A gel chromatography method for efficiently purifying a large amount of PCR products and its application

Abstract

The present invention relates to a method for efficiently purifying a large amount of PCR products and its application. Specifically, the present invention provides a method for purifying double-stranded DNA, wherein the filler for macromolecules is used for purification by gel chromatography, and in particular the filler used is Sepharose 6 Fast Flow. The purified double-stranded DNA can be used as a repair template for gene knock-in, for example, for integrating CAR genes into T cells to prepare CAR-T cells. By the method of the present invention, PCR products can be purified in large quantities, the purified double-stranded DNA has high purity and can effectively remove residual DNA polymerase, primers and dNTPs in the PCR system, the purification yield is high (up to 80%), and the operation is simple and the cost is low, especially the eluent can be PBS buffer, water and other neutral solutions, which is conducive to the subsequent application of purified double-stranded DNA.

Description

Technical Field

[0001] The present invention relates to the field of cell therapy, and in particular to a gel chromatography method for efficiently purifying a large amount of PCR products and its application Background Art

[0002] In recent years, with the continuous development of molecular biology, various innovative biotechnologies have been applied to the treatment or prevention of diseases, among which cell therapy is one of the most popular technologies. The inventors of this application have previously invented a new non-viral site-specific knock-in CAR-T cell therapy technology, which achieves a more efficient and precise transduction method, a simpler CAR-T cell preparation method, and a safer and more durable expression. The technical process is extremely simple, but requires a large amount of high-purity double-stranded DNA as a recombinant template for gene knock-in.

[0003] Among the current methods for obtaining double-stranded DNA in large quantities, PCR technology is the most common. However, in addition to the target amplified fragment, the PCR reaction product may also contain primers, primer dimers, DNA polymerase, and dNTPs. These non-target substances have a great impact on gene knock-in, so they need to be removed.

[0004] The principle of purification is to ensure the integrity of the primary structure of double-stranded DNA molecules and exclude contamination from other molecules. In order to purify the target double-stranded DNA from the PCR product, many methods for purifying DNA have been developed. According to the solubility, adsorption and charge properties of DNA, purification methods can be divided into precipitation, density gradient centrifugation, electrophoresis, magnetic bead adsorption, membrane adsorption and chromatography, etc.:

[0005] (1) Precipitation method: The more classic ones are phenol-chloroform extraction method, CTAB (cetyltrimethylammonium bromide) precipitation method, etc. This method is relatively simple and has low cost. The disadvantages are that the steps are complicated, time-consuming, and the use of organic reagents is not conducive to environmental protection and subsequent applications. It is not widely used at present and has a tendency to be gradually replaced.

[0006] (2) Electrophoresis: The electrophoresis method uses agarose gel electrophoresis technology to separate the target DNA fragments from the non-target DNA fragments, and combines the sol technology of the gel containing the target DNA fragment to obtain a high-purity target DNA. There are many kits based on this principle, such as Takara's agarose gel DNA purification kit. Using this method, not only the recovered purified DNA fragments are of high purity and good integrity, but the operation is also relatively efficient, fast and convenient, and the purified products are more suitable for experiments such as ligation reactions, PCR amplification, and DNA sequencing. The advantages are that it is suitable for micro-purification of target DNA and the method is mature. The disadvantages are that the cost is high and the recovery rate is low, and it is generally difficult to meet the needs of large-scale purification of target products.

[0007] (3) Magnetic bead adsorption method: Magnetic bead method nucleic acid purification technology uses nano-scale magnetic microbeads (such as patent CN206345846). The surface of this magnetic microbead is marked with a functional group that can react with DNA for adsorption. Magnetic silica particles refer to magnetic microbeads with a layer of silicon material coated on the surface to adsorb nucleic acids. Ion exchange magnetic beads refer to magnetic microbeads with a layer of ion exchangeable materials (such as diethylaminoethyl DEAE, COOH, etc.) coated on the surface to achieve the purpose of adsorbing nucleic acids. The purification principles corresponding to magnetic microbeads of different properties are not consistent. The biggest advantage of using magnetic beads to purify nucleic acids is automation. Magnetic beads can aggregate or disperse under magnetic field conditions, thereby completely getting rid of the manual operation process required by centrifugation, etc., and do not require the use of large-scale equipment, which is relatively easy to achieve. The disadvantages are that the process is cumbersome, the sample is not fully eluted, the eluted DNA contains more impurities, the cost is high, and it is generally difficult to meet the needs of large-scale target products.

[0008] (4) Silica matrix adsorption technology (such as QIAquick spin columns): For PCR products, they can be directly applied to the purification columns of the kit based on the principle of this technology, and the target product can be obtained through washing, elution and other steps. The advantage is simple operation. The disadvantage is incomplete recovery, low recovery rate, high cost, and generally difficult to meet the needs of large-scale target products.

[0009] (5) Combination technology: Some technologies are combined with the above technologies, such as combining electrophoresis with magnetic beads, such as patent CN104560954. Another example is the combination of electrophoresis and membrane adsorption technology. Although these technologies are also relatively easy to implement, these methods have disadvantages such as low yield and high cost, and there is still a need to find better methods to replace these traditional methods.

[0010] (6) Chromatography: Using high performance liquid chromatography technology system to purify nucleic acids is a different approach. High performance liquid chromatography is an important branch of chromatography. Liquid is used as the mobile phase. A high-pressure infusion system is used to pump a single solvent with different polarities or a mixed solvent with different proportions, a buffer solution and other mobile phases into a chromatography column containing a stationary phase. After the components in the column are separated, they enter the detector for detection, thereby realizing the analysis of the sample. This method has become an important separation and analysis technology in the fields of chemistry, medicine, industry, agronomy, commercial inspection and legal inspection.

[0011] In the application of nucleic acid purification, due to the physical characteristics of DNA itself, especially the many charged phosphate groups in its backbone, anion exchange chromatography has become the most commonly used technology by scientists. For example, in 1991, W Warren, J Doniger and others reported a method of using an anion exchange chromatography column to obtain a 344bp high-quality target DNA fragment through gradient elution; in 2004, Raquel Hernandez and others also reported the use of an anion exchange chromatography column to purify a 277bp target fragment with a recovery rate of up to 80%; even in 2008, Kim K and others tried to use this method for the purification of nucleic acid samples such as ancient human bones. The advantage is that it can be completed within 30 minutes and is relatively efficient. The disadvantages are that too many reagents are used, the operation is relatively cumbersome, and large fragments of DNA have not been verified.

[0012] In addition, due to the electrostatic effect of nucleic acid itself and the interaction of ions, the hydrophobic nature of nucleic acid is more prominent, and nucleic acid can also be purified by hydrophobic chromatography technology, especially for the purification of single-stranded DNA.

[0013] In recent years, the use of a hybrid chromatography technology has been gradually reported. This technology is not only applicable to DNA, but also can be used for the purification of antibodies and proteins. For example, in 2015, T Matos et al. reported the successful purification of single-stranded DNA from double-stranded DNA using a hybrid chromatography filler (capto adhere resin) from GE.

[0014] Although these existing chromatography technologies for DNA purification have played a certain role in gene cloning, gene editing, sequencing, viral DNA identification and other fields, they still need to be improved: for example, the ion exchange chromatography method is relatively complicated to operate, requires more reagents to be prepared, and beginners need to explore the experimental conditions a lot, which wastes manpower costs to a certain extent. The hybrid chromatography method has high requirements for fillers and is relatively expensive. Summary of the invention

[0015] Based on the current research progress, the present invention has found a better method that is convenient to obtain materials, simple to operate, saves manpower and can efficiently purify the target double-stranded DNA through continuous exploration and practice. This method relies on gel filtration chromatography, and uses the principle that the mesh structure of gel filtration chromatography filler can be separated according to molecular weight. It only uses a single neutral buffer and an unmodified chromatography filler that can form a gel cross-linked product with a certain pore size. The operation can be completed within 10 minutes. At present, this method has not been reported.

[0016] In one aspect, the present invention provides a method for purifying double-stranded DNA, wherein the purification is carried out by a gel chromatography method using a filler for macromolecules.

[0017] In some embodiments, the filler has a particle size of 45-165 μm.

[0018] In some embodiments, the filler has a stability working or cleaning pH of 2-12.

[0019] In some embodiments, the filler is Sepharose 6 Fast Flow. The filler Sepharose 6 Fast Flow is from Stovan, catalog number 17015901 (specification 1L) or 17015905 (specification 10L).

[0020] In some embodiments, the molecular weight of the double-stranded DNA is 10,000-4,000,000 Daltons or 270-10,800 bases (bp), such as 10,000-2,000,000 Daltons, 10,000-1,000,000 Daltons, 10,000-800,000 Daltons, 10,000-500,000 Daltons, 10,000-200,000 Daltons, 1000 0-100000 Daltons, 10000-80000 Daltons, 10000-50000 Daltons or 10000-20000 Daltons; or for example 300-10000 bp, 500-9000 bp, 600-8000 bp, 800-6000 bp, 1000-5000 bp, 2000-4000 bp, 2000-3000 bp.

[0021] In some embodiments, the eluent used in the gel chromatography method is a neutral buffer, preferably PBS buffer, Tris-HCl buffer and water, more preferably PBS buffer and water.

[0022] In some embodiments, the pH of the PBS buffer is 6.5-7.5, preferably 7.0-7.5, more preferably 7.4.

[0023] In some embodiments, the PBS comprises Na2HPO4, KH2PO4, NaCl and KCl. In a specific embodiment, the PBS comprises 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 1.76 mM KH2PO4.

[0024] In some embodiments, the method further comprises concentration after gel chromatography, preferably ultrafiltration concentration.

[0025] In some embodiments, the gel chromatogram of the method has two absorption peaks, the first absorption peak is the absorption peak of double-stranded DNA, and the subsequent absorption peak is the residual DNA polymerase, primer and dNTP of the PCT system.

[0026] In some embodiments, purified double-stranded DNA can be used as a repair template for gene knock-in.

[0027] In another aspect, the present invention provides a non-viral gene site-specific knock-in method, the method comprising:

[0028] 1) Template DNA is connected to the plasmid vector

[0029] 2) PCR amplification of template DNA

[0030] 3) Purification of template double-stranded DNA

[0031] 4) The purified template double-stranded DNA and gene editing material are transfected into the target cells through gene editing methods to knock in the gene at a specific site.

[0032] The purification of the template double-stranded DNA is carried out using the above method of the present invention.

[0033] In some embodiments, the gene editing methods used for gene knock-in include: CRISPR-Cas9, ZFN, ARCRS, TALEN and megaTAL, preferably CRISPR-Cas9.

[0034] In some embodiments, the gene editing materials used in the gene editing method include: plasmid, mRNA and protein.

[0035] In some embodiments, the DNA targeted by gene knock-in can be any DNA, such as genomic DNA.

[0036] In some embodiments, the gene editing material is transferred into the cell together with the template DNA using the following transfection methods: liposome, calcium phosphate, DEAE-dextran, electroporation, microinjection, and gene gun. After transfection, the gene editing nuclease first shears the genomic DNA to produce a broken double-stranded DNA nick. At this time, the purified template double-stranded DNA acts as a donor DNA and undergoes homologous recombination with the broken nick DNA, and the knocked-in DNA is successfully integrated into the target genomic site.

[0037] In some embodiments, the target cells may include: umbilical cord blood stem cells, bone marrow hematopoietic stem cells, adult stem cells, embryonic stem cells, T lymphocytes, B lymphocytes, NK cells, NK-92 and NK-92-derived cells, macrophages, DC cells, CHO and CHO-derived cells, 293 and 293-derived cells, and common cell lines.

[0038] In some embodiments, the template DNA includes a CAR gene and the target cell is a T cell, thereby site-specifically integrating the CAR gene into the T cell to prepare a CAR-T cell.

[0039] In another aspect, the present invention also provides the use of CAR-T cells for preparing drugs, wherein the drugs are used to treat leukemia (such as acute B lymphocytic leukemia, acute T lymphocytic leukemia, acute NK lymphocytic leukemia, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, acute myeloid leukemia, multiple myeloma, etc.), solid tumors (such as lung cancer, liver cancer, gastric cancer, breast cancer, colorectal cancer, prostate cancer, pancreatic cancer, glioma, esophageal cancer, bile duct cancer, endometrial cancer, ovarian cancer, mesothelioma and thymic cancer), AIDS, autoimmune diseases and other diseases treatable by CAR-T cells.

[0040] The beneficial technical effects of the present invention are: (1) large-scale purification of PCR products; (2) the purified double-stranded DNA has high purity and can effectively remove residual DNA polymerase, primers and dNTPs in the PCR system; (3) the purification yield is high, which can reach 80%; (4) the operation is simple and the cost is low; (5) the eluent can be PBS buffer, water or other neutral solutions, which is conducive to the subsequent application of the purified double-stranded DNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Schematic diagram of the gel chromatography purification method of the present invention: a. A large amount of PCR reaction products include: target double-stranded DNA, primers, DNA polymerase and dNTP; b. After loading on the gel chromatography column, the target double-stranded DNA is eluted first, while the primers, DNA polymerase and dNTP enter the interior of the gel particles and are eluted later; c. The target double-stranded DNA is concentrated by ultrafiltration.

[0042] Figure 2 Schematic diagram of knocking the CAR gene into the target site in Example 1.

[0043] Figure 3 The gel chromatography results in Example 4 are shown.

[0044] Figure 4 The results of DNA electrophoresis identification in Example 4 are shown.

[0045] Figure 5 This is the chromatogram of DNA polymerase, primers and dNTP loading in Example 5.

[0046] Figure 6 The gel chromatography results in Example 6 are shown.

[0047] Figure 7 The results of DNA electrophoresis identification in Example 6 are shown.

[0048] Figure 8 The flow cytometry results of Example 7 are shown. DETAILED DESCRIPTION

[0049] Although gene knockout technology has made great progress, the efficiency of knocking in large gene fragments has not been high. The difference between gene knock-in and gene knockout is whether a repair template DNA is added. The process of gene knock-in is: after nucleases (such as ZFN, TALEN, CRISPR-Cas9, etc.) shear the specific DNA, the repair template repairs the "damaged" DNA according to the principle of homologous recombination, and introduces the specific DNA fragment into a specific site after repair. The repair templates currently used mainly include plasmid DNA, double-stranded DNA, single-stranded DNA and adeno-associated virus vectors. The advantage of double-stranded DNA as a repair template is that it can be obtained through a large amount of PCR, which is cheaper and easier to obtain than other repair templates. The present invention provides a gel chromatography method for efficiently purifying a large amount of PCR products. The high-purity double-stranded DNA obtained according to the method can be used for CAR gene knock-in of specific T cell genomic sites, thereby obtaining CAR-T cells.

[0050] Figure 1 The specific technical scheme of the gel chromatography method for purifying double-stranded DNA is shown:

[0051] 1) Based on the commonly used reaction system in molecular biology, a large-scale PCR reaction mixture is prepared, packaged according to the micro-reaction system, and amplified using a PCR instrument according to the optimized amplification program to obtain a large-scale PCR product. The obtained PCR product contains primers, DNA polymerase and dNTP in addition to the target double-stranded DNA. The reagents used for PCR amplification can be kit products of any company, and the PCR amplifier or related equipment used for PCR amplification can also be from any manufacturer.

[0052] 2) Use commercial filler Sepharose 6Fast Flow (6% agarose highly cross-linked to form microbeads with a diameter of 90 μm) to pack the column. Load the above PCR product as the sample, use a non-toxic neutral buffer, preferably PBS, Tris-HCl and water, and ensure that the pH value is 7.0-7.5. According to the molecular sieve principle, the target double-stranded DNA is eluted first, while the small molecules (DNA polymerase, primers and dNTP) in the PCR product enter the gel particles due to their small molecular weight, and are eluted later, so that the target DNA can be fully separated from other small molecules;

[0053] 3) The double-stranded DNA eluted first is concentrated by ultrafiltration tube to increase the concentration of the target DNA for downstream needs.

[0054] Example

[0055] Example 1: Molecular design of repair template DNA

[0056] In this example, the CAR gene is knocked into the TRAC gene site of the T cell, and the CRISPR-Cas9 element and the modified repair template DNA are co-electroporated. First, the CRISPR-Cas9 element is used to make a cut in the TRAC gene site, and then the modified repair template DNA is used to perform homologous recombination repair on the gap, and finally the CAR gene is knocked into the target site. Figure 2 shown.

[0057] The repaired template DNA consists of two parts:

[0058] a. Left and right homology arms. The left and right homology arms are used to identify the target DNA and perform recombination exchange. In order to allow the knock-in gene to be correctly expressed, the knock-in gene is "landed" in the 5' end region of exon 1 of the TRAC gene. This embodiment designs a left and right homology arm of 300 bases, and the sequences are shown in SEQ ID NO: 1-2 respectively.

[0059] b. Knocked-in genes. Depending on the TCR-α promoter, the knocked-in genes include cleavage peptide genes, CAR genes, and polyA. In this embodiment, the cleavage peptide used is P2A, and the CAR gene adopts a second-generation structure, and the specific structure includes CD8 signal peptide, targeting CD19 scFv, CD8 hinge region, CD8 transmembrane region, 4-1BB co-stimulatory region, and CD3ζ activation region. In this embodiment, PolyA uses bGHpA polyA, and the gene structure sequences are as follows:

[0060]

[0061]

[0062] Example 2: Molecular cloning of repair template DNA

[0063] The repair template DNA of SEQ ID NO: 11 was synthesized and cloned into the pUC57 plasmid vector. The process is divided into the following steps:

[0064] 1) PCR preparation of target fragment. First, you need to prepare the PCR reaction system:

[0065] Reagents (Cat. No.: RO5OA, TAKARA) Amount used (μl) 5× primeSTAR GXL buffer 10 dNTP Mix 4 Primer F 1 Primer R 1 DNA fragments 0.5 Sterilized water 32.5 PrimeSTAR GXL DNA Polymerase 1 Total volume 50

[0066] Then place the PCR reaction system in a PCR amplification instrument and perform amplification under the following conditions:

[0067]

[0068] 2) Vector PCR preparation. First, you need to prepare the PCR reaction system:

[0069] Reagents Amount used (μl) 5× primeSTAR GXL buffer 10 dNTP Mix 4 Primer F 1 Primer R 1 pUC57 0.5 Sterilized water 31.5 PrimeSTAR GXL DNA Polymerase 2 Total volume 50

[0070] Then place the PCR reaction system in a PCR amplification instrument and perform amplification under the following conditions:

[0071]

[0072] 3) Recombination of target fragment and vector: Add the target gene and linearized vector to a centrifuge tube at a certain molar ratio on an ice box for recombination reaction, and react in a 50°C water bath for 10 minutes. The reaction system is shown in the following table:

[0073] Reagents (Cat. No.: NR001A, Nearshore Protein) Amount used (μl) Vector pUC57 3.5 DNA fragment (SEQ ID NO:11) 4 5× reaction buffer 2 NovoRec Plus Recombinase 0.5 Total volume 10

[0074] 4) Transformation plating of reaction products: melt a tube of 100 μl StbI3 competent cells (Cat. No.: DL1046, Vidi Biotech) on ice, tap the tube wall to resuspend the cells, add 10 μl of reaction solution to the competent cells, tap a few times, and place on ice for 30 minutes; heat shock in a 42°C water bath for 90 seconds and quickly place on ice for 5 minutes; add 500 μl LB liquid culture medium and incubate on a shaker at 37°C for 45-60 minutes; centrifuge at 5000 rpm for 3 minutes to collect the cells, discard 300 μl of the supernatant, and spread the remaining amount of cells on a plate containing ampicillin antibiotics, and incubate in a 37°C incubator overnight.

[0075] 5) Pick a single clone and extract the plasmid (Cat. No.: DP103-03, Tiangen Biochemical).

[0076] 6) Sequencing confirmation.

[0077] Example 3: Large-scale PCR amplification of repair template DNA.

[0078] The repair template DNA constructs the plasmid successfully, which is more conducive to PCR amplification. In this embodiment, the PCR reaction system mixture is prepared as follows:

[0079] Reagents Amount used (μl) 5× primeSTAR GXL buffer 200 dNTP Mix 80 Primer F 24 Primer R 24 pUC57-SEQ ID NO:11 30ng Sterilized water 652 PrimeSTAR GXL DNA Polymerase 20 Total volume 1000

[0080] Dispense into 20 common PCR tubes, then place the PCR reaction system in a PCR amplification instrument and perform amplification under the following conditions:

[0081]

[0082] After the PCR is completed, the PCR products are collected and merged in a 50ml centrifuge tube. At this time, the PCR products include double-stranded DNA products, dNTPs, DNA polymerase and primers, and reaction buffer. Transfer them to a 10ml syringe, exhaust the bubbles in the syringe, and prepare for sample loading.

[0083] Example 4: Purification of template double-stranded DNA by gel chromatography.

[0084] After large-scale PCR amplification of the template double-stranded DNA, it is purified. Innovative gel chromatography and ultrafiltration concentration are used. Gel chromatography is used to remove the DNA polymerase, primers and dNTPs remaining in the PCR reaction and purify the target double-stranded DNA. The purpose of ultrafiltration concentration is to concentrate the target double-stranded DNA to a certain concentration for subsequent use in gene knock-in.

[0085] Step 1: The steps of gel chromatography are as follows:

[0086] 1) Turn on AKTA pure 150 and the computer's supporting software UNICORE 7.1 at the same time, wait for the machine to self-check and communicate, and wait until the AKTA system flash light stops flashing, indicating that it can be used normally.

[0087] 2) System A / B pump cleaning: Pass 20% ethanol and water through the inlet tube in turn and clean the pump head at a maximum flow rate of 75 ml / min.

[0088] 3) Pipe cleaning: In the “system control” control software window, set the flow rate to 5 ml / min, the maximum column front pressure to 0.5 MPa, and the UV detection to 260 detection. AKTA executes the above commands and cleans the pipeline with water until the conductivity is 0 mS / cm.

[0089] 4) Install the chromatography column: The column body uses GE XK-16 / 10, the filler uses Sepharose 6Fast Flow, and the column volume is about 50ml. Install the gel chromatography column into the AKTA pure 150 system according to the column loading procedure, as follows: When connecting, connect in a drop-to-drop manner to avoid bubbles in this section of the pipeline. When operating, first connect the PEEK tube at the port above the original UV detector of the system (the other end is connected to the column valve port) to the lower end of the column, but do not tighten it, then wait for droplets to appear above, disconnect the connecting tube at the lower end of the chromatography column, connect the connecting tube above the UV detector to the upper port of the chromatography column, and then connect another section of PEEK tube to connect the lower end of the chromatography column to the upper port of the UV detector of the AKTA system. After loading the column, use deionized water to wash the chromatography column until the conductivity is stable to 0mS / cm.

[0090] 5) Loading buffer balance: Switch the pump head to neutral buffer PBS until the conductivity is stable (wash with PBS buffer until the conductivity is stable, about 16.2mS / cm). The composition of neutral buffer PBS is 137mM NaCl, 2.7mM KCl, 10mM Na2HPO4 and 1.76mM KH2PO4, pH 7.4.

[0091] 6) Preparation before loading: Clean the loading loop, use a 50ml syringe to draw water and loading buffer in turn and manually push them into the cleaning loading loop. The flushing volume should be more than 5 times the column volume. Prepare for loading, perform UV zero adjustment before loading (correct the 260nm UV absorption value to zero), and adjust the speed to 2ml / min.

[0092] 7) Sample loading: Make sure there are no bubbles in the syringe that injects the sample, then connect the 10 ml syringe that injects the sample to the column valve port of the AKTA device, and manually push 5 ml of the sample into the 5 ml sample loop.

[0093] 8) Collection: Switch the system to Injection mode. The first 260nm absorption peak appears about 11ml after the start of sample loading (-1 Figure 3 ), at the peak onset, switch the sample collection valve to Outlet mode, and when the peak decays to an absorbance of about 15, end the sample collection, and switch the switching valve to waste mode to collect the sample during this peak period. About 40ml after loading, a second 260nm absorption peak appears -2 ( Figure 3 ), collect the liquid in this peak period, which is the residual DNA polymerase, dNTP and primers in the PCR system. Repeat the loading procedure (6-7) above. After all samples are loaded, pool all the first peak samples collected and carry out the next step of concentration.

[0094] DNA electrophoresis was used to identify the purification effect of gel chromatography: In order to identify which of the absorption peaks 1 and 2 contained the target DNA, DNA electrophoresis was performed. First, 1% agarose gel was prepared, and then 1 μl of PCR product, 10 μl of absorption peak 1 product, and 10 μl of absorption peak 2 product were loaded. Then, 220V electrophoresis was performed, and the gel was illuminated by UV light. The results are as follows: Figure 4 As shown. It can be seen that the PCR product and the absorption peak-1 lane have bands at 2300bp, which is consistent with the expected fragment size; while there is no band at the corresponding position of the absorption peak-2 lane. This shows that the absorption peak-1 is the target fragment peak. In addition, 5ml of the sample obtained about 40ml of the absorption peak-1, and the brightness of the DNA bands of 1μl of PCR product and 10μl of absorption peak-1 product were almost the same. Based on this calculation, the yield of this purification method is about 80%.

[0095] Step 2: The steps of ultrafiltration concentration are as follows:

[0096] 1) Use a 30KDa ultrafiltration tube for ultrafiltration concentration. First rinse the ultrafiltration tube with ultrapure water, then centrifuge at 4000g for 5 minutes, and discard the water centrifuged at the bottom. Then add the first peak sample and centrifuge at 4000g for 5 minutes. Generally, as the number of centrifugations increases, the DNA retained on the membrane continues to increase, and the ultrafiltration time will also be increased as appropriate. Observe the volume of the liquid surface in the ultrafiltration tube. When it is about 500μl, add 15ml PBS buffer to wash and replace the liquid once, and then centrifuge at the same speed until it is concentrated in a volume of 500μl.

[0097] 2) In order to allow the DNA on the membrane to fully stretch and dissolve in the solvent, let it stand at 4°C for about 12 hours. Then use a 200μl pipette with a sterile pipette tip in a biosafety cabinet to carefully blow the side of the ultrafiltration tube 2-3 times. Then transfer all of it to a 1.5 ml EP tube and filter it with a 0.2μm filter membrane for sterilization.

[0098] 3) After the purified double-stranded DNA was concentrated by ultrafiltration and filtered, its concentration was detected by UV spectrophotometer to be 2.2 μg / ml.

[0099] Example 5: Verify the peak positions of DNA polymerase, primers and dNTPs respectively.

[0100] Take a small amount of samples of the DNA polymerase, primers and dNTPs used in Example 3 from the original commercial tubes, add water to 500 μl as the loading sample, use PBS (same as Example 4) as the eluent for chromatography, and record the peak position. Prepare samples of each component in the PCR system as shown in the following table:

[0101] Reagents Original concentration Amount used (μl) Add water (μl) DNA polymerase 1.25u / μl 20 480 Primers 10μM 100 490 dNTP 2.5mM 50 450

[0102] Place them in different EP tubes, then use three 1 ml syringes to draw out the liquids respectively and remove all bubbles in preparation for sampling.

[0103] The steps of gel chromatography are as follows:

[0104] Steps 1)-6) are consistent with Example 4.

[0105] 7) Primer loading: First load the PCR primer sample, make sure there are no bubbles in the 1ml syringe that injects the sample, then connect the 1ml syringe that injects 500μl of sample to the column valve port of the AKTA device, and manually push 500μl of sample into the 1ml loading loop. Observe the peak: switch the system to Injection mode. Elute at least 30ml after starting to load the sample, and observe the peak position. Wait until the first peak completely decays to the minimum (see the results). Figure 5 ). Switch the loading mode to manual mode, pull out the primer sample syringe from the loading valve, and rinse the loading loop with loading buffer.

[0106] 8) dNTP sample loading and observation: Insert the syringe containing the dNTP sample into the loading valve position, push the same volume of 500μl dNTP sample into the loading loop, then adjust the loading mode to Injection, the machine automatically injects the sample, elutes at 30ml, and observes the peak treatment (for results, see Figure 5 ). Wait until the first peak appears to completely decay to the minimum. Switch the loading mode to manual mode, pull out the primer sample syringe at the loading valve position, and rinse the loading loop with loading buffer.

[0107] 9) Loading of DNA polymerase sample: Insert the syringe containing DNA polymerase sample into the loading valve position, push 500μl DNA polymerase sample into the loading loop, and then adjust the loading mode to Injection. The machine automatically injects the sample, elutes at 30ml, and observes the peak treatment (for results, see Figure 5 ). Wait until the first peak appears to completely decay to the minimum. Switch the loading mode to manual mode, pull out the primer sample syringe at the loading valve position, and rinse the loading loop with loading buffer.

[0108] Figure 5 The chromatogram of DNA polymerase, primers and dNTP loading. Compared with Example 4, the peak positions of samples such as DNA polymerase, primers and dNTPs all correspond to the absorption peak-2 position in Example 4 (i.e., the position where the conductivity obviously decreases). This shows that the peak substances in absorption peak-2 are mainly PCR reaction components and do not contain the amplified target large fragment. This example further verifies that the absorption peak-1 in Example 2 is the peak of the target large fragment, and also shows that the method can completely separate the specifically amplified target double-stranded DNA from the PCR product.

[0109] Example 6: Using water instead of PBS buffer as the eluent.

[0110] Water, as a neutral solution, is often used as a solvent for dissolving DNA. This embodiment uses water instead of PBS buffer as an eluent for the key gel chromatography process in the present invention. The specific implementation steps are as follows:

[0111] The gel chromatography steps are as follows:

[0112] 1) Turn on AKTA pure 150 and the computer's supporting software UNICORE 7.1. Wait for the machine to self-check and connect to the communication, and wait until the AKTA system flashlight stops flashing, indicating that it can be used normally.

[0113] 2) System A / B pump cleaning: Pass 20% ethanol and water through the inlet tube in sequence and clean the pump head at a maximum flow rate of 75 ml / min.

[0114] 3) Pipe cleaning: In the "system control" control software window, set the flow rate to 5 ml / min, the maximum column pressure to 0.5 MPa, and the UV detection to 260. AKTA executes the above commands and cleans the pipeline with water until the conductivity is 0 mS / cm.

[0115] 4) Install the chromatography column: The column body uses GE XK-16 / 10, the filler uses Sepharose 6Fast Flow, and the column volume is about 50ml. Install the gel chromatography column into the AKTA pure 150 system according to the column loading procedure. The details are as follows: When connecting, connect in a droplet-to-droplet manner to avoid bubbles in this section of the pipeline. When operating, first connect the PEEK tube at the port above the original UV detector of the system (the other end is connected to the column valve port) to the lower end of the column, but do not tighten it, then wait for droplets to appear above, disconnect the connecting tube at the lower end of the chromatography column, connect the connecting tube above the UV detector to the upper port of the chromatography column, and then connect another section of PEEK tube to connect the lower end of the chromatography column to the upper port of the UV detector of the AKTA system. After loading the column, use deionized water to wash the chromatography column until the conductivity is stable to 0mS / cm.

[0116] 5) Preparation before loading: Clean the sample loop, use a 50ml syringe to draw water and sample buffer in turn and manually push them into the clean sample loop. The flushing volume should be more than 5 times the column volume. Prepare for loading. Before loading, perform UV zero adjustment (correct the 260nm UV absorption value to zero) and adjust the speed to 2ml / min.

[0117] 6) Sample loading: Ensure that there are no bubbles in the 1 ml syringe injected with the sample, then connect the 1 ml syringe injected with 500 μl of sample to the column valve port of the AKTA device, and manually push 500 μl of sample into the sample loop.

[0118] 7) Observe the peak: switch the system to Injection mode. After the sample is loaded, the first 260nm absorption peak 1 is eluted with 15ml. At the peak start time, switch the sample collection valve to Outlet mode. When the peak decays to the lowest absorption, end the sample collection and switch the switching valve to waste mode to collect the sample during this peak period. About 40ml after loading, the second 260nm absorption peak 2 appears, and the liquid during this peak period is collected. Figure 6 The column chromatography results of gel chromatography are shown. Figure 6It can be seen that using water as the eluent, the PCR product also has two peaks after loading, namely absorption peak 1 and absorption peak 2. The peak position of absorption peak 1 is close to that of Example 2, but the conductivity of absorption peak 2 is different from the conductivity of the second peak loaded with PBS buffer (the conductivity is slightly decreased), and the conductivity is slightly increased. Secondly, the separation of absorption peak 1 and absorption peak 2 is relatively obvious, and the baseline in the middle is close to the baseline eluted with PBS buffer. Comparison of the chromatograms shows that the two peaks can also be separated using water.

[0119] DNA electrophoresis was used to identify the purification effect of gel chromatography: In order to identify which of the absorption peaks 1 and 2 contained the target DNA, DNA electrophoresis was performed. First, 1% agarose gel was prepared, and then 1 μl of PCR product, 10 μl of absorption peak 1 product, and 10 μl of absorption peak 2 product were loaded. Then, 220V electrophoresis was performed, and the gel was illuminated by UV light. The results are as follows: Figure 7 As shown. It can be seen that the PCR product and the absorption peak-1 lane have a band at 2300bp, which is consistent with the expected fragment size; while there is no band at the corresponding position of the absorption peak-2 lane. This indicates that the absorption peak-1 is the target fragment peak. This result shows that the target amplified fragment can also be separated from the PCR product by using water as the eluent.

[0120] Example 7: Purified double-stranded DNA for preparation of non-viral site-directed knock-in CAR-T cells

[0121] The double-stranded DNA purified by gel chromatography in Example 4 and the double-stranded DNA purified by the large amount of DNA product purification kit (Cat. No.: DP205) of Tiangen Biochemical Technology Co., Ltd. (silicon matrix adsorption technology, comparative example) were used as repair templates to mediate the site-specific integration of the CAR gene after CRISPR-Cas9 cutting. The specific steps are as follows:

[0122] 1) Cell preparation

[0123] PBMC extraction: healthy volunteers without cold or fever symptoms were recruited, and professional medical personnel collected 100 ml of blood from the median cubital vein and connected it to an anticoagulant tube (Cat. No.: 367886, Becton Dickinson); after blood collection, the blood was mixed with an equal amount of PBS buffer [containing 2% fetal bovine serum (Cat. No.: SE100-B, Vistech)]; PBMC separation tube Sepmate-50 (Cat. No.: 86450, STEMCELL) was taken, 15 ml of Ficoll buffer was carefully added, and then the blood PBS mixture was added, and about 30 ml was carefully added to each tube; after centrifugation at 1200g for 10 minutes, the supernatant was quickly poured into a new 50 ml tube, and then centrifuged at 200g for 8 minutes, the supernatant was discarded, 10 ml of PBS buffer was added to resuspend the precipitate, and then the supernatant was discarded and 10 ml of PBS buffer was added to resuspend the precipitate, and the cell precipitate was resuspended in 10 ml PBS buffer after centrifugation and discarding the supernatant; the resuspended cells were counted, 10 μl of the suspension was taken and 10 μl 0.1% trypan blue (Cat. No. 15250061, Gibco) was added and mixed, and the cell number and viability were counted on a computer.

[0124] T cell purification: Take a small amount of PBMC cells and calculate the proportion of CD3 positive T cells by flow cytometry; according to the experimental needs and the proportion of CD3 positive cells, take out the required PBMC cells, and the required cells should be 70% of the cells taken (loss will occur during the operation); use the kit Release Human CD3 Positive Selection Cocktail (Cat. No.: 17751, STEMCELL) for sorting, first resuspend the total PBMC cells with positive selection buffer to make the total cell concentration 1×10 8 / ml; every 1×10 8 100 μl CD3 antibody was added to the cells; incubated at room temperature for 3 minutes; magnetic beads (Releasable Rapid Sphenes) were mixed in advance with a vortex mixer for 30 seconds, and 1×10 8 Add 100 μl of magnetic beads to the cells and incubate at room temperature for 3 minutes; transfer the cell suspension to a special sorting tube, and dilute to 2.5 ml with positive selection buffer, place it on a magnetic rack and incubate at room temperature for 5 minutes; carefully grasp the magnetic rack, pour the sorting tube, discard the unbound cell suspension, resuspend the attached magnetic beads and cells with 2.5 ml of positive selection buffer, place it on the magnetic rack again and incubate at room temperature for 3 minutes, and discard the unbound cell suspension again; remove the sorting tube from the magnetic rack, add "Release buffer" to the suspension, and incubate it at room temperature for 3 minutes; place it on a magnetic rack and incubate at room temperature for 5 minutes; carefully grasp the magnetic rack, pour the sorting tube, and collect the unbound cell suspension in a clean and sterile 15 ml centrifuge tube. These cells are the extracted T cells.

[0125] T cell activation: Take 1×10 purified T cells from above 7 Activate with anti-CD3 / anti-CD28 magnetic beads (Cat. No. 40203D, Thermo): Take 3×10 anti-CD3 / anti-CD28 magnetic beads 7 The washed beads were added to T cells, mixed well, and cultured at 37 degrees for two days. After two days, the magnetic beads were taken out, the T cells were resuspended several times with a pipette, and the cell suspension was placed in a magnetic pole. After standing for two minutes, the magnetic beads on the tube wall were discarded. The cell number and survival rate were counted on a computer.

[0126] Electroporation: 3×10 6 The cell suspension was placed in a centrifuge tube (2 tubes in total) and centrifuged at 200g for 5 minutes. After centrifugation, the culture medium was completely removed for use. Lonza electroporation buffer (Cat. No.: V4XP-3024, Lonza) was prepared, and then 15 μg of spCas9 protein (Cat. No.: A36499, Thermo, SEQ ID NO: 12) and 1 μg of designed and synthesized sgRNA-0001 (SEQ ID NO: 13) were added. After mixing at room temperature for 10 minutes, 6 μg of SEQ ID NO: 11 repair template DNA purified by silica matrix and gel chromatography were added respectively. After 10 minutes, the mixture was mixed with the cell pellet and added to the electroporation cup (Cat. No.: V4XP-3024, Lonza), and the EH115 program was used for electroporation. After the electroporation was completed, 400 μl of culture medium was added immediately and placed in a 37-degree incubator for 15 minutes, and then added to 5 ml of preheated cell culture medium.

[0127] 2) Flow cytometry

[0128] After electroporation, the control T cells and electroporated CAR-T cells were replaced with a medium every other day. After the medium was replaced, the cell density was adjusted to 1×10 6 During the culture process, 10 ng / ml recombinant IL-2 (Cat. No.: 200-02-1000, PeproTech) was added, and the cells were cultured in the well plate throughout the process. 1×10 cells were taken from each well plate 7 days after the completion of electroporation. 6 After washing once with PBS, add CD19-FITC (Cat. No.: CD9-HF251, Biopsy) for staining for 25 minutes, wash once with PBS, and finally resuspend the cells with PBS and analyze them on flow cytometer. The results are as follows: Figure 8 As shown, it can be seen from the flow cytometry results that both the silica matrix purification and the gel chromatography purification of the present invention can recombinantly transfer the template DNA to the target site, thereby expressing the CAR protein, and the gel chromatography group has a higher integration efficiency, suggesting that it can be used for CRISPR-Cas9-mediated site-specific gene knock-in.

Claims

1. A non-viral gene site-specific knock-in method, the method comprising: 1) The template DNA is connected to the plasmid vector; 2) PCR amplification of template DNA; 3) purifying the template double-stranded DNA by gel chromatography, wherein the gel chromatography method uses a filler for macromolecules for purification, The filler is Sepharose 6 Fast Flow, The particle size of the filler is 45-165 µm. The pH value of the packing for stability working or cleaning is 2-12. The eluent used in the gel chromatography method is a neutral PBS buffer, and Gel chromatography was followed by ultrafiltration concentration; 4) Transfecting the purified template double-stranded DNA and the gene editing material into the target cells by a gene editing method to knock in the gene at a specific site, wherein the gene editing method is CRISPR-Cas9, the template double-stranded DNA includes the CAR gene and the target cells are T cells, thereby integrating the CAR gene into the T cells at a specific site to prepare CAR-T cells.

2. The method according to claim 1, wherein the molecular weight of the double-stranded DNA is 10,000-4,000,000 Daltons.

3. The method of claim 1, wherein the molecular weight of the double-stranded DNA is 10,000-2,000,000 Daltons, 10,000-1,000,000 Daltons, 10,000-800,000 Daltons, 10,000-500,000 Daltons, 10,000-200,000 Daltons, 10,000-100,000 Daltons, 10,000-80,000 Daltons, 10,000-50,000 Daltons or 10,000-20,000 Daltons.

4. The method of claim 1, wherein the PBS buffer comprises Na2HPO4, KH2PO4, NaCl and KCl.

5. The method according to claim 1, wherein the pH of the PBS buffer is 6.5-7.

5. The method according to claim 1 , wherein the pH of the PBS buffer is 7.0-7.

5.

7. The method of claim 1, wherein the pH of the PBS buffer is 7.

4.

8. The method of claim 1, wherein the PBS buffer comprises 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 1.76 mM KH2PO4.

9. The method according to claim 1 or 2, wherein the gel chromatogram of the method has two absorption peaks, and the absorption peak that appears first is the absorption peak of double-stranded DNA.

10. The method according to claim 1 or 2, wherein the purified double-stranded DNA is used as a repair template for gene knock-in.

11. The method according to claim 1, wherein the gene editing substance comprises: Plasmids, mRNA, and proteins.

12. The method of claim 1, wherein the DNA targeted by gene knock-in is genomic DNA.

13. The method of claim 1, wherein the transfection method comprises: Liposomes, calcium phosphate, DEAE-dextran, electroporation, microinjection, and gene gun.

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