skin, and may be, for example, a biological substance, a genetically recombinant substance, or a synthetic compound.

[0048] The step of confirming the phase separation phenomenon in the above step 2) is a step of confirming the phase separation phenomenon, specifically the formation of stress granules, by comparing it with a control group that was not treated with the test substance, and the method of confirming the phase separation phenomenon is not particularly limited as long as it is a method that can confirm the formation of stress granules with the naked eye.

[0049]

[0050] Hereinafter, the present invention will be described in more detail using examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0051]

[0052] Example 1. Production of a CRISPR / Cas9-based gene insertion vector (knock-in vector) for introducing a fluorescent marker into the phase separation marker (G3BP1) gene.

[0053] 1) Production of CRISPR / Cas9-sgRNA vector for CRISPR / Cas9-based gene editing

[0054] To construct a CRISPR / Cas9-sgRNA vector targeting G3BP1 exon 12, a DNA oligomer for sgRNA was constructed and complementarily synthesized. The sequence of sgRNA is shown in SEQ ID NO: 1 in Table 1 below.

[0055] The pX330 plasmid was used as the Cas9 vector, and this was digested with the restriction enzyme BbsI to obtain the pX330 / BbsI vector. Afterwards, the obtained annealed sgRNA was ligated to the pX330 / BbsI vector, transformed, and cloned. Afterwards, the culture was plated on LB agar medium containing ampicillin and cultured at 37°C. Colony formation was confirmed, and DNA was extracted from each colony and sequenced to confirm that the sgRNA had entered the correct position in the vector.

[0056] Sequence number sgRNA sequence 15'-CGACGAGATAATCGCCTTCG-3'

[0057]

[0058] 2) Production of a gene insertion vector for introducing a fluorescent marker into the phase separation marker (G3BP1) gene

[0059] To produce a targeting vector to induce endogenous expression by introducing a fluorescent marker (EGFP) just before the stop codon at the 3'-terminal of the G3BP1 gene, a 550-bp 5' homology arm (5'HA) and an 800-bp 3' homology arm (3'HA) were designed based on the stop codon sequence located in exon 12 of the G3BP1 gene, and the EGFP gene was inserted between them.

[0060] Specifically, a donor vector and insert were secured for the construction of a G3BP1-EGFP gene knock-in vector. The primer sequences used for the construction of the homology arm vector are as disclosed in SEQ ID NOs: 2 to 7 of Table 2 below. The promoter-less pDsRed-Express2-1 plasmid was used as the donor vector, and the DsRed-Express 2 site was removed by digestion with BglII and NotI, thereby securing the pDsRed / BglII / NotI vector.

[0061] To produce the insert, three fragments, G3BP1 5'HA with BglII restriction enzyme sequence, G3BP1 3'HA with EGFP and NotI restriction enzyme sequences, were obtained through PCR, and then ligated through overlap extension PCR to obtain the final BglII-G3BP1 5'HA_EGFP_G3BP1 3'HA-NotI insert.

[0062] Afterwards, the insert (BglII-G3BP1 5'HA_EGFP_G3BP1 3'HA-NotI) was ligated into the constructed vector (pDsRed / BglII / NotI), transformed, and cloned. Afterwards, the culture was spread on LB agar medium containing kanamycin and cultured at 37℃. After confirming the formation of colonies, DNA was extracted from each colony to obtain the G3BP1-EGFP gene insertion vector (G3BP1-EGFP knock-in vector), and sequence analysis confirmed that the sequence of the obtained vector was 100% identical to the intended sequence.

[0063] A schematic diagram of a CRISPR / Cas9-based gene insertion (Knock-in) system for introducing a fluorescent marker into the phase separation marker gene (G3BP1) of the present invention is as disclosed in Fig. 1.

[0064] SEQ ID NO: primer sequence 2BglII-G3BP1 5' HA forward5'-ATATAGATCTCAATGGCGTGATCTTGGCT-3'3G3BP1 5' HA reverse5'-CTGTCGTGGCGCAAGC-3'4G3BP1 5' HA-GFP forward5'-AAGGGGGCTTGCGCCACGACAGATGGTGAGCAAGGGCGA-3'5GFP-G3BP1 3' HA reverse5'-GCATGAAGATCCATGAAGATTTACTTGTACAGCTCGTCCATG-3'6G3BP1 3' HA forward5'-ATCTTCATGGATCTTCATGCAG-3'7G3BP1 3' HA-NotI reverse5'-TAGCGGCCGCAGGCACAACAGTTTTGCTC-3'

[0065]

[0066] Example 2. Analysis of fluorescent protein and G3BP1 protein expression in human skin cell lines with a phase separation marker (G3BP1) gene-specific fluorescent marker introduced - Western Blotting

[0067] In HaCaT cells, GFP fluorescent protein and G3BP1 protein expression were analyzed to confirm whether phase separation marker gene-specific fluorescent markers were introduced.

[0068] To introduce phase separation marker gene-specific fluorescent markers into HaCaT cells, Amaxa® Cell Line Nucleofector ® 1×10 according to the manufacturer's protocol using Kit T (Lonza, Basel, Switzerland) 6Cells were transfected with 2 μg of the knock-in construct (CRISPR / Cas9-sgRNA vector + G3BP1-EGFP gene insertion vector) per cell using Nucleofector program X-01. ® Transfection was performed using a 2b device, and after transfection, cells were transferred to 100 mm dishes and cultured in a humidified 37°C, 5% CO2 incubator until analysis.

[0069] Afterwards, proteins were extracted from human skin cell line HaCaT (hereinafter referred to as HaCaT_G3BP1-EGFP) into which a phase separation marker gene-specific fluorescent marker was introduced using M-PER™ mammalian protein extraction reagent (Thermo Scientific, USA, Cat# 78501) containing a protease inhibitor cocktail (Roche Applied Science, Basel, Switzerland), denatured and reduced with SDS and β-mercaptoethanol, and separated by protein size through SDS-polyacrylamide gel electrophoresis, and then transferred to a nitrocellulose membrane (Pall Life Science, Port Washington, NY, USA). After blocking with 5% (w / v) skim milk containing 0.05% (v / v) Tween-20 (Rockland Immunochemicals, West Grove, PA, USA) for 1 hour at room temperature, the membrane was incubated with primary antibodies (GFP, Santa Cruz Biotechnology, sc-9996; G3BP1, Santa Cruz Biotechnology, #sc-365338; GAPDH, Meridian Life Science) diluted in 5% (w / v) skim milk containing 0.05% (v / v) Tween-20 overnight at 4°C. The membrane was then washed and incubated with secondary antibodies (Horseradish peroxidase-conjugated secondary antibodies, Cell Signaling Technology) for 1 hour at room temperature (20±5°C) and then washed with TBST.

[0070] Proteins were detected using a WSE-6200H LuminoGraph II (ATTO, Tokyo, Japan) with a SuperSignal system (Thermo Fisher Scientific, Waltham, MA, USA).

[0071] As a result, as disclosed in Fig. 2, it was confirmed that the skin cell line into which the fluorescent reporter of the present invention was introduced normally expressed G3BP1-GFP without modification of the G3BP1 protein.

[0072]

[0073] Example 3. PCR to confirm the introduction of a specific fluorescent marker for the phase separation marker (G3BP1) gene into a human skin cell line.

[0074] PCR was performed to confirm the introduction of a specific fluorescent marker for the phase separation marker (G3BP1) gene into human skin cell lines.

[0075] After extracting total gDNA using the G-spin™ Total DNA Extraction Mini kit (iNtRON Biotechnology, Korea), PCR was performed using GoldHotStart Taq PCR premix (Bioneer, Daejeon, Korea) using a PCR machine (SimpliAmp thermal cycler PCR machine; Applied Biosystems, Waltham, MA, USA) with a forward primer located outside the G3BP1 5'HA and a reverse primer located within EGFP.

[0076] The specificity of each PCR product was assessed by agarose gel electrophoresis. A 942-bp band was observed only in clones introduced into the correct location within the cell.

[0077] The sequence information of the primers used in gDNA PCR is as disclosed in Table 3 below.

[0078] Sequence number primer sequence 8G3BP1-GFP forward 5'-AGAAGAAGACTCGAGCTGCC-3' 9G3BP1-GFP reverse 5'-TTCATGTGGTCGGGGTAG-3' 10GAPDH forward 5'-CCATGGAGAAGGCTGGGG-3' 11GAPDH reverse 5'-CAAAGTTGTCATGGATGACC-3'

[0079] As a result, as disclosed in Fig. 3, the skin cell line of the present invention into which the fluorescent reporter was introduced showed a band at 942 bp, confirming that GFP was introduced into the correct location within the cell.

[0080]

[0081] Example 4. Confirmation of expression of intrinsic phase separation marker (G3BP1) and fluorescent marker in established skin cell lines following NaAsO2 (Ars.) treatment

[0082] The expression of G3BP1 and fluorescent markers and stress granule formation were analyzed in the human skin cell line HaCaT (hereinafter referred to as HaCaT_G3BP1-EGFP) into which a phase separation marker gene-specific fluorescent marker was introduced.

[0083] HaCaT_G3BP1-EGFP cell culture medium (DMEM + 10% FBS + 1% P / S) was treated with 400 μM NaAsO2 for 1 hour, washed with DPBS, fixed with 4% PFA fixative for 10 minutes, and permeabilized with 0.5% Triton X-100 for 15 minutes. After washing with DPBS, cells were blocked with 5% (v / v) goat serum and 0.1% (v / v) bovine serum for 1 hour, and primary antibodies (G3BP1, Santa Cruz, #sc-365338; GFP, MBL, #598) were diluted 1:500 each and reacted overnight at 4℃, and then washed with DPBS. Secondary antibodies (Alexa Fluor 488- and 594-conjugated goat antibodies, Thermo Fisher Scientific, #A11008 and #A11005) were then diluted 1:2,000 and reacted at room temperature for 1 hour, and washed with DPBS. Nuclear counterstaining was performed by staining with DAPI diluted 1:1,000 in DPBS for 20 minutes at room temperature, and GFP and G3BP1 were observed using a Zeiss LSM 880 confocal microscope, and the presence or absence of stress granule formation in the cytoplasm was analyzed and observed.

[0084] As a result, it was confirmed that the fluorescently labeled G3BP1 was well expressed as disclosed in Fig. 4, and when NaAsO2 (Ars.) was treated, a phase separation phenomenon, specifically stress granules, were formed. Stress granules were also well formed in the HaCaT_G3BP1-EGFP cells of the present invention, and this could also be confirmed through GFP.

[0085]

[0086] Example 5. Confirmation of the expression of an endogenous phase separation marker (G3BP1) and a fluorescent marker in established skin cell lines following BPA treatment using live cell imaging.

[0087] After treating the human skin cell line constructed in the present invention with BPA, the phase separation phenomenon, specifically the formation of stress granules, was observed in real time in live cells. For real-time analysis of stress granule formation, the G3BP1-GFP gene-inserted human skin cell line (HaCat_G3BP1-EGFP) was treated with BPA. Live cell imaging was performed at 37°C in a humidified 5% CO2 atmosphere, and fluorescence was excited with 488 laser diodes and detected with a Zeiss LSM 880 confocal laser scanning microscope.

[0088] As a result, as disclosed in Fig. 5, it was confirmed that phase separation, specifically stress granule formation, occurred after BPA treatment, and it was confirmed in real time that the number of stress granules formed increased as the treatment time passed.

[0089] Therefore, the human skin cell line into which the fluorescent reporter of the present invention has been introduced can be used to evaluate skin irritant substances by detecting the phase separation phenomenon in real time.

Claims

1. A human skin cell line with a fluorescent reporter for skin irritation evaluation that detects phase separation phenomenon in real time.

2. A human skin cell line having a fluorescent reporter for skin irritation evaluation introduced, characterized in that the phase separation phenomenon in the first paragraph is stress granule formation.

3. In the first paragraph, the human skin cell line into which the fluorescent reporter for skin irritation evaluation has been introduced is characterized in that a fluorescent label is introduced into the 3'-end of the G3BP1 gene using the CRISPR-Cas9 system.

4. A human skin cell line having a fluorescent reporter for skin irritation evaluation introduced therein, wherein the fluorescent reporter for skin irritation evaluation in the first paragraph is any one selected from the group consisting of firefly luciferase, renilla luciferase, green fluorescent protein (GFP), modified green fluorescent protein, enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP), enhanced red fluorescent protein (ERFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), cyan fluorescent protein (CFP), enhanced cyan fluorescent protein (ECFP), and DsRed.

5. In the third paragraph, the human skin cell line into which the fluorescence reporter for skin irritation evaluation is introduced is prepared by using the CRISPR-Cas9 system to introduce firefly luciferase, renilla luciferase, green fluorescent protein (GFP), modified green fluorescent protein, enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP), enhanced red fluorescent protein (ERFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), cyan fluorescent protein (CFP), enhanced cyan fluorescent protein (ECFP), and A human skin cell line introduced with a fluorescent reporter for skin irritation evaluation, characterized in that any one fluorescent marker selected from the group consisting of DsRed is introduced.

6. A composition for evaluating skin irritation comprising a human skin cell line according to any one of claims 1 to 5.

7. A composition for evaluating skin irritation in accordance with claim 6, wherein the skin irritation evaluation is characterized by evaluating irritation caused by any one of pesticides; household chemical products or industrial chemicals; and biological hazards including bacteria or viruses.

8. In paragraph 7, the household chemical product or industrial chemical substance is sodium arsenite (NaAsO 2 ) or BPA (Bisphenol A) for skin irritation evaluation.

9. A composition for screening for skin irritant-causing substances, comprising a human skin cell line according to any one of claims 1 to 5.

10. A composition for screening for skin irritant-causing substances, characterized in that in clause 9, the skin irritant-causing substance is any one of pesticides; household chemical products or industrial chemicals; and biological hazards including bacteria or viruses.

11. A method for producing a human skin cell line having a fluorescent reporter for skin irritation evaluation, comprising the step of introducing a fluorescent label to the 3'-end of the G3BP1 gene using the CRISPR-Cas9 system.

12. In claim 11, the manufacturing method comprises a step of transfecting skin cells with a CRISPR-Cas9-sgRNA vector including sgRNA; and a gene insertion (Knock-in) vector for introducing a fluorescent marker into the G3BP1 gene; and introducing a fluorescent marker into the 3'-end of the G3BP1 gene; A method for manufacturing a human skin cell line into which a fluorescent reporter for skin irritation evaluation has been introduced.

13. A method for producing a human skin cell line having a fluorescent reporter for skin irritation evaluation, characterized in that the sgRNA in claim 12 consists of a base sequence of sequence number 1. 14.1) A step of treating a test substance for skin irritation evaluation to a human skin cell line according to any one of clauses 1 to 5; and 2) A method for screening a skin irritant substance, comprising: a step of confirming a real-time phase separation phenomenon after the above step 1).

15. A method for screening a skin irritant substance in claim 14, wherein the step of confirming the real-time phase separation phenomenon is characterized by confirming the degree of stress granule formation compared to a control group that was not treated with the test substance.

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