CN113969276A - Gel chromatography method for efficiently purifying large amount of PCR products and application thereof - Google Patents

Abstract

The invention relates to a method for efficiently purifying a large amount of PCR products and application thereof. In particular, the invention provides a method for purifying double-stranded DNA, wherein the purification is carried out by means of gel chromatography using a filler for macromolecules, in particular Sepharose6Fast Flow. The purified double-stranded DNA can be used as a repair template for gene knock-in, e.g., for site-directed integration of a CAR gene into a T cell to make a CAR-T cell. The method can purify PCR products in large batch, the purity of the purified double-stranded DNA is high, residual DNA polymerase, primers and dNTP in a PCR system can be effectively removed, the purification yield is high (can reach 80 percent), the operation is simple, the cost is low, particularly, eluent can be PBS buffer solution, water and other neutral solutions, and the subsequent application of purifying the double-stranded DNA is facilitated.

Description

Gel chromatography method for efficiently purifying large amount of PCR products and application thereof

Technical Field

The invention relates to the field of cell therapy, in particular to a gel chromatography method for efficiently purifying a large amount of PCR products and application thereof

Background

In recent years, with the continuous development of molecular biology, various innovative biotechnology is applied to the treatment or prevention of diseases, of which cell therapy is one of the hottest technologies. The present inventors have previously invented a novel non-viral site-directed knock-in CAR-T cell therapy technology that enables more efficient and precise transduction, simpler CAR-T cell production, safer and longer lasting expression. The technical process is extremely simple, but a large amount of high-purity double-stranded DNA is required to be used as a recombination template for knocking-in genes.

At present, most of methods for obtaining double-stranded DNA are methods using PCR technology. However, the PCR reaction product may contain a primer, a primer dimer, a DNA polymerase, a dNTP, and the like in addition to the target amplified fragment. These non-target substances have a great influence on gene knock-in and therefore need to be removed.

The principle of purification is to ensure the integrity of the primary structure of the double-stranded DNA molecule and to exclude contamination by other molecules. In order to purify the objective double-stranded DNA from the PCR product, many methods for purifying DNA have been developed. According to the nature of DNA such as solubility, adsorptivity and electric charge, the purification method can be divided into precipitation, density gradient centrifugation, electrophoresis, magnetic bead adsorption, membrane adsorption and chromatography:

(1) a precipitation method: the comparative methods are typically phenol chloroform extraction, CTAB (cetyltrimethylammonium bromide) precipitation, and the like. The method is simple and low in cost. The disadvantages are that the steps are complicated, the time consumption is long, and the use of organic reagents is not beneficial to environmental protection and subsequent application. The current use is not very widespread and there is a trend towards replacement.

(2) Electrophoresis method: the electrophoresis method separates target DNA fragments from non-target DNA fragments by an agarose gel electrophoresis technology, and combines a sol technology of gel containing the target DNA fragments to obtain high-purity target DNA. At present, many kits based on this principle are available, such as agarose gel DNA purification kit from Takara. By using the method, the recovered and purified DNA fragments have high purity and good integrity, the operation is more efficient, faster and more convenient, and the purified products are suitable for experiments such as ligation reaction, PCR amplification, DNA sequencing and the like. The method has the advantages of being suitable for micro-purification of target DNA and mature. The disadvantages are higher cost, low recovery rate and generally difficult to meet the requirement of large-scale purification of target products.

(3) Magnetic bead adsorption method: the magnetic bead method adopts nanometer magnetic bead microbeads (such as patent CN206345846), the surface of the magnetic bead microbeads is marked with a functional group which can perform adsorption reaction with DNA. The silicon Magnetic particles (Magnetic Silica particles) refer to Magnetic bead microbeads coated with a layer of silicon material to adsorb nucleic acids. The ion exchange magnetic beads are magnetic beads, wherein the surfaces of the magnetic beads are coated with a layer of material (such as diethylaminoethyl DEAE, COOH and the like) capable of generating ion exchange, so that the purpose of adsorbing nucleic acid is achieved. The purification principles corresponding to magnetic bead microbeads of different properties are not consistent. The greatest advantage of using the magnetic bead method to purify nucleic acid is automation, and magnetic beads can be aggregated or dispersed under the condition of a magnetic field, so that manual operation processes such as centrifugation can be thoroughly eliminated, large-scale equipment is not needed, and the method is relatively easy to implement. The disadvantages are that the process is complicated, the sample elution is not sufficient, the eluted DNA contains more impurities, the cost is higher, and the requirement of large-scale target products is generally difficult to meet.

(4) Silicon matrix adsorption techniques (e.g., QIAquick spin columns): the PCR product can be directly applied to a purification column matched with the kit based on the technical principle, and the target product is obtained through the steps of washing, eluting and the like. The method has the advantage of simple operation. The disadvantages are incomplete recovery, low recovery rate, high cost and difficult meeting the requirement of large-scale target products.

(5) The combination technology comprises the following steps: some techniques are combined with the above techniques, such as combining electrophoresis with magnetic beads, as disclosed in patent CN 104560954. Further, for example, electrophoresis is combined with membrane adsorption, although these techniques are also easier to implement, these methods have the disadvantages of low yield, high cost, etc., and thus it is still necessary to find a better method to replace these conventional methods.

(6) Chromatography: the purification of nucleic acids by means of high performance liquid chromatography systems is a different approach. High performance liquid chromatography is an important branch of chromatography, liquid is used as a mobile phase, a high-pressure infusion system is adopted, the mobile phases such as single solvents with different polarities or mixed solvents, buffer solutions and the like with different proportions are pumped into a chromatographic column filled with a fixed phase, and after components in the column are separated, the components enter a detector for detection, so that the analysis of a sample is realized. The method becomes an important separation and analysis technology in the subject fields of chemistry, medicine, industry, agriculture, commodity inspection, law inspection and the like.

In nucleic acid purification applications, anion exchange chromatography is the most used technique by scientists due to the physical characteristics of DNA itself, especially the presence of many charged phosphate groups in its backbone. For example, in 1991, W Warren, J Doniger et al reported that a 344bp high-quality target DNA fragment was obtained by gradient elution using an anion exchange chromatography column; 2004 Raquel Hernandez et al also reported that 277bp target fragments were purified using an anion exchange chromatography column with a recovery rate as high as 80%; even in 2008 Kim K et al tried to use this method for purification of nucleic acid samples such as ancient human bone. The method has the advantages of being capable of being completed within 30 minutes and being relatively efficient. The disadvantages are excessive reagent, relatively complicated operation and no verification of large-fragment DNA.

In addition, due to the electrostatic effect of nucleic acid itself and the interaction of ions, the nucleic acid hydrophobic properties are more outstanding, through hydrophobic chromatography technology can also be nucleic acid purification, especially for single-stranded DNA purification.

In recent years, it has been reported that a mixed chromatography technique is used for purification of not only DNA but also antibody and protein. For example, T Matos et al reported that single-stranded DNA was successfully purified from double-stranded DNA in 2015 using a mixed chromatography packing (capto adhere resin) from GE.

Although these existing chromatographic techniques for DNA purification have already played a role in the fields of gene cloning, gene editing, sequencing, viral DNA identification, etc., there is still a need for improvement: for example, the ion exchange chromatography method is relatively complex in operation, more in reagent preparation, and a beginner needs to grope a lot of experimental conditions, which wastes labor cost to a certain extent. The requirements for the filler in the mixed chromatography are high, and the cost is relatively high.

Disclosure of Invention

On the basis of the current research progress, the invention finds a more optimal method which is convenient to obtain materials, simple to operate, labor-saving and capable of efficiently purifying the target double-stranded DNA through continuous groping practice. The method relies on gel filtration chromatography, only uses a single neutral buffer solution and a chromatography filler which is not modified and can form gel cross-linked substances with certain pore diameters by utilizing the principle that the reticular structure of the gel filtration chromatography filler can be separated according to the molecular weight, and can complete the operation within 10 minutes, and the method has not been reported at present.

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

In some embodiments, the filler has a particle size of 45 to 165 μm.

In some embodiments, the pH at which the filler is worked or washed for stability is from 2 to 12.

In some embodiments, the filler is Sepharose6Fast Flow. The packing Sepharose6Fast Flow is from Situofen, Cat # 17015901 (Specification 1L) or 17015905 (Specification 10L).

In some embodiments, the molecular weight of the double-stranded DNA is 10000-; or for example, 300-.

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

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

In some embodiments, the PBS comprises Na₂HPO₄、KH₂PO₄NaCl and KCl. In a specific embodiment, PBS contains 137mM NaCl, 2.7mM KCl, 10mM Na₂HPO₄And 1.76mM KH₂PO₄。

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

In some embodiments, the gelchromatography profile of the method presents two absorption peaks, the first appearing absorption peak being that of double-stranded DNA. The absorption peaks appeared thereafter as residual DNA polymerase, primers and dNTPs from the PCT system.

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

In another aspect, the present invention provides a non-viral gene site-directed knock-in method, comprising:

1) ligation of template DNA to plasmid vector

2) PCR amplification template DNA

3) Purification of template double-stranded DNA

4) Transfecting the purified template double-stranded DNA and a gene editing substance into a target cell by a gene editing method to knock in a gene at a fixed point,

wherein the purification of the template double-stranded DNA is carried out using the above-mentioned method of the present invention.

In some embodiments, the gene editing methods used for gene typing include: CRISPR-Cas9, ZFN, ARCRS, TALEN and megaTAL, preferably CRISPR-Cas 9.

In some embodiments, the gene-editing materials employed by the gene-editing methods include: plasmids, mRNA and proteins.

In some embodiments, the DNA targeted for knock-in can be any DNA, such as genomic DNA.

In some embodiments, the gene-editing material is transferred into the cell along with the template DNA using the following transfection method: liposomes, calcium phosphate, DEAE-dextran, electroporation, microinjection and gene gun. After transfection, the gene editing nuclease cleaves the genomic DNA to nick it into a broken double-stranded DNA, at which time the purified template double-stranded DNA undergoes homologous recombination with the broken nicked DNA as donor DNA, and the knocked-in DNA is successfully integrated into the desired genomic site.

In some embodiments, the target cell may comprise: 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.

In some embodiments, wherein the template DNA comprises a CAR gene and the target cell is a T cell, site-specific integration of the CAR gene into the T cell to make the CAR-T cell.

In another aspect, the invention also provides the use of CAR-T cells for the preparation of a medicament for the treatment of leukemia (e.g., 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 (e.g., lung cancer, liver cancer, stomach cancer, breast cancer, colorectal cancer, prostate cancer, pancreatic cancer, brain glioma, esophageal cancer, cholangiocarcinoma, endometrial cancer, ovarian cancer, mesothelioma, and thymus cancer), aids, autoimmune diseases, and other CAR-T cell treatable diseases.

The invention has the beneficial technical effects that: (1) PCR products can be purified in large batch; (2) the purified double-stranded DNA has high purity, and residual DNA polymerase, primers and dNTP in a PCR system can be effectively removed; (3) the purification yield is high and can reach 80 percent; (4) the operation is simple, and the cost is low; (5) the eluent can be PBS buffer solution, water and other neutral solutions, which is beneficial to the subsequent application of purified double-stranded DNA.

Drawings

FIG. 1 is a schematic diagram of a gel chromatography purification process of the present invention: a. the bulk of the PCR reaction product comprises: target double-stranded DNA, a primer, DNA polymerase and dNTP; b. after the gel chromatography column is used for loading, the target double-stranded DNA is eluted first, and the primer, the DNA polymerase and the dNTP enter the interior of the gel particles, so that the elution time is later; c. the double-stranded DNA of interest is concentrated by ultrafiltration.

FIG. 2 is a schematic diagram of knocking-in the CAR gene into a target site in example 1.

FIG. 3 shows the results of gel chromatography in example 4.

FIG. 4 shows the results of the electrophoretic identification of DNA in example 4.

FIG. 5 is a chromatogram of the DNA polymerase, primers and dNTPs in example 5.

FIG. 6 shows the results of gel chromatography in example 6.

FIG. 7 shows the results of the electrophoretic identification of DNA in example 6.

Fig. 8 shows the streaming detection result of example 7.

Detailed Description

Although the gene knockout technology has been developed, the efficiency of gene knockout for large fragments has been low. Gene knock-in and gene knock-out differ by whether a repair template DNA is added. The process of gene knock-in is: after a nuclease (such as ZFN, TALEN, CRISPR-Cas9 and the like) cuts a specific DNA, a repair template repairs the damaged DNA according to the homologous recombination principle, and a specific DNA fragment is introduced into a specific site after repair. The repair templates currently used are mainly 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 by bulk PCR, which is much cheaper and more readily available than other repair templates. The invention provides a gel chromatography method for efficiently purifying a large amount of PCR products. The high-purity double-stranded DNA obtained by the method can be used for knocking in the CAR gene into a specific T cell genome site, so that the CAR-T cell is obtained.

FIG. 1 shows a specific embodiment of a gel chromatography method for purifying double-stranded DNA:

1) based on a reaction system commonly used in molecular biology, a large-batch PCR reaction mixed solution is prepared, subpackaging is carried out according to a micro reaction system, a PCR instrument is utilized to carry out amplification according to an optimized amplification program to obtain a large-batch PCR product, and the obtained PCR product contains a primer, DNA polymerase and dNTP besides target double-stranded DNA. The reagent used for PCR amplification can be a kit product of any company, and a PCR amplification instrument or related equipment used for PCR amplification can also be of any manufacturer.

2) The column was packed with commercial filler Sepharose6Fast Flow (beads 90 μm in diameter formed by highly cross-linking 6% agarose). And taking the PCR product as a sample to load, and using a non-toxic neutral buffer solution, preferably PBS, Tris-HCI and water, wherein the pH value is ensured to be 7.0-7.5. According to the principle of molecular sieve, target double-stranded DNA is preferentially eluted, and small molecular substances (DNA polymerase, primer and dNTP) in a PCR product enter the interior of gel particles due to small molecular weight, so that the elution time is later, and the target DNA can be fully separated from other small molecular substances;

3) the double-stranded DNA eluted first is concentrated in an ultrafiltration tube to increase the concentration of the target DNA for downstream use.

Examples

Example 1: molecular design of repair template DNA

In this example, the CAR gene knocks into the T cell TRAC gene site and a method of co-electrotransformation with modified repair template DNA using a CRISPR-Cas9 element was employed. Firstly, a CRISPR-Cas9 element is utilized to generate a nick on a TRAC gene locus, then the modified repair template DNA is utilized to carry out homologous recombination repair on the nick, and finally, the CAR gene is knocked into a target locus as shown in figure 2.

The repaired template DNA contains two parts:

a. left and right homology arms. The left and right homology arms are used to identify the DNA of interest and to perform recombination exchanges. In order to allow for the correct expression of the knock-in gene, the knock-in gene is "logged in" to the 5' end region of exon 1 of the TRAC gene. In this example, the left and right homology arms of 300 bases were designed, and the sequences are shown in SEQ ID NO 1-2, respectively.

b. A knock-in gene. The knocked-in genes included splicing peptide gene, CAR gene and polyA, depending on the TCR-alpha promoter. In this example, the cleavage peptide used was P2A, and the CAR gene used was a secondary structure, specifically comprising the CD8 signal peptide, targeting CD19 scFv, CD8 hinge region, CD8 transmembrane region, 4-1BB costimulatory region, and CD3 zeta activating region. In this example, the polyA was bGHpA polyA, and the structural sequences of the genes were as follows:

example 2: molecular cloning of repair template DNA

The repair template DNA of SEQ ID NO. 11 was synthesized and cloned into a pUC57 plasmid vector. The process comprises the following steps:

1) and (3) preparing a target fragment by PCR. Firstly, a PCR reaction system needs to be prepared:

reagent (cargo number: RO5OA, TAKARA)	Amount used (ul)
		5 XPrimeSTAR GXL buffer solution	10
dNTP Mix	4
		Primer F	1
Primer R	1
		DNA fragment	0.5
Sterilized water	32.5
		PrimeSTAR GXL DNA polymerase	1
Total volume	50

Then, the PCR reaction system is placed in a PCR amplification instrument for amplification according to the following conditions:

2) and (3) preparing a vector by PCR. Firstly, a PCR reaction system needs to be prepared:

reagent	Amount used (ul)
		5 XPrimeSTAR GXL buffer solution	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

Then, the PCR reaction system is placed in a PCR amplification instrument for amplification according to the following conditions:

3) and (3) recombining the target fragment with a vector: adding a target gene and a linearized vector into a centrifuge tube on an ice box according to a certain molar ratio for recombination reaction, and reacting for 10 minutes in a water bath kettle at 50 ℃, wherein the reaction system is shown in the following table:

reagent (cat # NR001A, near shore protein)	Amount used (ul)
		Vector pUC57	3.5
DNA fragment (SEQ ID NO:11)	4
		5 × reaction buffer	2
NovoRec Plus recombinase	0.5
		Total volume	10

4) And (3) conversion coating of reaction products: melting a tube of 100. mu.l StbI3 competent cells (Cathaya, DL1046, Withania) on ice, flicking the tube wall to resuspend the cells, adding 10. mu.l of the reaction solution to the competent cells, and ice-cooling for 30 min under flicking; quickly putting the mixture on ice for 5 minutes after heat shock is carried out for 90 seconds in a water bath kettle at 42 ℃; adding 500 mul LB liquid culture medium, and incubating for 45-60 minutes at 37 ℃ by a shaking table; the cells were collected by centrifugation at 5000rpm for 3 minutes, 300. mu.l of the supernatant was discarded, and the remaining cell amount was applied to a plate containing ampicillin and incubated overnight in an incubator at 37 ℃.

5) Single clones were picked and subjected to plasmid extraction (cat #: DP103-03, Tiangen Biochemical).

6) And (5) sequencing and confirming.

Example 3: and (3) performing large-batch PCR amplification on the repair template DNA.

The plasmid constructed by repairing template DNA is successful, and PCR amplification is facilitated. This example prepares a PCR reaction mixture as follows:

reagent	Amount used (ul)
		5 XPrimeSTAR GXL buffer solution	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

Subpackaging the mixture into 20 common PCR tubes, then placing the PCR reaction system into a PCR amplification instrument, and carrying out amplification according to the following conditions:

after the PCR was completed, the PCR products were collected and pooled in a 50ml centrifuge tube. In this case, the PCR product includes a double-stranded DNA product, dNTPs, DNA polymerase and primers, and reaction buffer. The sample was transferred to a 10ml syringe, and the syringe was emptied of air bubbles to prepare for loading.

Example 4: purifying template double-stranded DNA by gel chromatography.

After PCR amplification of template double-stranded DNA in large scale, it is purified. Adopts innovative gel chromatography and ultrafiltration concentration. Gel chromatography is used for removing residual DNA polymerase, primers and dNTP in PCR reaction and purifying the target double-stranded DNA. The purpose of ultrafiltration concentration is to concentrate the target double-stranded DNA to a certain concentration, which is convenient for subsequent gene knock-in.

The first step is as follows: the gel chromatography procedure was as follows:

1) and (3) opening the AKTA pure 150, simultaneously opening the coordination software UNICORE 7.1 of the computer, waiting for the self-checking and communication connection of the machine, and indicating that the flash lamp of the AKTA system can be normally used when the flash lamp of the AKTA system does not flash.

2) Cleaning a system A/B pump: the inlet tube was passed through 20% ethanol, water in turn and the pump head was cleaned at a high flow rate of 75 ml/min.

3) Cleaning a pipeline: the "system control" control software window set flow rate at 5ml/min, pre column pressure set at 0.5Mpa maximum, UV detection select 260 run, AKTA perform the above command, wash line with water to conductivity 0 mS/cm.

4) Installing a chromatographic column: the column was GE XK-16/10, and Sepharose6Fast Flow was used as the packing, and the column volume was about 50 ml. The gel chromatography column was installed into the AKTA pure 150 system according to the column packing procedure as follows: when in connection, the connection is carried out in a liquid drop-to-liquid drop mode, so that bubbles in the section of pipeline are avoided. When in operation, the PEEK pipe (the other end is connected with the port of the column position valve) at the port position above the original UV detector of the system is connected with the lower end of the column without being screwed, then a connecting pipe at the lower end of the chromatographic column is disconnected after liquid drops appear above the connecting pipe, the connecting pipe above the UV detector is connected with the port above the chromatographic column, and then another section of PEEK pipe is connected with the lower end of the chromatographic column and the upper port of the UV detector of the AKTA system. After the column is filled, the chromatographic column is washed by deionized water until the electric conductivity is stabilized to 0 mS/cm.

5) And (3) balancing loading buffer solution: the pump head was switched to neutral buffer PBS until the conductivity stabilized (PBS buffer wash to smooth conductance, approximately 16.2 mS/cm). Wherein the composition of the neutral buffer PBS is 137mM NaCl, 2.7mM KCl and 10mM Na₂HPO4 and 1.76mM KH₂PO₄The pH was 7.4.

6) Preparation before sample loading: and (3) cleaning the loading ring, sequentially sucking water and loading buffer solution by using a 50ml syringe, and manually pushing the sample into the cleaning loading ring, wherein the washing volume is more than 5 times of the column volume. The sample is prepared, and before the sample is loaded, ultraviolet zero setting (the UV absorption value is zero at the positive 260 nm) is carried out, and the speed is adjusted to be 2 ml/min.

7) Loading: ensure that the syringe into which the sample was injected is free of air bubbles, then connect the 10ml syringe into which the sample was injected to the AKTA device column position valve port, and manually push the sample into the 5ml to 5ml loading ring.

8) Collecting: the conversion system proceeds to Injection mode. About 11ml after the start of sample loading begins to appear a first 260nm absorption peak-1 (fig. 3), at the time of peak starting, the sample collection valve is switched to an Outlet mode, when the peak is attenuated to about 15 of absorption value, the collection of the sample is ended, and simultaneously, the switching valve is switched to a waste mode, and the sample output of the peak output period is collected. About 40ml after loading, a second 260nm absorption peak-2 (FIG. 3) appears, and the liquid in this peak period is collected, namely the residual DNA polymerase, dNTP, primer and the like in the PCR system. The loading can be repeated once according to the above loading procedure (6-7). After all samples were loaded, all the first peak samples collected were pooled together for further concentration.

The purification effect of gel chromatography is identified by DNA electrophoresis: in order to identify which of the absorption peak-1 and the absorption peak-2 contained the target DNA, it was subjected to DNA electrophoresis. First, 1% agarose gel was prepared, and then loaded: 1. mu.l of PCR product, 10. mu.l of absorption peak-1 product, and 10. mu.l of absorption peak-2 product. After that, electrophoresis was carried out at 220V, and after completion, UV-irradiation was carried out, and the results are shown in FIG. 4. As can be seen, the PCR product and the lane of the absorption peak-1 have bands at 2300bp, which are relatively consistent with the expected fragment size; while the position corresponding to lane-2 of the absorption peak has no band. The absorption peak-1 is indicated as the peak of the fragment of interest. In addition, about 40ml of absorption peak-1 was obtained from 5ml of the sample loaded, and the DNA band brightness of 1. mu.l of PCR product loaded was almost identical to 10. mu.l of absorption peak-1 product, from which the yield of this purification method was calculated to be about 80%.

The second step is that: the steps of ultrafiltration and concentration are as follows:

1) ultrafiltration concentration was performed using a 30KDa ultrafiltration tube. The ultrafiltration tube was rinsed with ultrapure water and then centrifuged at 4000g for 5 minutes, the water centrifuged at the bottom being decanted off. The first off-peak sample is then added and centrifuged at 4000g for 5 minutes, typically with increasing number of centrifugations, the DNA retained on the membrane increasing, the ultrafiltration time also increasing as appropriate, the volume of the liquid surface in the ultrafiltration tube is observed, about 500. mu.l of the liquid surface is washed once with 15ml PBS buffer and centrifuged again at the same speed until the concentration is reached in a volume of 500. mu.l.

2) In order to fully extend and dissolve the DNA on the membrane in the solvent, the membrane is left to stand at 4 ℃ for about 12 hours, then a 200-microliter pipette is used to suck the sterile pipette tip to operate in a biological safety cabinet, the side surface of the ultrafiltration tube is carefully blown and punched for 2-3 times, then the whole membrane is transferred to a 1.5-milliliter EP tube, and then a 0.2-micrometer filter membrane is used for filtration and sterilization.

3) After the purified target double-stranded DNA is subjected to ultrafiltration concentration and filtration, the concentration of the purified target double-stranded DNA is detected to be 2.2 mu g/ml by an ultraviolet spectrophotometer.

Example 5: and respectively verifying the peak positions of the DNA polymerase, the primer and the dNTP.

A small amount of each of the DNA polymerase, the primer and the dNTP used in example 3 was taken from a commercial tube, and the sample was prepared by adding water to 500. mu.l, and subjected to chromatography using PBS (same as in example 4) as an eluent to record the peak position. Samples of the components of the PCR system were prepared as follows:

reagent	Original concentration	Amount used (ul)	Water (ul)
				DNA polymerase	1.25u/μl	20	480
Primer and method for producing the same	10μM		100					490
				dNTP	2.5mM	50	450

Put into different EP tubes and then aspirate separately with 3 1ml syringes in sequence and expel air bubbles for loading.

The gel chromatography procedure was as follows:

steps 1) -6) correspond to example 4.

7) And (3) primer loading: the PCR primer sample was first loaded to ensure that there were no air bubbles in the 1ml syringe into which the sample was injected, and then the 1ml syringe into which 500. mu.l of the sample was injected was connected to the AKTA instrument column position valve port and manually pushed into the sample 500. mu.l to 1ml loading ring. The peak condition was observed: the conversion system proceeds to Injection mode. At least 30ml eluted after the start of the loading, and the peak positions were observed. The first peak decays completely to the lowest (see results in fig. 5). And switching the loading mode to a manual mode, pulling out the primer sample injector at the loading valve position, and washing the loading ring clean by using loading buffer.

8) dNTP sample loading and observation: the syringe containing the dNTP sample was inserted into the loading valve position, 500. mu.l of the same volume of dNTP sample was pushed into the loading loop, and then the loading mode was adjusted to Injection, machine-automated sampling, elution at 30ml, and peak placement was observed (see results in FIG. 5). Etc. the first peak to appear decays completely to the lowest. And switching the loading mode to a manual mode, pulling out the primer sample injector at the loading valve position, and washing the loading ring clean by using loading buffer.

9) Loading of DNA polymerase samples: the syringe containing the DNA polymerase sample was inserted into the loading valve position, 500. mu.l of the DNA polymerase sample was pushed into the loading loop, and the loading mode was adjusted to Injection. The machine was automatically injected and eluted at 30ml and peak disposition was observed (see results in FIG. 5). Etc. the first peak to appear decays completely to the lowest. And switching the loading mode to a manual mode, pulling out the primer sample injector at the loading valve position, and washing the loading ring clean by using loading buffer.

FIG. 5 is a chromatogram of DNA polymerase, primers and dNTPs being loaded. In comparison with example 4, the peak positions of the samples such as DNA polymerase, primer and dNTP all correspond to the absorption peak-2 position (i.e., the position where the conductivity is significantly decreased) in example 4. This indicates that the peak-appearing substance in absorption peak-2 is mainly a PCR reaction component and does 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 target double-stranded DNA which is specifically amplified from the PCR product.

Example 6: water was used as eluent instead of PBS buffer.

Water acts as a neutral solution, often as a solvent for dissolving DNA. This example uses water as an eluent instead of PBS buffer for the key gel chromatography process of the present invention. The specific implementation steps are as follows:

the gel chromatography procedure was as follows:

1) and opening the AKTA pure 150 and simultaneously opening the coordination software UNICORE 7.1 of the computer. Waiting for machine self-checking and communication connection, and waiting for the flash lamp of the AKTA system not to flash, indicating normal use.

2) Cleaning a system A/B pump: the pump head was cleaned by passing the inlet tube through 20% ethanol, water in sequence and at a high flow rate of 75 ml/min.

3) Cleaning a pipeline: the "system control" control software window set the flow rate at 5ml/min, the maximum pre-column pressure at 0.5MPa, UV detection selection 260 detection. AKTA executes the above command, and the line is cleaned with water to a conductivity of 0 mS/cm.

4) Installing a chromatographic column: the column was GE XK-16/10, and Sepharose6Fast Flow was used as the packing, and the column volume was about 50 ml. The gel chromatography column was installed into the AKTA pure 150 system according to the column packing procedure. The method comprises the following specific steps: when in connection, the connection is carried out in a liquid drop-to-liquid drop mode, so that bubbles in the section of pipeline are avoided. When in operation, the PEEK pipe (the other end is connected with the port of the column position valve) at the port position above the original UV detector of the system is connected with the lower end of the column without being screwed, then a connecting pipe at the lower end of the chromatographic column is disconnected after liquid drops appear above the connecting pipe, the connecting pipe above the UV detector is connected with the port above the chromatographic column, and then another section of PEEK pipe is connected with the lower end of the chromatographic column and the upper port of the UV detector of the AKTA system. After the column is filled, the chromatographic column is washed by deionized water until the electric conductivity is stabilized to 0 mS/cm.

5) Preparation before sample loading: and (3) cleaning the loading ring, sequentially sucking water and loading buffer solution by using a 50ml syringe, and manually pushing the sample into the cleaning loading ring, wherein the washing volume is more than 5 times of the column volume. And (4) preparing for loading. Before loading, ultraviolet zero setting (correcting UV absorption value at 260nm to be zero) is carried out, and the speed is adjusted to be 2 ml/min.

6) Loading: ensure that there are no air bubbles in the 1ml syringe that is injected with the sample, then connect the 1ml syringe that is injected with 500. mu.l of sample to the AKTA instrument column position valve port, and manually push 500. mu.l of sample into the loading ring.

7) The peak condition was observed: the conversion system proceeds to Injection mode. The first 260nm absorption peak 1 is eluted 15ml after the sample loading is started, the sample collection valve is switched to an Outlet mode at the time of the peak starting, the sample collection is finished when the peak is attenuated to the lowest absorption, and meanwhile, the switching valve is switched to a waste mode, and the sample collection in the peak discharging period is carried out. About 40ml after loading, a second 260nm absorption peak 2 appeared and the liquid was collected during this peak period. Fig. 6 shows the column chromatography results of gel chromatography. From FIG. 6, it can be seen that two peaks, absorption peak 1 and absorption peak 2, were observed after the PCR product was loaded using water as the eluent. The position of the peak of absorption peak 1 was closer to that of example 2, but the conductivity of absorption peak 2 was different from that of the second peak of the sample loaded with PBS buffer (slightly decreased conductivity) and slightly increased. Secondly, the two peaks of absorption peak 1 and absorption peak 2 are clearly separated, and the middle baseline is close to the baseline eluted by PBS buffer. From the comparison of the chromatogram, it is shown that the two peaks can also be separated using water.

The purification effect of gel chromatography is identified by DNA electrophoresis: in order to identify which of the absorption peak-1 and the absorption peak-2 contained the target DNA, it was subjected to DNA electrophoresis. First, 1% agarose gel was prepared, and then loaded: 1. mu.l of PCR product, 10. mu.l of absorption peak-1 product, and 10. mu.l of absorption peak-2 product. After that, electrophoresis was carried out at 220V, and after completion, UV-irradiation was carried out, and the results are shown in FIG. 7. As can be seen, the PCR product and the lane of the absorption peak-1 have bands at 2300bp, which are relatively consistent with the expected fragment size; while the position corresponding to lane-2 of the absorption peak has no band. The absorption peak-1 is indicated as the peak of the fragment of interest. This result indicates that the desired amplified fragment can also be isolated from the PCR product using water as an eluent.

Example 7: purified double-stranded DNA of interest for the preparation of non-viral site-directed knock-in CAR-T cells

The double-stranded DNA of interest purified by gel chromatography in example 4 and the double-stranded DNA purified by a large-scale DNA product purification kit (cat # DP205) from Tiangen Biochemical technology Ltd (silica matrix adsorption technique, comparative example) were used as repair templates to mediate site-specific integration of the CAR gene cleaved by CRISPR-Cas9, respectively. The method comprises the following specific steps:

1) cell preparation

PBMC extraction: healthy volunteers were recruited, who did not have symptoms of cold and fever, and 100ml of blood was drawn from the median vein of the elbow by medical professionals and connected to an anticoagulation vessel (cat No. 367886, Becton Dickinson); after the blood collection, the blood was mixed with an equal amount of PBS buffer (containing 2% fetal bovine serum (cat # SE100-B, Vistech)); taking a PBMC separation tube Sepmate-50 (cat # 86450, STEMCELL), carefully adding 15ml of Ficoll buffer solution, then adding the mixed solution of blood PBS, and carefully adding about 30ml of the mixed solution into each tube; centrifuging 1200g for 10 minutes, quickly pouring the supernatant into a new 50ml tube, centrifuging 200g for 8 minutes, discarding the supernatant, adding 10ml PBS buffer solution for resuspension and precipitation, discarding the supernatant, adding 10ml PBS buffer solution for resuspension, centrifuging and discarding the supernatant, and then resuspending the cell precipitation by 10ml PBS buffer solution; the resuspended cells were counted, 10. mu.l of the suspension was added to 10. mu.l of 0.1% trypan blue (cat # 15250061, Gibco) and mixed, and the cell count and viability were counted on the machine.

T cell purification: taking a small amount of PBMC cells obtained, and calculating the proportion of CD3 positive T by a flow cytometer; the required PBMC cells are taken out according to the proportion of CD3 positive cells required by the experiment, and the required cells are 70% of the taken cells (which are lost in the operation process); sorting was performed using a kit Release Human CD3 Positive Selection Cocktail (CatCELL, cat: 17751), and total PBMC cells were first resuspended in Positive Selection buffer to a total cell concentration of 1X 10⁸Per ml; each 1 × 10⁸Cells were added with 100 μ l of CD3 antibody; incubation for 3 minutes at room temperature; the magnetic beads (reusable Rapid Sphenes) were mixed together in advance by a vortex mixer for 30 seconds at 1X 10 intervals⁸Cells were added to 100. mu.l of magnetic beads and incubated at room temperature for 3 minutes; transferring the cell suspension into a special sorting tube, metering the volume to 2.5ml by using positive sorting buffer solution, and placing the cell suspension on a magnetic frame for incubation for 5 minutes at room temperature; carefully grasping the magnetic frame, dumping the sorting tube, discarding the unbound cell suspension, re-suspending the attached magnetic beads and cells with 2.5ml of positive selection buffer solution, placing on the magnetic frame again, incubating at room temperature for 3 minutes, and discarding the unbound cell suspension again; taking down the sorting tube from the magnetic frame, adding a 'Release buffer solution' into the suspension, and incubating for 3 minutes at room temperature; placing on a magnetic frame and incubating for 5 minutes at room temperature; carefully grasping the magnetic frame, pouring the sorting tube, and collecting the unbound cell suspension in a clean sterile 15ml centrifuge tube, wherein the cell is the extracted T cell.

Activation of T cells: taking the purified T cells 1X 10⁷Then, anti-CD 3/anti-CD 28 magnetic beads (cat # 402)03D, Thermo) activation: 3X 10 magnetic beads of anti-CD 3/anti-CD 28 were used⁷Resuspend with PBS buffer (containing 2mM EDTA and 1% fetal calf serum), add to the magnetic pole, stand for 2 minutes, and carefully discard the supernatant; repeating the above process; taking the washed magnetic beads, adding the magnetic beads into T cells, uniformly mixing, and culturing for two days at 37 ℃; taking out the magnetic beads after two days, firstly re-suspending the T cells for multiple times by using a pipette, then placing the cell suspension in a magnetic pole, standing for two minutes, and then removing the magnetic beads on the tube wall; cell number and survival rate were measured on the machine.

Electric conversion: each taking 3X 10⁶Placing the cell suspension in a centrifuge tube (2 tubes in total) for centrifugation at the rotation speed of 200g for 5 minutes, and completely removing the culture medium for later use after centrifugation; a Lonza electrotransfer buffer (cat # V4XP-3024, Lonza) was prepared, 15. mu.g of spCas9 protein (cat # A36499, Thermo, SEQ ID NO:12) and 1. mu.g of synthetic sgRNA-0001(SEQ ID NO:13) were added thereto, and after mixing at room temperature for 10 minutes, 6. mu.g of silica matrix-purified and gel chromatography-purified repair template DNA of SEQ ID NO:11 were added thereto, respectively. After 10 minutes, the cell pellet was mixed well and added to an electric rotor (cat # V4XP-3024, Lonza), and EH115 was programmed to shock, and 400. mu.l of the medium was added immediately after completion of the shock and left in a 37 ℃ incubator for 15 minutes, and then added to 5ml of the preheated cell medium.

2) Flow assay

After the completion of electrotransformation, the control T cells and the electrotransformed CAR-T cells were incubated once every 1 day, and the cell density was adjusted to 1X 10 after incubation⁶Each ml of the culture medium was supplemented with 10ng/ml of recombinant IL-2 (cat # 200-02-1000, PeproTech), and the cells were cultured in well plates throughout the culture. Cells were harvested at 1X 10 days after completion of electroporation⁶After one time of PBS washing, CD19-FITC (cat # CD9-HF251, Poppsies) is added for staining for 25 minutes, PBS is washed again, and finally the cells are re-suspended by PBS and analyzed by an up-flow cytometer, and the result is shown in FIG. 8, and it can be seen from the flow result that template DNA can be recombined to the target site to express CAR protein no matter silica matrix purification or gel chromatography purification of the invention, and the gel chromatography group integration efficiency is higher, which indicates that the method can be used for CRISPR-Cas9 mediated gene site-directed knock-in.

Claims (15)

1. A method for purifying double-stranded DNA, wherein purification is carried out by a gel chromatography method using a filler for macromolecules,

preferably, the filler has a particle size of 45 to 165 μm, and

preferably, the pH of the stable working or washing of the filler is 2-12.

2. The method of claim 1, wherein the filler is Sepharose6Fast Flow.

3. The method of claim 1 or 2, wherein the molecular weight of the double-stranded DNA is 10000-.

4. The method according to claim 1 or 2, wherein the eluent used in the gel chromatography method is a neutral buffer, preferably PBS buffer, Tris-HCI buffer and water, more preferably PBS buffer and water.

5. The method of claim 4, wherein the PBS buffer comprises Na₂HPO₄、KH₂PO₄The concentration of NaCl and KCl,

preferably, the pH of the PBS buffer is 6.5-7.5, preferably 7.0-7.5, more preferably 7.4,

preferably, the PBS buffer comprises 137mM NaCl, 2.7mM KCl, 10mM Na₂HPO₄And 1.76mM KH₂PO₄。

6. The method according to claim 1 or 2, wherein the method further comprises concentration after gel chromatography, preferably ultrafiltration concentration.

7. The method according to claim 1 or 2, wherein the gel chromatogram of the method presents two absorption peaks, the first appearing absorption peak being the absorption peak of double-stranded DNA.

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

9. A method of non-viral gene site-directed knock-in, the method comprising:

1) connecting the template DNA to a plasmid vector;

2) amplifying template DNA by PCR;

3) purifying the template double-stranded DNA by the gel chromatography method according to any one of claims 1 to 8;

4) the purified template double-stranded DNA and the gene-editing substance are transfected into the target cell by the gene-editing method to knock in the gene at a site.

10. The method of gene site-directed typing according to claim 9, wherein the gene editing method comprises: CRISPR-Cas9, ZFN, ARCRS, TALEN and megaTAL, preferably CRISPR-Cas 9.

11. The method of gene site-directed knock-in according to claim 9, wherein the gene-editing material comprises: plasmids, mRNA and proteins.

12. The method of gene site-directed knock-in according to claim 9, wherein the DNA targeted for knock-in is genomic DNA.

13. The method of gene site-directed knock-in according to claim 9, wherein the transfection method comprises: liposomes, calcium phosphate, DEAE-dextran, electroporation, microinjection and gene gun.

14. The method of claim 9, wherein the target cell comprises: 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.

15. The gene site-directed knock-in method of claim 9, wherein the template DNA comprises a CAR gene and the target cell is a T cell, whereby the CAR gene is site-directed integrated into the T cell to make a CAR-T cell.

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