Deafness gene mutation cell line, construction method and detection quality control product

Abstract

The invention provides a deafness gene mutation cell line, a construction method and a detection quality control product, and belongs to the technical field of gene detection. The invention provides an sgRNA group for constructing a deafness gene mutation cell line, the sgRNA group comprises an sgRNA group designed for mutation sites of deafness genes GJB2, GJB3 and SLC26A4, and 35delG, 176-191del16, 235delC and 299-300delAT which carry the deafness genes GJB2 are obtained through gene editing; 538Cgt in GJB3; t and 547 Ggt; a; in SLC26A4, IVS7-2Agt is added; and extracting genome DNA of the editing cells, and mixing the genome DNA with wild type genome DNA to obtain the deafness gene mutation detection quality control product. The deafness gene mutation detection quality control product provided by the invention can simulate the deafness gene mutation frequency of a real sample, and can more accurately simulate real mutation occurring in vivo.

Description

Technical Field

[0001] This invention belongs to the field of gene detection technology, specifically relating to a deafness gene mutation cell line, its construction method, and detection quality control products. Background Technology

[0002] The proportion of people carrying hereditary deafness genes in the general population is approximately 6%, and it has been determined that about 60% of congenital deafness is related to genetic factors, with most pathogenic genes detectable through screening. Meanwhile, 50% of hearing-impaired individuals experience late-onset deafness, which cannot be detected by traditional hearing screenings. This permanent hearing loss is caused by environmental factors, and this tragedy can be completely avoided through deafness gene screening. Hearing impairment in children is often discovered after the age of two, resulting in irreparable damage to hearing and speech functions. Early detection and medical intervention can prevent deafness from leading to muteness, allowing children to participate in normal social life. Hereditary deafness accounts for up to 50% of birth hearing defects. Statistics show that the incidence of deafness in newborns is 1‰, with about half of these cases related to genetic factors. The main genes influencing this are GJB2, GJB3, and SLC26A4. Therefore, mutation testing for common deafness genes such as GJB2, SLC26A4, and MT-RNR1 can determine whether parents are recessive carriers. If both parents carry the same recessive disease-causing gene, their offspring have a 25% chance of inheriting the condition. Early screening can help families mitigate risks and reduce the incidence of hereditary deafness through prenatal diagnosis and assisted reproductive technologies.

[0003] Currently, there are many deafness gene mutation detection kits on the market, but they all lack effective quality control products or standards to control the quality of the detection methods and processes. Summary of the Invention

[0004] This invention provides a deafness gene mutation cell line, its construction method, and a detection quality control product, which simulates the frequency of deafness gene mutations in real samples and can more accurately simulate real mutations occurring in vivo.

[0005] This invention provides an sgRNA group for constructing deafness gene mutant cell lines, including an sgRNA group designed for mutation sites of deafness genes GJB2, GJB3 and SLC26A4; The mutation sites of the GJB2 mentioned therein include at least one of the following: 35delG, 176-191del16, 235delC and 299-300delAT; The mutation sites of GJB3 include at least one of the following: 538C>T and 547G>A; The mutation sites of SLC26A4 include at least one of the following: 281C>T, IVS7-2A>G, and 2168A>G.

[0006] In one specific embodiment of the present invention, the mutation sites of GJB2 are: 35delG, 176-191del16, 235delC and 299-300delAT; The mutation sites of GJB3 are: 538C>T and 547G>A; The mutation site of SLC26A4 is IVS7-2A>G.

[0007] In one specific embodiment of the present invention, the nucleotide sequences of the targeting sequences containing the mutation sites 35delG, 176-191del16, 235delC and 299-300delAT of GJB2 are shown in SEQ ID No. 1 to SEQ ID No. 4, respectively. The nucleotide sequences of the targeting sequences containing the GJB3 mutation sites 538C>T and 547G>A are shown in SEQ ID No. 5 to SEQ ID No. 6, respectively. The nucleotide sequence of the targeting sequence containing the mutation site IVS7-2A>G of SLC26A4 is shown in SEQ ID No. 7.

[0008] In one specific embodiment of the present invention, the nucleotide sequence of the sgRNA designed for the mutation site of GJB2 is shown in SEQ ID No. 8 to SEQ ID No. 15 in sequence; The nucleotide sequences of the sgRNA designed targeting the mutation site of GJB3 are shown in SEQ ID No. 16~SEQ ID No. 19. The nucleotide sequences of the sgRNA designed targeting the mutation site of SLC26A4 are shown in SEQ ID No. 20~SEQ ID No. 21.

[0009] The present invention also provides a method for constructing a deafness gene mutant cell line, comprising refolding the above-mentioned sgRNA group and ligating it into a gene editing vector, transfecting cells respectively, and constructing a deafness gene mutant cell line.

[0010] The present invention also provides a deafness gene mutant cell line constructed using the above-described construction method.

[0011] The present invention also provides a quality control product for detecting deafness gene mutations, comprising genomic DNA of the aforementioned deafness gene mutation cell line.

[0012] In one specific embodiment of the present invention, the deafness gene mutation detection quality control product further includes genomic DNA of wild-type cells, and the gene mutation frequency of each mutation site of each gene in each deafness gene mutation detection quality control product is 5%.

[0013] In one specific embodiment of the present invention, it includes a nucleic acid quality control product positive for GJB2 mutation of the deafness gene, a nucleic acid quality control product positive for GJB3 mutation of the deafness gene, and a nucleic acid quality control product positive for SLC26A4 mutation of the deafness gene. The deafness gene GJB2 mutation-positive nucleic acid quality control product contains GJB2 mutation sites 35delG, 176-191del16, 235delC and 299-300delAT; The GJB3 mutation-positive nucleic acid quality control sample for the deafness gene contains the GJB3 mutation sites 538C>T and 547G>A. The SLC26A4 mutation-positive nucleic acid quality control product for the deafness gene contains the SLC26A4 mutation site IVS7-2A>G.

[0014] This invention also provides the application of the above-mentioned quality control material for detecting deafness gene mutations in the preparation of a kit for detecting deafness gene mutations.

[0015] Beneficial Effects: This invention provides an sgRNA genome for constructing deafness gene mutation cell lines, comprising sgRNA genomes designed targeting mutation sites in the deafness genes GJB2, GJB3, and SLC26A4. Through gene editing, cell lines carrying mutation sites in the deafness genes GJB2 (35delG, 176-191del16, 235delC, and 299-300delAT), GJB3 (538C>T and 547G>A), and SLC26A4 (IVS7-2A>G) are obtained. Genomic DNA from the edited cells is then extracted and mixed with wild-type genomic DNA to obtain a quality control product for deafness gene mutation detection. The deafness gene mutation detection quality control product of this invention can simulate the frequency of deafness gene mutations in real samples, and can more accurately simulate real mutations occurring in vivo. Attached Figure Description

[0016] Figure 1 This is a sequencing result of GA0183-1 at the mutation site; Figure 2 This is a diagram showing the sequencing results of GA0183-2 at the mutation site; Figure 3 This is a graph showing the sequencing results of GA0183-3 at the mutation site; Figure 4 This is a sequencing result diagram of GA0183-4 at the mutation site; Figure 5 This is a sequencing result of GA0186-1 at the mutation site; Figure 6 This is a graph showing the sequencing results of GA0186-2 at the mutation site; Figure 7This is a graph showing the sequencing results of GA0189-1 at the mutation site; Figure 8 1D plot of digital PCR detection results for GJB2-35delG, GJB2-176_191del16, GJB2-235delC and GJB2-299_300delAT with an expected mutation frequency of 5%; Figure 9 2D plot of digital PCR detection results for GJB2-35del with an expected mutation frequency of 5%; Figure 10 2D plot of digital PCR detection results for GJB2-176_191del16 with an expected mutation frequency of 5%; Figure 11 2D image of digital PCR detection results for GJB2-235delC with an expected mutation frequency of 5%; Figure 12 2D image of digital PCR detection results for GA0183-4 GJB2-299_300delAT with an expected mutation frequency of 5%; Figure 13 1D plot of digital PCR detection results for GJB3-538C>T with an expected mutation frequency of 5%; Figure 14 2D plot of digital PCR detection results for GJB3-538C>T with an expected mutation frequency of 5%; Figure 15 1D plot of digital PCR detection results for GJB3-547G>A with an expected mutation frequency of 5%; Figure 16 2D plot of digital PCR detection results for GJB3-547G>A with an expected mutation frequency of 5%; Figure 17 1D plot of digital PCR detection results for SLC26A4-IVS7-2A>G with an expected mutation frequency of 5%; Figure 18 2D plot of digital PCR detection results for SLC26A4-IVS7-2A>G with an expected mutation frequency of 5%. Detailed Implementation

[0017] This invention provides an sgRNA group for constructing deafness gene mutant cell lines, including an sgRNA group designed for mutation sites of deafness genes GJB2, GJB3 and SLC26A4; The mutation sites of the GJB2 mentioned therein include at least one of the following: 35delG, 176-191del16, 235delC and 299-300delAT; The mutation sites of GJB3 include at least one of the following: 538C>T and 547G>A; The mutation sites of SLC26A4 include at least one of the following: 281C>T, IVS7-2A>G, and 2168A>G.

[0018] In this invention, the mutation sites of GJB2 are: 35delG, 176-191del16, 235delC, and 299-300delAT; the mutation sites of GJB3 are: 538C>T and 547G>A; and the mutation site of SLC26A4 is IVS7-2A>G. Based on the above mutation sites, this invention constructs a total of 7 targeting sequences, each approximately 500 bp upstream and downstream of the gene mutation site, totaling approximately 1000 bp, and ligates them into a cloning vector, in one embodiment of which is pUC57.

[0019] The nucleotide sequence of the targeting sequence containing the GJB2 mutation site 176_191del16 is shown in SEQ ID No. 2: Atgacactccccagcacagcaaatttttatgatgtgtttaaagattgggtgaattactcaggtgaacaagctactttttatcagagaacacctaaaaacacgttcaagagggtttgggaactatacatttaatcctatgacaaactaagttggttctgtcttcacctgttttggtgaggttgtgtaagagttggtgtttgctcaggaagagatttaagcatgcttgcttacccagactcagagaagtctccctgttctgtcctagctagtgattcctgtgttgtgtgcattcgtcttttccagagcaaaccgcccagagtagaagatggattggggcacgctgcagacgatcctggggggtgtgaacaaacactccaccagcattggaaagatctggctcaccgtcctcttcatttttcgcattatgatcctcgttgtggctgcaaaggaggtgtggggagatgagcaggccgactttgtctgcaacaccctgcagccagctacgatcactacttccccatctcccacatccggctatgggccctgcagctgatcttcgtgtccacgccagcgctcctagtggccatgcacgtggcctaccggagacatgagaagaagaggaagttcatcaagggggagataaagagtgaatttaaggacatcgaggagatcaaaacccagaaggtccgcatcgaaggctccctgtggtggacctacacaagcagcatcttcttccgggtcatcttcgaagccgccttcatgtacgtcttctatgtcatgtacgacggcttctccatgcagcggctggtgaagtgcaacgcctggccttgtcccaacactgtggactgctttgtgtcccggcccacggagaagactgtcttcacagtgttcatgattgcagtgtctggaatttgcatcctgctgaatgtcactgaattgtgttatttgctaattagatattgttctgggaagtcaaaaaagccagtttaacgcattgccc; The nucleotide sequence of the targeting sequence containing the GJB2 mutation site 235delC is shown in SEQ ID No. 3: Aggtgaacaagctactttttatcagagaacacctaaaaacacgttcaagagggtttgggaactatacatttaatcctatgacaaactaagttggttctgtcttcacctgttttggtgaggttgtgtaagagttggtgtttgctcaggaagagatttaagcatgcttgcttacccagactcagagaagtctccctgttctgtcctagctagtgattcctgtgttgtgtgcattcgtcttttccagagcaaaccgcccagagtagaagatggattggggcacgctgcagacgatcctggggggtgtgaacaaacactccaccagcattggaaagatctggctcaccgtcctcttcatttttcgcattatgatcctcgttgtggctgcaaaggaggtgtggggagatgagcaggccgactttgtctgcaacaccctgcagccaggctgcaagaacgtgtgctacgatcactacttccccatctcccacatccggctatgggcctgcagctgatcttcgtgtccacgccagcgctcctagtggccatgcacgtggcctaccggagacatgagaagaagaggaagttcatcaagggggagataaagagtgaatttaaggacatcgaggagatcaaaacccagaaggtccgcatcgaaggctccctgtggtggacctacacaagcagcatcttcttccgggtcatcttcgaagccgccttcatgtacgtcttctatgtcatgtacgacggcttctccatgcagcggctggtgaagtgcaacgcctggccttgtcccaacactgtggactgctttgtgtcccggcccacggagaagactgtcttcacagtgttcatgattgcagtgtctggaatttgcatcctgctgaatgtcactgaattgtgttatttgctaattagatattgttctgggaagtcaaaaaagccagtttaacgcattgcccagttgttagattaagaaatagacagcatgagagggatgaggcaa; The nucleotide sequence of the GJB2 mutation site 299_300delAT targeting sequence is shown in SEQ ID No. 4: Tacatttaatcctatgacaaactaagttggttctgtcttcacctgttttggtgaggttgtgtaagagttggtgtttgctcaggaagagatttaagcatgcttgcttacccagactcagagaagtctccctgttctgtcctagctagtgattcctgtgttgtgtgcattcgtcttttccagagcaaaccgcccagagtagaagatggattggggcacgctgcagacgatcctggggggtgtgaacaaacactccaccagcattggaaagatctggctcaccgtcctcttcatttttcgcattatgatcctcgttgtggctgcaaaggaggtgtggggagatgagcaggccgactttgtctgcaacaccctgcagccaggctgcaagaacgtgtgctacgatcactacttccccatctcccacatccggctatgggccctgcagctgatcttcgtgtccacgccagcgctcctagtggccatgcacgtggcctaccggagacgagaagaagaggaagttcatcaagggggagataaagagtgaatttaaggacatcgaggagatcaaaacccagaaggtccgcatcgaaggctccctgtggtggacctacacaagcagcatcttcttccgggtcatcttcgaagccgccttcatgtacgtcttctatgtcatgtacgacggcttctccatgcagcggctggtgaagtgcaacgcctggccttgtcccaacactgtggactgctttgtgtcccggcccacggagaagactgtcttcacagtgttcatgattgcagtgtctggaatttgcatcctgctgaatgtcactgaattgtgttatttgctaattagatattgttctgggaagtcaaaaaagccagtttaacgcattgcccagttgttagattaagaaatagacagcatgagagggatgaggcaacccgtgctcagctgtcaaggctcagtcgctagcatttcccaacacaaagattctgaccttaaatg; The nucleotide sequence containing the 538C>T targeting sequence of the GJB3 mutation site is shown in SEQ ID No. 5: The nucleotide sequence containing the 547G>A targeting sequence of the GJB3 mutation site is shown in SEQ ID No. 6: The nucleotide sequence containing the IVS7-2A>G targeting sequence at the SLC26A4 mutation site is shown in SEQ ID No. 7:

[0020] Based on the above-mentioned targeting sequence, this invention identifies the PAM sequence near the mutation site, designs an sg sequence near the PAM sequence, and performs off-target analysis on the sg sequence using the website https: / / crispr.cos.uni-heidelberg.de / to screen for sg primers with low off-target rates. A "CACCGG" BbsI restriction enzyme complementary sequence is added to the 5' end of the sg primer as the forward sg primer, and "AAAC" is added to the 5' end and "CC" BbsI restriction enzyme complementary sequence is added to the 3' end of the reverse sg primer.

[0021] Table 1 sgRNA sequences in embodiments of the present invention

[0022] The present invention also provides a method for constructing a deafness gene mutant cell line, comprising refolding the above-mentioned sgRNA group and ligating it into a gene editing vector, transfecting cells respectively, and constructing a deafness gene mutant cell line.

[0023] This invention commissioned a biotechnology company to synthesize the forward and reverse sg primers described in Table 1, achieving HPLC purification. During renaturation of sgRNA, each forward and reverse sg primer was diluted with water to 10 μM, and a renaturation system was prepared by shaking and centrifugation. The renaturation system, in 20 μL increments, comprised: 5 μL of forward sg primer, 5 μL of reverse sg primer, 4 μL of Pfubuffer (full-Gold), and 6 μL of ddH2O. The renaturation system after shaking and centrifugation was renatured at 98°C for 3 min, allowed to cool naturally for 3–5 min, and then allowed to reach room temperature.

[0024] This invention connects renatured sgRNA to a gene editing vector. In one embodiment, the gene editing vector is a px458 vector. The ligation is performed by connecting the linearized px458 plasmid obtained after Bbs I digestion to the renatured sgRNA to construct a gene-edited recombinant plasmid, which is then transfected into cells.

[0025] This invention uses HEK-293T cells as host cells. After the HEK-293T cells are completely confined in the culture flask, they are digested with trypsin. The digested cells are then resuspended in DMEM complete medium containing 10% FBS and transfected. During the transfection, CAS9 protein and the recombinant plasmid are mixed and transfected, and transfected positive cells are screened.

[0026] The present invention also provides a deafness gene mutant cell line constructed using the above-described construction method.

[0027] In this embodiment of the invention, a total of 7 cell lines were constructed. The mutation sites of the cell lines and the corresponding naming relationships of the cell lines are shown in Table 2.

[0028] Table 2. Mutation sites and nomenclature of cell lines

[0029] The present invention also provides a quality control product for detecting deafness gene mutations, comprising genomic DNA of the aforementioned deafness gene mutation cell line.

[0030] The deafness gene mutation detection quality control product of the present invention contains the genomic DNA of the cell line and the genomic DNA of wild-type cells, and the gene mutation frequency of each mutation site of each gene in each deafness gene mutation detection quality control product is 5%.

[0031] In this invention, the deafness gene mutation detection quality control products can be classified according to the gene, including deafness gene GJB2 mutation positive nucleic acid quality control products, deafness gene GJB3 mutation positive nucleic acid quality control products and deafness gene SLC26A4 mutation positive nucleic acid quality control products; The deafness gene GJB2 mutation-positive nucleic acid quality control product contains GJB2 mutation sites 35delG, 176-191del16, 235delC and 299-300delAT; The GJB3 mutation-positive nucleic acid quality control sample for the deafness gene contains the GJB3 mutation sites 538C>T and 547G>A. The SLC26A4 mutation-positive nucleic acid quality control product for the deafness gene contains the SLC26A4 mutation site IVS7-2A>G.

[0032] This invention also provides the application of the above-mentioned quality control material for detecting deafness gene mutations in the preparation of a kit for detecting deafness gene mutations.

[0033] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, provides a deafness gene mutation cell line, its construction method, and its detection quality control products, but these should not be construed as limiting the scope of protection of the present invention.

[0034] Example 1 1. Design Oligo DNA sequences (sg primers and sg sequencing primers) 1.1 sg sequencing primers This primer is a universal sequencing primer for the px458 plasmid, used to determine whether the subsequent sg sequence is effectively ligated to the px458 restriction plasmid. The nucleotide sequence is shown in SEQ ID No. 22: ATTTCTTGGGTAGTTTGCAG.

[0035] 1.2 sg primers The cDNA and gDNA sequences of the GJB2, GJB3, and SLC26A4 genes were searched on the NCBI website. Based on the mutation information of the GJB2 gene (35delG, 176-191del16, 235delC, 299-300delAT), the GJB3 gene (538C>T, 547G>A), and the SLC26A4 gene (IVS7-2A>G), primers as shown in Table 1 were designed.

[0036] 1.3 Target Sequence Selection and Synthesis (1) GJB2 gene targeting sequence The sequences containing approximately 500 bp upstream and downstream of the mutation sites 35delG, 176-191del16, 235delC, and 299-300delAT on the GJB2 gDNA sequence, totaling about 1000 bp, were used as the target sequences, as shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, and SEQ ID No. 4, respectively.

[0037] (2) GJB3 gene targeting sequence The sequences containing approximately 500 bp upstream and downstream of the 538C>T and 547G>A mutation sites in the GJB3 gene, totaling about 1000 bp, were used as the target sequences, as shown in SEQ ID No. 5 and SEQ ID No. 6, respectively.

[0038] (3) SLC26A4-IVS7-2A>G The sequence containing approximately 500 bp upstream and downstream of the 919-2A>G mutation site in the SLC26A4 gene, totaling about 1000 bp, was used as the target sequence, as shown in SEQ ID No. 7.

[0039] 1.4 Synthesis of primers and targeting sequences The designed sequence was synthesized, and the SG sequencing primers, forward SG primers, and reverse SG primers were purified by HPLC. The targeting sequence was constructed into the PUC57 vector with Kana resistance and a concentration >1 μg / μL.

[0040] 2. SG primer annealing 2.1 Dilute the sg sequencing primers, forward sg primers, and reverse sg primers with water to 10 μM.

[0041] 2.2 Prepare the renaturation system (20 μL): 5 μL of forward SG primer, 5 μL of reverse SG primer, 4 μL of Pfu buffer (full gold) and 6 μL of ddH2O, and centrifuge with shaking.

[0042] 2.3 PCR reaction: 98℃ for 3 min, after which the PCR reaction was allowed to cool naturally for 3-5 min, and then allowed to return to room temperature.

[0043] 3. Target sequence digestion and recovery 3.1 Prepare the enzyme digestion system for the target sequence (200 μL): CutSmart 20 μL, target sequence 20 μg, MluⅠ 5 μL and the remainder ddH2O.

[0044] 3.2 Water bath at 37℃ for 2~3 hours.

[0045] 3.3 Add 200 μL of DNA extraction buffer (containing chloroform) to the enzyme digestion reaction system, vortex to mix for 15 s, centrifuge at 13000 rpm for 2 min, transfer the supernatant to a new centrifuge tube, add 0.6 times the volume of isopropanol to precipitate the DNA, flick to mix, centrifuge at 13000 rpm for 10 min at 4 °C, and discard the supernatant.

[0046] 3.4 Add 1 mL of 75% ethanol to the centrifuge tube, gently invert to mix once, centrifuge at 13000 rpm for 10 min at 4℃, and discard the supernatant.

[0047] 3.5 Add 1 mL of anhydrous ethanol to a centrifuge tube, centrifuge at 13000 rpm for 10 min at 4 °C, discard the supernatant, being careful not to touch the white precipitate, air dry for 5-10 min until it becomes transparent, add 30 μL of purified water preheated to 65 °C, let stand for 3-5 min, mix by pipetting, and measure the concentration to be approximately 455.2 ng / μL for subsequent cell transfection.

[0048] 4.px458 plasmid digestion and recovery 4.1 Prepare the px458 plasmid digestion system (100 μL): CutSmart 10 μL, px458 4 μg, BbsⅠ 2 μL and the remainder ddH2O.

[0049] 4.2 Water bath at 37℃ for 2~3 hours.

[0050] 4.3 The enzyme digestion products were recovered using a DNA purification and recovery kit (centrifuge column) (Tiangen, DP214), eluted with 30 μL of purified water preheated to 65℃, and the concentration was determined (20 ng / μL).

[0051] 5. Ligation, transfection, and selection of positive clones 5.1 Ligation system of sg primer annealing product and px458 enzyme digestion product (10 μL): 6 μL (approximately 120 ng) of px458 digestion product, 2 μL of 1:200 diluted sg primer renaturation product, 1 μL of 10X T4 buffer, and 1 μL of T4 ligase.

[0052] 5.2 PCR program: 16℃ for 60 min, store at 4℃.

[0053] 5.3 Remove DH5α competent cells from the -80℃ freezer, place on ice for 4 min, thaw, add 10 μL of the above ligation system, mix well, and place on ice for another 30 min.

[0054] 5.4 Heat shock at 42℃ for 90s in a water bath, then place on ice for 2.5min. Add 200μL of LB medium (antibiotic-free), and incubate at 37℃ and 220r / min for 40min on a shaker. Then centrifuge at 3000rpm for 5min, aspirate 100μL of supernatant, transfer to LB solid medium (Amp resistance, 100μg / mL), plate, and incubate upside down at 37℃ overnight.

[0055] 5.5 Pick a single clone and culture it in 1 mL of LB liquid medium (Amp resistant, 100 μg / mL) at 37 °C and 220 r / min for 4 h on a shaker. Aliquot 200 μL of the culture and send it to fresh bacterial culture for Sanger sequencing with sg sequencing primers (10 μM).

[0056] 5.6 Based on the returned sequencing results, select qualified bacterial cultures for large-scale plasmid DNA preparation, requiring a final plasmid concentration of 1 μg / μL or higher, which can be used for subsequent cell transfection.

[0057] 6. Cell transfection 6.1 Remove the frozen HEK-293T cells from the liquid nitrogen container and place them in a 37°C water bath. After the cells have completely thawed, transfer them to DMEM + 10% FBS complete medium, centrifuge at 300g for 5 min, discard the supernatant, resuspend the cells in 10 mL of DMEM + 10% FBS complete medium, and seed them in 25 cm... 2 Place the culture flasks in a 37°C, 5% CO2 incubator for incubation.

[0058] 6.2 Wait until the cells have completely covered a 25cm area. 2 After removing the culture medium from the culture flask, wash the cells with 10 mL of PBS, add 3 mL of 0.05% trypsin, and incubate at 37°C for 3 min to digest. Resuspend the digested cells in 5 mL of complete culture medium, centrifuge at 300 g for 5 min, discard the supernatant, resuspend the cells in 10 mL of DMEM + 10% FBS complete culture medium, and count them.

[0059] 6.3 Based on the cell count results, seed the cells into 6-well plates at a seeding density of 2 × 10⁶ cells / well. 5 Cells / well: Place the inoculated cells into an incubator and culture for 24 hours.

[0060] 6.4 Prepare reagent 1 according to the ratio of Opti-MEM:Lipo3000 125μL:5μL, and prepare reagent 2 according to the ratio of Opti-MEM:pX458-sgRNA plasmid:targeting sequence:P3000 125μL:1.25μg:125μg:5μL.

[0061] 6.5 Slowly add reagent 2 to reagent 1 at a volume ratio of 1:1, gently mix by pipetting, and let stand at room temperature for 10 minutes to prepare the reagent mix. Discard the culture medium in the 6-well plate, add 2 ml of fresh serum-free DMEM culture medium, then add 250 μL of the reagent mix to each well, and incubate in an incubator. After 10 hours, discard the culture medium and add DMEM + 10% FBS again.

[0062] 6.6 After 72 hours, the culture medium in the 6-well plate was discarded, the cells were digested with 0.1% trypsin and resuspended in DMEM + 10% FBS complete medium. The cells were seeded into 96-well plates using the infinite dilution method, and the culture wells with single cells were observed under a microscope and labeled.

[0063] 6.7 After the labeled wells reached confluence, the cells were transferred to 6-well plates, and genomic DNA was extracted from the cells. The DNA was then sequenced, and the single clones of successfully edited positive cells were selected as cell lines, as shown in Table 2. The sequencing results are as follows: Figures 1-7 As shown.

[0064] Example 2 1. Preparation of nucleic acid quality control material for deafness genes 1.1 Cell Culture Seven types of deafness gene mutation-positive cells and one type of unedited wild-type cells were removed from the liquid nitrogen tank and quickly placed in a 37°C water bath. After the cells were completely thawed, they were transferred to DMEM + 10% FBS complete medium, centrifuged at 300g for 5 minutes, the supernatant was discarded, and the cells were resuspended in 10mL of DMEM + 10% FBS complete medium and seeded in 25cm... 2 The culture flasks were placed in an incubator at 37°C with 5% CO2 and incubated. After 48–72 hours, the samples were observed at a depth of 25 cm. 2 The cells at the bottom of the culture flask were confluent. After digestion with 0.1% trypsin, the cells were transferred to a 182 cm⁻¹ culture flask. 2 Cells were resuspended in 80 ml of DMEM + 10% FBS complete medium in culture flasks and cultured at 37°C in a 5% CO2 incubator for 48 h. An additional 80 ml of DMEM + 10% FBS complete medium was added, and the flasks were incubated at 37°C in a 5% CO2 incubator. After 72 h, the cells had spread to a depth of 182 cm⁻¹. 2 Collect cells from the bottom of the culture flask for counting, and take 5 × 10⁶ cells. 6Cells for later use.

[0065] 1.2 Extraction of genomic DNA Use the Quick-DNA Miniprep Kit (brand: ZYMO, catalog number D3024) to sample 5 × 10⁵ DNA molecules. 6 Genomic DNA was extracted from the cells, yielding genomic DNA from eight different cell types.

[0066] 1.3 DNA quality testing and concentration calibration OD measured using Nanodrop 260 / OD 280 A ratio within the range of 1.8 to 2.0 indicates acceptable DNA purity. The integrity of DNA fragments was checked using an Agilent 4150 bioanalyzer; DNA fragments larger than 48 kb without diffuse bands indicated DNA integrity. The DNA concentration was adjusted to 30 ng / μL using the Qubit assay.

[0067] 1.4 Preparation of quality control materials The copy number concentrations of the GJB2, GJB3, and SLC26A4 genes in eight DNA samples were detected using Bio-Rad QX200 droplet digital PCR. Based on the test results, and according to the expected gene mutation frequency of 5%, GA0183-1, GA0183-2, GA0183-3, and GA0183-4 were mixed with wild-type cell genomic DNA in a specific ratio to prepare a GJB2 mutation-positive nucleic acid quality control sample. GA0186-1 and GA0186-2 were mixed with wild-type cell genomic DNA in a specific ratio to prepare a GJB3 mutation-positive nucleic acid quality control sample. GA0189-1 was mixed with wild-type cell genomic DNA in a specific ratio to prepare an SLC26A4 mutation-positive nucleic acid quality control sample.

[0068] 1.5 The mutation frequencies of the GJB2, GJB3, and SLC26A4 genes in the three prepared quality control samples were detected using Bio-Rad QX200 droplet digital PCR. The measured mutation frequencies of the quality control samples are shown in Table 3. The detection results are as follows: Figures 8-18 As shown.

[0069] Table 3 Quality Control Parameters

[0070] 2. Performance evaluation of quality control products for deafness gene testing. 2.1 Uniformity Evaluation Ten sets of quality control samples from the smallest packaging units were randomly selected. Each quality control sample from each packaging unit was measured once on the detection system. The average value of the test results for each component was calculated according to equations (1), (2), and (3). XStandard deviation (s) and coefficient of variation (CV) ,(1); (2); (3); Table 4. Evaluation of the uniformity of GJB2-35delG mutation in GJB2-35 delG quality control sample positive for deafness gene mutation.

[0071] Table 5. Evaluation of the uniformity of the GJB2-176_191del16 mutation in the GJB2 mutation-positive nucleic acid quality control material for the deafness gene.

[0072] Table 6. Evaluation of the uniformity of the GJB2-235delC mutation in the GJB2 mutation-positive nucleic acid quality control material for the deafness gene.

[0073] Table 7. Evaluation of the uniformity of mutation in GJB2-299_300delAT quality control material, which is positive for GJB2 mutations in the deafness gene.

[0074] Table 8. Evaluation of the uniformity of GJB3-538C>T mutation in GJB3-538 nucleic acid quality control material positive for deafness gene mutation.

[0075] Table 9. Evaluation of the uniformity of the GJB3-547G>A mutation in the GJB3 mutation-positive nucleic acid quality control material for deafness gene.

[0076] Table 10 Evaluation of the uniformity of SLC26A4-IVS7-2A>G mutation in the SLC26A4-IVS7-2A>G quality control sample for SLC26A4 mutation-positive nucleic acid for deafness gene.

[0077] 2.2 Stability Evaluation That is, the stability evaluation during the effective period is performed by testing the significance of the slope trend according to the formula of the t-test table, and the t-value is calculated. p,n-2 × s ( b 1 For a 95% inclusion probability, compare b 1 The absolute value of t 0.95,n-2 × s ( b1 The size is used to evaluate stability.

[0078] Regression standard error: (5); Slope annotation deviation: , (6).

[0079] Table 11. Stability evaluation of GJB2-35delG mutation-positive nucleic acid control material for deafness gene GJB2.

[0080] Table 12. Stability evaluation of GJB2-176_191del16 mutation in GJB2-176_191del16 quality control material for GJB2 mutation-positive nucleic acid for deafness gene.

[0081] Table 13. Evaluation of the stability of the GJB2-235delC mutation in the GJB2 mutation-positive nucleic acid control material for the deafness gene.

[0082] Table 14. Evaluation of the stability of GJB2-299_300delAT mutation-positive nucleic acid control material for the deafness gene GJB2.

[0083] Table 15. Stability evaluation of GJB3-538C>T mutation-positive nucleic acid control material for deafness gene GJB3.

[0084] Table 16. Stability evaluation of GJB3-547G>A mutation-positive nucleic acid control material for deafness gene GJB3.

[0085] Table 17. Stability evaluation of SLC26A4-IVS7-2A>G mutation-positive nucleic acid control material for deafness gene SLC26A4.

[0086] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An sgRNA genome for constructing a cell line with a deafness gene mutation, characterized in that, This includes sgRNA sets designed targeting mutation sites in the deafness genes GJB2, GJB3, and SLC26A4; The mutation sites of the GJB2 mentioned therein include at least one of the following: 35delG, 176-191del16, 235delC and 299-300delAT; The mutation sites of GJB3 include at least one of the following: 538C>T and 547G>A; The mutation sites of SLC26A4 include at least one of the following: 281C>T, IVS7-2A>G, and 2168A>G.

2. The sgRNA group according to claim 1, characterized in that, The mutation sites of GJB2 are: 35delG, 176-191del16, 235delC and 299-300delAT; The mutation sites of GJB3 are: 538C>T and 547G>A; The mutation site of SLC26A4 is IVS7-2A>G.

3. The sgRNA group according to claim 2, characterized in that, The nucleotide sequences of the targeting sequences containing the GJB2 mutation sites 35delG, 176-191del16, 235delC and 299-300delAT are shown in SEQ ID No. 1 to SEQ ID No. 4, respectively. The nucleotide sequences of the targeting sequences containing the GJB3 mutation sites 538C>T and 547G>A are shown in SEQ ID No. 5 to SEQ ID No. 6, respectively. The nucleotide sequence of the targeting sequence containing the mutation site IVS7-2A>G of SLC26A4 is shown in SEQ ID No.

7.

4. The sgRNA group according to claim 3, characterized in that, The nucleotide sequences of the sgRNA designed targeting the mutation site of GJB2 are shown in SEQ ID No. 8 to SEQ ID No. 15, respectively. The nucleotide sequences of the sgRNA designed targeting the mutation site of GJB3 are shown in SEQ ID No. 16 to SEQ ID No. 19, respectively. The nucleotide sequences of the sgRNA designed targeting the mutation site of SLC26A4 are shown in SEQ ID No. 20~SEQ ID No.

21.

5. A method for constructing a cell line with a deafness gene mutation, characterized in that, This includes refolding the sgRNA group described in any one of claims 1 to 4 and ligating it into a gene editing vector, then transfecting cells to construct a deafness gene mutant cell line.

6. The deafness gene mutant cell line constructed using the construction method described in claim 5.

7. A quality control product for detecting deafness gene mutations comprising genomic DNA of the deafness gene mutation cell line of claim 6.

8. The quality control product for detecting deafness gene mutations according to claim 7, characterized in that, The deafness gene mutation detection quality control products also include wild-type cell genomic DNA, and the gene mutation frequency of each mutation site of each gene in each deafness gene mutation detection quality control product is 5%.

9. The quality control product for detecting deafness gene mutations according to claim 8, characterized in that, Including nucleic acid quality control products positive for GJB2 mutation in the deafness gene, nucleic acid quality control products positive for GJB3 mutation in the deafness gene, and nucleic acid quality control products positive for SLC26A4 mutation in the deafness gene; The deafness gene GJB2 mutation-positive nucleic acid quality control product contains GJB2 mutation sites 35delG, 176-191del16, 235delC and 299-300delAT; The GJB3 mutation-positive nucleic acid quality control sample for the deafness gene contains the GJB3 mutation sites 538C>T and 547G>A. The SLC26A4 mutation-positive nucleic acid quality control product for the deafness gene contains the SLC26A4 mutation site IVS7-2A>G.

10. The use of the deafness gene mutation detection quality control material according to any one of claims 7 to 9 in the preparation of a kit for detecting deafness gene mutations.

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