CN111944848A

CN111944848A - Method for improving gene site-specific integration into cell based on electrotransformation technology and CRISPR-Cas9 technology

Info

Publication number: CN111944848A
Application number: CN201910413839.0A
Authority: CN
Inventors: 王甜
Original assignee: Hangzhou Puketing Biomedical Co ltd
Current assignee: Hangzhou Puketing Biomedical Co ltd
Priority date: 2019-05-17
Filing date: 2019-05-17
Publication date: 2020-11-17

Abstract

The invention discloses a method for improving fixed-point integration of genes into cells based on an electrotransfer technology and a CRISPR-Cas9 technology, which comprises the following steps: aiming at a certain specific tumor antigen, a target gene is designed and synthesized, and homologous arms are synthesized on two sides of the target gene; constructing the synthesized target gene and the homologous arm on an adeno-associated virus vector to produce viruses; synthesizing guide RNA for recognizing the insertion site, and expressing and purifying the Cas9 protein; after the guide RNA and the Cas9 protein are combined in vitro, the mixture is mixed with cells, and is subjected to electrotransformation, and adeno-associated virus with a target gene is added to detect the expression condition of the target gene and the functions of the cells. The invention is a method for improving the fixed-point integration of genes into cells based on an electrotransformation technology and a CRISPR-Cas9 technology, and can realize a high-efficiency fixed-point insertion effect by taking adeno-associated viruses as a vector for inserting the genes.

Description

Method for improving gene site-specific integration into cell based on electrotransformation technology and CRISPR-Cas9 technology

Technical Field

The invention relates to site-specific insertion of a gene, in particular to a method for improving site-specific integration of the gene into cells by combining an electrotransfer technology with a CRISPR-Cas9 technology, which is used for treating leukemia.

Background

The current method for integrating genes into cells is mainly realized by traditional lentiviruses and retroviruses, but the insertion of the method is random insertion, and if the aim of treating diseases by inserting exogenous genes into cells at a fixed point can be achieved, the method can play a greater role clinically. The CRISPR-Cas9 system has made great success in achieving gene knockout, but the site-directed insertion ratio for genes is still low, whether in a stable cell line or a primary cell.

If a target gene can be inserted into a cell at a fixed point by modifying a CRISPR-Cas9 technology, so that the fixed-point integration of the target gene on the cell is realized, and especially if a Chimeric Antigen Receptor (CAR) for recognizing a tumor antigen can be integrated into a T cell, the effectiveness and safety of CAR-T therapy can be greatly improved.

Disclosure of Invention

The invention aims to provide a method for improving the site-specific integration of a gene into a cell based on an electrotransformation technology and a CRISPR-Cas9 technology, so as to solve the defects of the prior art.

The invention adopts the following technical scheme:

a method for improving the site-specific integration of a gene into a cell based on an electrotransformation technology and a CRISPR-Cas9 technology comprises the following steps:

step 1, aiming at a certain specific tumor antigen, designing and synthesizing a target gene, and synthesizing homology arms at two sides of the target gene;

step 2, constructing the target gene and the homology arm synthesized in the step 1 on an adeno-associated virus vector, and producing viruses;

step 3, synthesizing guide RNA for identifying the insertion site, and expressing and purifying Cas9 protein;

step 4, combining the guide RNA and the Cas9 protein in the step 3 in vitro, mixing with cells, and performing electric transfer;

step 5, after cell electrotransformation, adding the adeno-associated virus with the target gene into the electrotransformed cell so as to integrate the target gene into the cell at a fixed point;

and 6, detecting the expression condition of the target gene and the function of the cell.

Further, the target gene in step 1 is a CAR gene, the insertion site in step 3 is a TCR site, and the cell in step 4 is a human primary T cell.

Further, the size of the homology arm in step 1 is 300-800 bp.

Further, in step 4 guide RNA and Cas9 protein were bound at room temperature or 37 ℃ for 10-20min in vitro.

Further, in step 4, the CeletrixTM electrotransfer system is used for electrotransfer.

Further, step 6 adopts flow cytometry to detect the expression of the target gene and detect the function of the cell.

The invention has the beneficial effects that:

the invention is a method for improving the fixed-point integration of genes into cells based on an electrotransformation technology and a CRISPR-Cas9 technology, and can realize a high-efficiency fixed-point insertion effect by taking adeno-associated viruses as a vector for inserting the genes.

The invention can realize the site-specific integration of the target gene CAR to the TCR of the human primary T cell, greatly improves the site-specific insertion ratio of CAR-T cells, and has better effect than the traditional method which relies on lentivirus and retrovirus to insert the CAR gene.

Drawings

FIG. 1 is a schematic diagram of the CAR gene structure of example 1.

FIG. 2 is a schematic diagram of reprogramming of the TCR α gene in example 1.

Fig. 3 is a vector schematic of Cas9 of example 1.

Fig. 4 is a diagram of Cas9 protein purification of example 1.

Figure 5 is a graph of flow analysis of CAR gene expression in primary T cells after modification in example 1.

Fig. 6 shows the proliferation of engineered primary T cells encountering tumor antigens.

Fig. 7 is a killing ability test of engineered primary T cells encountering tumor antigens.

Detailed Description

The invention is explained in more detail below with reference to exemplary embodiments and the accompanying drawings. The following examples are provided only for illustrating the present invention and are not intended to limit the scope of the present invention.

step 1, designing and synthesizing a CAR gene aiming at a specific tumor antigen, and synthesizing homology arms at two sides of the CAR gene, wherein the size of the homology arms is 300-800 bp; the size of the homology arm is not limited to 300-800bp, and the homology arms with other lengths can be used;

step 2, constructing the CAR gene and the homology arm synthesized in the step 1 on an adeno-associated virus vector, and producing viruses;

step 3, synthesizing and identifying guide RNA inserted into the TCR locus, and expressing and purifying Cas9 protein;

step 4, combining the guide RNA and the Cas9 protein in the step 3 in vitro at room temperature or 37 ℃ for 10-20min, mixing with human primary T cells, and performing electrotransformation by adopting a CeletrixTM electrotransfer system;

step 5, after cell electrotransformation, adding the adeno-associated virus with the CAR gene into the electrotransformed cells so as to integrate the target gene into the cells in a fixed point manner;

and 6, detecting the expression condition of the CAR gene by adopting flow cytometry, and detecting the function of the cell.

Example 1

Synthesis of CAR Gene and homology arm Gene

Taking a target CD19-CAR for treating leukemia, which is clinically applied at present, as an example, the target CD19-CAR is constructed in an adeno-associated virus (AAV) vector, and the amino acid sequence of a CAR gene is shown as SEQ ID NO: 1, the structural schematic diagram is shown in figure 1, and mainly comprises an ScFv recognizing a specific tumor antigen CD19, an extracellular CD28 transmembrane region, a costimulatory signal CD28 and an activation signal CD3 zeta. The left homologous arm gene sequence is shown as SEQ ID NO: 2, the right homologous arm gene sequence is shown as SEQ ID NO: 3, respectively. Synthesizing CAR gene and homologous arm gene fragment by PCR, and synthesizing forward primer by PCR as shown in SEQ ID NO: 4, the reverse primer is shown as SEQ ID NO: 5, respectively.

Secondly, the CAR gene and the homologous arm gene are constructed on an adeno-associated virus vector and produce viruses

After the adeno-associated virus vector was digested with MluI-HF and RsrII, the synthesized CAR gene and the homologous arm gene fragment were ligated to the adeno-associated virus vector by T4 ligase to produce virus (Shandong Wei Zhen Biotech Co., Ltd.).

Thirdly, synthesizing and recognizing guide RNA inserted into TCR locus and expressing and purifying Cas9 protein

1. The site used in this example for knocking out the TCR α gene was the TRAC site, and reprogramming of the TRAC gene with the CRISPR-Cas9 system is shown in figure 2. The TRAC guide RNA sequence is shown as SEQ ID NO: and 6.

2. The Cas9 protein used in this example is mainly divided into the following two steps:

(1) constructing a Cas9 expression vector, expressing Cas9 protein by using an escherichia coli expression vector system, amplifying the Cas9 gene from a PX330 vector through PCR, wherein the sequence of a forward primer is shown as SEQ ID NO: 7, the reverse primer sequence is shown as SEQ ID NO: 8, then constructed into KS expression vector, and linked with T4DNA ligase, the cleavage sites are BamHI and NotI, and the vector schematic is shown in FIG. 3. The amino acid sequence of the Cas9 protein is shown in SEQ ID NO: shown at 9.

(2) Purifying Cas9 protein, purifying protein by using a GE protein purification system, amplifying Escherichia coli of a Cas9 vector, splitting bacterial liquid, binding Cas9 protein by using GST label, then cutting the GST label by using GST enzyme, and passing through a Heparin HP affinity column and Superdex^TMAfter 200 Increate column, Cas9 protein with higher purity is obtained, as shown in figure 4; FIG. 4 shows the electrophoresis of Cas9 protein on the left side after Heparin HP affinity column, Cas9 protein size is 180KD, and Superdex on the right side^TMThe narrower the peak, the higher the purity of the protein, and the height of the first peak representing the yield of Cas9 protein.

Fourthly, after the guide RNA and the Cas9 protein are combined in vitro, the mixture is mixed with human primary T cells and is electrically transferred, the adeno-associated virus with the target gene is added into the electrically transferred cells, so as to integrate the CAR gene into the cells in a fixed point manner

1. Acquisition of human Primary T cells

Separating peripheral blood mononuclear cells from peripheral blood, extracting 10ml of human venous blood, diluting the venous blood by PBS for one time, adding 20ml of ficoll lymphocyte separation liquid to separate the peripheral blood mononuclear cells, culturing the cells by a 1640 culture medium, adding 10% fetal calf serum, 1% streptomycin mixed solution (P/S) and 200U/ml IL2 into the 1640 culture medium, stimulating the proliferation of the peripheral blood mononuclear cells by a CD3/CD28 antibody, wherein the concentration of CD3 and CD28 is 1ug/ml, the peripheral blood mononuclear cells are paved in a 6-well plate one day in advance, and the cell clustering effect is good after 72 hours of stimulation, wherein more than 70% of cells are CD3 positive cells.

2. Electrotransformation guide RNA and Cas9 protein

Collecting the human primary T cells cultured in the step 1, washing the human primary T cells for 1-2 times by using a transfer fluid or a serum-free 1640 (primary culture) culture medium after centrifugal precipitation, and finally suspending the cells into the 1640 culture medium. In vitro, 30pmol of guide RNA and 30pmol of Cas9 protein are combined, placed at room temperature or 37 ℃ for 10min to form an RNP complex, mixed with human primary T cells, and subjected to electrotransfer by using a Celetrix (TM) electrotransfer system, wherein the electrotransfer system is 20ul, the electrotransfer cells are 0.5-1million, the electrotransfer voltage is 620v, and the pulse time is 20 ms. After electrotransformation, the T cells are continuously cultured in 1640 culture medium + 10% fetal calf serum and 1% P/S overnight, then added with 200U/ml IL2 for continuous culture, and electrotransformed for 24h, and added with the adeno-associated virus with CAR gene.

And fifthly, detecting the expression condition of the target gene and the function of the cell by adopting flow cytometry.

1. After 7 days of electroporation cell culture, the TCR knockout and CAR gene insertion ratios were measured by flow, KO of TCR by CD3 and site-directed insertion ratio of CAR gene into cells by coat IgG. As shown in figure 5, the ordinate represents the expression of CD3 on T cells and the abscissa represents the expression of CAR. Normally, peripheral blood mononuclear cells stimulated by CD3/CD28 magnetic beads became T cells expressing CD3, and fig. 5 shows that approximately 90% of the TCR was knocked out, while 60% of the cells expressed the CAR gene in the case of TCR knocked out.

2. Proliferation capacity of T cells infected by CRISPR-Cas9-AAV to encounter tumor antigens in vitro was examined.

3T3-CD19 cells were plated in 24-well plates, and after overnight culture in DMEM medium, CAR-T cells were adjusted to 1million/ml, after one week of co-culture, 3T3-CD19 cells were continued to stimulate proliferation of CAR-T cells, and after one week of stimulation, proliferation of CAR-T cells was examined, as shown in FIG. 6.

3. Killing ability of T cells infected by CRISPR-Cas9-AAV when co-cultured with the target leukemia tumor cell line NALM 6-GFP-Luciferase.

NALM6-GFP-Luciferase tumor cells and CAR-T cells were placed in cell culture plates as described by Effect: adding the Target in a ratio of 2:1, 1:1, 1:2, 1:4, 1:8, 1:16 and 1:32, co-culturing for 18 hours, adding a fluorescein substrate, measuring the luminescence of the fluorescein by using an enzyme-labeling instrument, and calculating the killing capacity of the CAR-T cells according to the luminescence of the fluorescein. As shown in figure 7, the ordinate represents tumor cell lysis and the abscissa represents the ratio of CAR-T cells to tumor cells.

Sequence listing

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Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr

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Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro

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Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe

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Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp

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Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile

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Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu

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Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys

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Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys

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Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp

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Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile

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Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly

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Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp

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Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser

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Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu

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Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp

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Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile

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Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu

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Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu

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Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala

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Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg

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Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu

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Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser

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Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val

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Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp

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Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His

1220 1225 1230

Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr

1235 1240 1245

Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp

1250 1255 1260

Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr

1265 1270 1275 1280

Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu

1285 1290 1295

Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr

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Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala

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Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys

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Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys

1345 1350 1355 1360

Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys

1365 1370 1375

Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val

1380 1385 1390

Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys

1395 1400 1405

Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn

1410 1415 1420

Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp

1425 1430 1435 1440

Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly

1445 1450 1455

Arg Lys Arg Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu

1460 1465 1470

Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His

1475 1480 1485

Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu

1490 1495 1500

Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile

1505 1510 1515 1520

Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys

1525 1530 1535

Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln

1540 1545 1550

Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro

1555 1560 1565

Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr

1570 1575 1580

Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr

1585 1590 1595 1600

Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Gly

1605 1610 1615

Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser

1620 1625 1630

Claims

1. A method for improving the site-specific integration of a gene into a cell based on an electrotransformation technology and a CRISPR-Cas9 technology is characterized by comprising the following steps:

2. The method for improving the site-directed integration of a gene into a cell based on an electrotransfer technology and a CRISPR-Cas9 technology according to claim 1, wherein the target gene in step 1 is a CAR gene, the insertion site in step 3 is a TCR site, and the cell in step 4 is a human primary T cell.

3. The method for improving the site-specific integration of a gene into a cell based on the electrotransformation technique and the CRISPR-Cas9 technique as claimed in claim 1 or 2, wherein the size of the homology arm in step 1 is 300-800 bp.

4. The method for improving the site-directed integration of a gene into a cell based on the electrotransfer technology and the CRISPR-Cas9 technology according to claim 1 or 2, wherein the guide RNA and the Cas9 protein are bound in step 4 at room temperature or 37 ℃ for 10-20min in vitro.

5. The method for improving the site-directed integration of a gene into a cell based on the electrotransfer technology and the CRISPR-Cas9 technology according to claim 1 or 2, wherein the CeletrixTM electrotransfer system is used for electrotransfer in the step 4.

6. The method for improving the site-specific integration of the gene into the cell based on the electrotransfer technology and the CRISPR-Cas9 technology as claimed in claim 1 or 2, wherein step 6 is to detect the expression of the target gene by flow cytometry and to detect the function of the cell.