Bax2.6 cell strain and application thereof in improving AAV virus titer

Abstract

The invention relates to the technical field of gene therapy, in particular to a method for knocking out a Bax gene on the basis of an HEK293 cell line by utilizing a CRISPR / Cas9 technology, constructing a stable cell line expressed by foreign protein and improving the AAV packaging efficiency. The invention discloses a Bax2.6 cell strain. A pro-apoptosis factor Bax gene is knocked out from an HEK293T cell. Compared with a wild type HEK293T cell, the Bax2.6 cell strain provided by the invention shows higher cell activity under various stress conditions, the cell survival rate of the Bax2.6 cell strain is improved by at least 20% under the treatment conditions of small molecule inhibitors ZV-FMK, Nec-1, puromycin (Puro), cis-platinum (CDDP) and rotenone, and the Bax2.6 cell strain shows excellent environmental tolerance and production robustness. When the Bax2.6 cell strain provided by the invention is used for transient transfection and packaging of an adeno-associated virus (AAV) vector, the copy number of the obtained AAV virus can be up to 2 * 10 copies / mL, and the AAV packaging efficiency is improved by more than 10 times compared with that of a conventional HEK293T cell line.

Description

Technical Field

[0001] This invention relates to the field of gene therapy technology, specifically to the use of CRISPR / Cas9 technology to knock out the Bax gene on the basis of the HEK293 cell line, thereby constructing a stable cell line expressing exogenous proteins and improving AAV packaging efficiency. Background Technology

[0002] Adeno-associated virus (AAV), belonging to the family Parvovirus and genus Dependent Virus, is a non-enveloped, single-stranded linear DNA virus. Due to its safety and clinical efficacy in Phase I and II clinical trials, broad host cell range (infecting both dividing and non-dividing cells), low immunogenicity, and stable and long-term in vivo transgenic expression, it is widely used in in vivo experimental and clinical gene therapy research. However, achieving high packaging rates, high titers, high purity, and high quality AAV remains an unresolved challenge in clinical practice.

[0003] HEK293 cells, derived from human embryonic kidney cells, express very little of the endogenous receptors required for extracellular ligands, are easily transfected, and are a commonly used cell line for expressing and studying exogenous genes. HEK293T cells, transfected from HEK293 cells with the adenovirus E1A gene, express the SV40 large T antigen and contain the SV40 replication origin and promoter region. As one of the main chassis cell lines for AAV production, HEK293T cells require modification to obtain high AAV titers and stable expression. Summary of the Invention

[0004] This invention addresses the current low AAV packaging rate, which fails to meet clinical requirements. It introduces a mammalian cell line by knocking out the pro-apoptotic gene Bax in HEK293T cells, thereby inhibiting apoptosis, increasing cell viability, reducing apoptosis, and improving AAV packaging rate by more than 10 times, making it suitable for clinical production.

[0005] The present invention first provides a Bax2.6 cell line that improves AAV packaging efficiency, which is achieved by knocking out the pro-apoptotic factor Bax gene in HEK293T cells.

[0006] The Bax knockout cell line constructed in this invention enables stable expression of AAV virus and significantly improves packaging efficiency. Attached Figure Description

[0007] Figure 1 This is a graph showing the sequencing results of Bax- / - cell lines.

[0008] in, Figure 1A This is a graph showing the sequencing alignment results; Figure 1BThis refers to the specific sequencing results near the sgRNA.

[0009] Figure 2 To detect Bax protein expression using Western blotting.

[0010] Figure 3 To detect the mTOR and AKT pathways in Bax-deficient cells.

[0011] Figure 4 Bright-field and fluorescence images of HEK293T and Bax- / - cell lines transfected with AAV virus under a fluorescence microscope.

[0012] Figure 5 The iodixanol gradient before ultracentrifugation.

[0013] Figure 6 This is a diagram of the reaction system. Detailed Implementation

[0014] This invention relates to improving the packaging efficiency of AAV virus and comprises a mammalian cell line. The technical solution of this invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the invention and should not be construed as limiting the scope of protection of this invention. All technologies implemented based on the above content of this invention are covered within the scope of protection intended by this invention.

[0015] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. Experimental methods in the following examples that do not specify specific conditions are generally performed under standard conditions or as recommended by the manufacturer.

[0016] Example 1. Bax-sgRNA Design

[0017] Bax-sgRNA design principles: Bax-sgRNA is located before the PAM region, Bax-sgRNA is located at the front rather than the back, and off-target efficiency is reduced.

[0018] The Bax-sgRNA sequence is as follows (transcript, targeting):

[0019] Bax-sgRNA1 (F): CCTAGCTCGTCCCGCTTACC.

[0020] Bax-sgRNA2 (F): TCCTAGCTCGTCCCGCTTAC.

[0021] Bax-sgRNA3 (F): CCTGGGCCACGGAGTCCTAC.

[0022] Example 2. Obtaining Bax knockout monoclonal cell lines

[0023] Bax- / - cells were constructed by transfecting Bax-sgRNA into the HEK293T cell line, selecting cells with puromycin, expanding the culture, and then performing DNA sequencing and protein immunoprecipitation to detect Bax protein expression, thereby obtaining the Bax- / - cell line.

[0024] The three sgRNA plasmids containing the aforementioned Bax gene were transfected into the middle molecule according to the transfection instructions. The transfection process is as follows:

[0025] HEK293T cells were passaged in 10cm cell culture dishes until the cells were in good condition, so that the cell concentration reached 90-95% confluence at the time of transfection the next day.

[0026] For each transfection sample, a DAN-Lip2000 complex needs to be prepared. In a centrifuge tube, gently mix 600 μL of serum-free and antibiotic-free DMEM medium with 24 μg of DNA to prepare a DNA dilution. In another centrifuge tube, add 600 μL of serum-free and antibiotic-free DMEM medium and 60 μL of Lip2000 (mix well before use), gently mix, and incubate at room temperature for 5 min. Combine the DNA dilution and the Lip2000 dilution, gently mix, and incubate at room temperature for 30 min to form the DNA-Lip2000 complex. The DNA-Lip2000 complex is stable at room temperature for 6 hours.

[0027] Add the DNA-Lip2000 complex to a 10 cm culture dish inoculated with cells. Gently shake the culture dish back and forth to disperse and mix the complex. Incubate at 37°C in a CO2 incubator for 4-6 hours, then change the medium and add 10% FBS DMEM complete medium and continue incubation for 24-48 hours.

[0028] Forty-eight hours after transfection into HEK293T cells, 10 μg / ml puromycin was added for selection for 3 days. After 3 days, the cell suspension was collected and single cells were sorted into 96-well plates by flow cytometry, with 200 μl of culture medium in each well. After 10-15 days, the single clones were gradually expanded to 24-well plates and then 6-well plates, eventually yielding a total of 154 single-clonal cell lines.

[0029] Example 3 Construction of Bax- / - cells

[0030] The genomes of the 154 Bax- / - HEK293T cell line monoclonal cells obtained above were extracted, and nested PCR was performed on them using synthesized primers. Monoclonal cells were then selected for verification by culture PCR.

[0031] The obtained DNA sequencing results were compared with the wild-type sequence for sequence and amino acid alignment. This was to check for additions or deletions at the gene level. Through genome extraction and DNA sequencing verification, we finally obtained 12 knockout cell lines (Bax1.1, Bax1.2, Bax1.3, Bax1.5, Bax1.6, Bax2.1, Bax2.2, Bax2.3, Bax2.5, Bax2.6, Bax3.1, Bax3.2). The sequencing results are shown in Table 1. After screening out cells with deletions of 3 folds and heterozygous cells, two editing methods remained: one was adding 1 bp, and the other was deleting 8 bp. Since the cell line with the 1 bp addition had instability during subsequent passaging, to avoid this, we selected the Bax2.6 cell line with the 8 bp deletion. Genome alignment showed that it terminated prematurely at position 70, thus confirming at the gene level that the Bax gene deletion cell line was successfully constructed. Figure 1 ).

[0032] Table 1. Gene editing results of Bax knockout monoclonal cell lines

[0033]

[0034] The primers designed for nested PCR are:

[0035] Primer 1F: CAGGGACTCAGTTGTCTGGG.

[0036] Primer 1R: CCACAACTCAGCGCAAACAT.

[0037] Primer 2F: TTCAGGGGTGAGTTTGAGGT.

[0038] Primer 2R: CTCCCCTAAGAACTAGGAGT.

[0039] Example 4: Verification of the absence of expression of the pro-apoptotic factor Bax

[0040] Take cells that have filled a 6-well plate, discard the culture medium, digest the cells, wash the pellet twice with PBS pre-cooled at 4°C, and count the cells to 1 million.

[0041] (1) Add 100 μL of TGH lysis buffer to every million cells to lyse the cells (the amount can be adjusted according to different protein expression levels).

[0042] (2) Add half the volume of 3×Loading buffer to the cell lysis buffer and vortex. Heat in a 96℃ metal bath for 30 min and centrifuge at 13000 rpm at room temperature for 15 min. The supernatant is the whole protein extract, which can be directly used for SDS-PAGE loading.

[0043] (3) The remaining sample was stored in a -20℃ refrigerator.

[0044] (4) Gel preparation: If using pre-made gels, unpack the pre-made gels, remove the seal at the bottom, and place the gel in the electrophoresis tank. If preparing your own gel, choose a thicker gel plate to prevent breakage. Select the gel concentration according to the protein size (8% gel for proteins larger than 80 kDa, and 13-15% gel for proteins smaller than 80 kDa). Fill approximately 13 mL of separating gel to the graduation mark, and add an appropriate amount of pure water to form a liquid seal. Gel for 1 hour, then drain the water and air dry. Prepare a 5% stacking gel (approximately 2 mL). Slowly add the stacking gel along the gel plate wall until it just barely doesn't overflow. Insert a comb of the appropriate width and thickness for the gel plate and gel for 0.5 hours.

[0045] (5) Load equal amounts of the prepared test samples. The loading order from left to right is Marker, control group, and experimental group. If the number of samples is less than the number of wells, use Loading to fill in the loading wells to ensure the gel is free of voids (this will make the gel run more smoothly). Load the samples as far as possible into the middle of the gel. The loading volume is 30 μL. Set the electrophoresis conditions as follows: stacking gel 80 V for 30 min and separating gel 120 V for 2 h. After electrophoresis, cut the tails of the separating gel and stacking gel (to make the gel smoother).

[0046] (6) Prepare 6 sheets of filter paper, soak them in wet transfer buffer, and activate the PVDF membrane in methanol for 5 min.

[0047] (7) Place three moistened filter papers, an SDS-PAGE gel, a PVDF membrane activated in methanol, and three moistened filter papers in the electroporator from cathode to anode, in that order. Try to avoid leaving air bubbles between each layer. If there are air bubbles, gently remove them with a glass rod. If the PVDF membrane is slightly large, you can trim it slightly. Secure the sandwich structure and place it in the electroporator along with an ice box (to absorb heat during electroporation and prevent overheating). Set the current to a constant 200 mA for 30 min.

[0048] (8) After electroplating, rinse the transfer tank with pure water to reduce the corrosion of the platinum wire in the transfer tank by ions in the transfer solution. Seal the PVDF membrane with a 5% skim milk powder solution and place it on a decolorizing shaker at room temperature (80 rpm) for 1 hour.

[0049] (9) Cut the membrane appropriately according to the protein size, seal the PVDF membrane into the Yijie membrane, add an appropriate volume of primary antibody and incubate overnight at 4°C or at room temperature for 1 h.

[0050] (10) To recover the primary antibody, cut a corner off the plastic film and pour the primary antibody into a prepared 15 mL centrifuge tube. When pouring the primary antibody, let it flow out naturally and do not squeeze the bag to prevent leaving marks on the PVDF membrane. Store the recovered primary antibody at -20 degrees Celsius.

[0051] (11) Rinse the PVDF membrane three times in TBST for 6-8 min each time at 120 r (TBST buffer preparation: 50× to 10×, then diluted to 1×, add 5% Tween. That is, 2000 mL× plus 1 mL Tween. The Tween content can be adjusted according to the degree of nonspecific binding of the primary antibody. If there is more nonspecific binding, the Tween content can be increased). Both membranes can be washed at the same time. Turn them over during the second wash.

[0052] (12) Add the corresponding secondary antibody according to the properties of the primary antibody, place the membrane in the secondary antibody and incubate at room temperature for 1 h at 120 r.

[0053] (13) After incubation, pick up the PVDF membrane with tweezers, rinse it in TBST buffer, and then rinse it for 5 min at 80 r, 3 times. Both membranes can be washed at the same time, and the membranes can be turned over during the second wash.

[0054] (14) Turn on the chemiluminescence imaging instrument, as it requires 3-5 minutes to pre-cool.

[0055] (15) Prepare the luminescent solution in centrifuge tubes, first 400 μL of the white bottle (developing solution), then 400 μL of the black bottle (enhancing solution). If the membrane has been cut, halve the volumes of both the developing and enhancing solutions. Take a plastic membrane and drop a small amount of rinsing solution on the lab bench to help flatten the membrane. Add the developing solution to the plastic membrane in small, repeated drops. Use tweezers to pick up the rinsed PVDF membrane, allowing one corner to be slightly drained of rinsing solution on tissue paper. Then place it on the plastic membrane, immerse the luminescent solution into the PVDF membrane, clamp it into the Yijie membrane, and place it in the luminescence imaging device to emit light.

[0056] (16) Use the appropriate software to calculate the grayscale value of the acquired image.

[0057] (17) If the same membrane needs to be incubated with other primary antibodies after luminescence is completed, wash the membrane with TBST at 80 r for 30 min. Repeat the above steps of incubating with primary antibody, rinsing, incubating with secondary antibody, rinsing, and luminescence.

[0058] (18) If the same membrane needs to be incubated with the internal control after luminescence, wash the membrane with TBST at 80 r for 30 min. Incubate with the primary antibody for the internal control at 4℃ for 3 h, and repeat the above steps of rinsing, incubating with the secondary antibody, rinsing, and luminescence. Try to incubate the target protein first and then incubate the internal control to prevent excessive impurities.

[0059] like Figure 2 As shown in the figure, the experimental group showed no obvious fluorescence of Bax, indicating the expression of the pro-apoptotic gene Bax, and finally a cell line without Bax expression was obtained.

[0060] Example 5: Detection and verification of cellular mTOR and AKT pathways

[0061] Take cells that have filled a 6-well plate, discard the culture medium, digest the cells, wash the pellet twice with PBS pre-cooled at 4°C, and count the cells to 1 million.

[0062] (1) Add 100 μL of TGH lysis buffer to every million cells to lyse the cells (the amount can be adjusted according to different protein expression levels).

[0063] (2) Add half the volume of 3×Loading buffer to the cell lysis buffer and vortex. Heat in a 96℃ metal bath for 30 min and centrifuge at 13000 rpm at room temperature for 15 min. The supernatant is the whole protein extract, which can be directly used for SDS-PAGE loading.

[0064] (3) The remaining sample was stored in a -20℃ refrigerator.

[0065] (4) Gel preparation: If using pre-made gels, unpack the pre-made gels, remove the seal at the bottom, and place the gel in the electrophoresis tank. If preparing your own gel, choose a thicker gel plate to prevent breakage. Select the gel concentration according to the protein size (8% gel for proteins larger than 80 kDa, and 13-15% gel for proteins smaller than 80 kDa). Fill approximately 13 mL of separating gel to the graduation mark, and add an appropriate amount of pure water to form a liquid seal. Gel for 1 hour, then drain the water and air dry. Prepare a 5% stacking gel (approximately 2 mL). Slowly add the stacking gel along the gel plate wall until it just barely doesn't overflow. Insert a comb of the appropriate width and thickness for the gel plate and gel for 0.5 hours.

[0066] (5) Load equal amounts of the prepared test samples. The loading order from left to right is Marker, control group, and experimental group. If the number of samples is less than the number of wells, use Loading to fill in the loading wells to ensure the gel is free of voids (this will make the gel run more smoothly). Load the samples as far as possible into the middle of the gel. The loading volume is 30 μL. Set the electrophoresis conditions as follows: stacking gel 80 V for 30 min and separating gel 120 V for 2 h. After electrophoresis, cut the tails of the separating gel and stacking gel (to make the gel smoother).

[0067] (6) Prepare 6 sheets of filter paper, soak them in wet transfer buffer, and activate the PVDF membrane in methanol for 5 min.

[0068] (7) Place three moistened filter papers, an SDS-PAGE gel, a PVDF membrane activated in methanol, and three moistened filter papers in the electroporator from cathode to anode, in that order. Try to avoid leaving air bubbles between each layer. If there are air bubbles, gently remove them with a glass rod. If the PVDF membrane is slightly large, you can trim it slightly. Secure the sandwich structure and place it in the electroporator along with an ice box (to absorb heat during electroporation and prevent overheating). Set the current to a constant 200 mA for 30 min.

[0069] (8) After electroplating, rinse the transfer tank with pure water to reduce the corrosion of the platinum wire in the transfer tank by ions in the transfer solution. Seal the PVDF membrane with a 5% skim milk powder solution and place it on a decolorizing shaker at room temperature (80 rpm) for 1 hour.

[0070] (9) Cut the membrane appropriately according to the protein size, seal the PVDF membrane into the Yijie membrane, add an appropriate volume of primary antibody and incubate overnight at 4°C or at room temperature for 1 h.

[0071] (10) To recover the primary antibody, cut a corner off the plastic film and pour the primary antibody into a prepared 15 mL centrifuge tube. When pouring the primary antibody, let it flow out naturally and do not squeeze the bag to prevent leaving marks on the PVDF membrane. Store the recovered primary antibody at -20 degrees Celsius.

[0072] (11) Rinse the PVDF membrane three times in TBST for 6-8 min each time at 120 r (TBST buffer preparation: 50× to 10×, then diluted to 1×, add 5% Tween. That is, 2000 mL× plus 1 mL Tween. The Tween content can be adjusted according to the degree of nonspecific binding of the primary antibody. If there is more nonspecific binding, the Tween content can be increased). Both membranes can be washed at the same time. Turn them over during the second wash.

[0073] (12) Add the corresponding secondary antibody according to the properties of the primary antibody, place the membrane in the secondary antibody and incubate at room temperature for 1 h at 120 r.

[0074] (13) After incubation, pick up the PVDF membrane with tweezers, rinse it in TBST buffer, and then rinse it for 5 min at 80 r, 3 times. Both membranes can be washed at the same time, and the membranes can be turned over during the second wash.

[0075] (14) Turn on the chemiluminescence imaging instrument, as it requires 3-5 minutes to pre-cool.

[0076] (15) Prepare the luminescent solution in centrifuge tubes, first 400 μL of the white bottle (developing solution), then 400 μL of the black bottle (enhancing solution). If the membrane has been cut, halve the volumes of both the developing and enhancing solutions. Take a plastic membrane and drop a small amount of rinsing solution on the lab bench to help flatten the membrane. Add the developing solution to the plastic membrane in small, repeated drops. Use tweezers to pick up the rinsed PVDF membrane, allowing one corner to be slightly drained of rinsing solution on tissue paper. Then place it on the plastic membrane, immerse the luminescent solution into the PVDF membrane and clamp it into the Yijie membrane, then place it in the luminescence imaging device to emit light.

[0077] (16) Use the appropriate software to calculate the grayscale value of the acquired image.

[0078] (17) If the same membrane needs to be incubated with other primary antibodies after luminescence is completed, wash the membrane with TBST at 80 r for 30 min. Repeat the above steps of incubating with primary antibody, rinsing, incubating with secondary antibody, rinsing, and luminescence.

[0079] (18) If the same membrane needs to be incubated with the internal control after luminescence, wash the membrane with TBST at 80 r for 30 min. Incubate with the primary antibody for the internal control at 4℃ for 3 h, and repeat the above steps of rinsing, incubating with the secondary antibody, rinsing, and luminescence. Try to incubate the target protein first and then incubate the internal control to prevent excessive impurities.

[0080] like Figure 3 As shown in the figure, the experimental group showed no obvious fluorescence of Bax, indicating the expression of the pro-apoptotic gene Bax, and finally a cell line without Bax expression was obtained.

[0081] Example 6: Cell growth properties - cell viability assay

[0082] (1) Divide approximately 8,000 cells into 96-well plates, add 100 μL of LMEM complete medium to each well, and incubate the 96-well plates in a cell culture incubator overnight;

[0083] (2) Before the test, discard the DMEM complete medium and replace it with phenol red-free medium. Add 100 μL to each well and perform three replicates for each cell line.

[0084] (3) Prepare MTS / PMS at a ratio of 20:1 with MTS and PMS. Add 20 μl of MTS / PMS to each well and measure the absorbance at a wavelength of 492 nm using an enzyme-linked immunosorbent assay (ELISA) reader. Measure every 30 min (depending on the color change) until the OD value reaches about 0.8, then stop measuring and analyzing the results.

[0085] Table 2

[0086]

[0087] Bax- / - cell lines showed over 20% increased cell viability compared to HEK293T cells treated with different small molecule inhibitors ZV-FMK, Nec-1, Puro, CDDP, and rotenone. (A: ZV-FMK, Nec-1, Puro; B: CDDP, rotenone.)

[0088] Example 7: AAV triple plasmid transfection of HEK293T and Bax- / - HEK293T

[0089] (1) HEK293T and Bax- / - HEK293T cells were passaged in 10cm cell culture dishes until the cells were in good condition, so that the cell concentration reached 90-95% confluence when transfected the next day.

[0090] (2) For each transfection sample, a DAN-Lip2000 complex needs to be prepared. In a centrifuge tube, add 600 μL of serum-free and antibiotic-free DMEM medium and 24 μg of DNA, and gently mix to prepare a DNA dilution. In another centrifuge tube, add 600 μL of serum-free and antibiotic-free DMEM medium and 60 μL of Lip2000 (mix well before use), and gently mix to prepare a Lip2000 dilution. Let stand at room temperature for 5 min. Mix the DNA dilution and the Lip2000 dilution, gently mix, and let stand at room temperature for 30 min to form the DNA-Lip2000 complex. The DNA-Lip2000 complex is stable at room temperature for 6 hours.

[0091] (3) Add the DNA-Lip2000 complex to a 10cm culture dish inoculated with cells, and gently shake the culture dish back and forth to disperse and mix the complex. After culturing in a CO2 incubator at 37℃ for 4-6 hours, change the culture medium and add 10% FBS DMEM complete medium and continue culturing for 3-5 days.

[0092] Depend on Figure 4 Bright-field and fluorescence images of HEK293T and Bax- / - cell lines transfected with AAV virus, showing a transfection efficiency of 95%.

[0093] Example 8: AAV triple plasmid transfection of HEK293T and collection of Bax- / - HEK293T.

[0094] (1) Transfer the cells into a 15ml centrifuge tube, centrifuge at 1600 rpm for 4 min, discard the supernatant, resuspend the cells in 1mL DMEM medium in an EP tube, and mix by pipetting.

[0095] (2) Place the sample in liquid nitrogen to freeze for 8 minutes, then quickly place it in 37 degrees Celsius for 8 minutes, vortex for 5 minutes, and repeat 8 times.

[0096] (3) Add 1 μL of MgCl2 and 1 μL of omnipotent nuclease, and react at 37 degrees Celsius for 30 min.

[0097] (4) Centrifuge at 10000g for 10 min at 4 degrees Celsius. Aspirate the supernatant to obtain the virus suspension.

[0098] Example 9: Iodixanol ultracentrifugation purification of HEK293T and Bax- / - AAV harvested from HEK293T.

[0099] (1) Using a 10mL syringe and an 18g needle, add each iodixanol gradient to the sealed tube in the following order, taking care to avoid air bubbles. Figure 6 The iodixanol gradient before ultracentrifugation.

[0100] (2) Carefully add 5 mL of AAV obtained from HEK293T and Bax- / - HEK293T to the gradient. Fill the top of the tube with 1×PBS cell lysis buffer and seal the cap (the two sealed centrifuge tubes need to be leveled).

[0101] (3) Centrifuge at 200,000 rcf / min for 2 h in a T70i rotor at 18℃.

[0102] (4) Carefully remove the quick-sealing centrifuge tubes from the rotor and place them in a stable rack, making sure not to disturb the stratification.

[0103] (5) Carefully puncture the quick-seal centrifuge tube at the interface of the 60% and 40% gradients using an 18g syringe, and puncture the top of the quick-seal centrifuge tube with another 18g needle. Begin collecting the purified AAV in a 10mL centrifuge tube and store temporarily at 4°C. Avoid collecting proteins at the 40%–25% interface.

[0104] Example 9: Real-Time PCR determination of AAV titers of HEK293T and Bax- / - HEK293T

[0105] (1) Take eight tubes, each containing 20 microliters of reaction system, and set up four parallel experiments.

[0106] Mix: 10 microliters

[0107] Primer F: 10 μL

[0108] Primer R: 10 μL

[0109] AAV template: 5 microliters

[0110] (2) The reaction system is as follows Figure 6 As shown.

[0111] The copy number harvested from AAV transfection in HEK293T and Bax- / - cell lines was more than 10 times higher in the Bax- / - cell line than in the HEK293T cell line.

[0112] Table 3

[0113]

Claims

1. Bax2.6 cell line: The pro-apoptotic factor Bax gene was knocked out in HEK293T cells.

2. The method for constructing the Bax2.6 cell line according to claim 1, characterized in that, Bax-sgRNA was designed to target the Bax gene for multi-gene editing in cells.

3. The method for constructing the Bax2.6 cell line according to claim 2, characterized in that, The sequence of the Bax-sgRNA is TCCTAGCTCGTCCCGCTTAC.

4. The application of the Bax2.6 cell line as described in claim 1 in increasing AAV virus titer.

Images