WO2023102712A1 - Genetic biological preparation, and preparation method therefor and use thereof - Google Patents

Abstract

The present invention relates to the technical field of biological cells, and in particular to a method of knocking an IN044 sequence into a target gene by means of using a gene editing technique, so as to silence the target gene and achieve a gene knockout effect. After the target gene is edited by means of using the method of the present invention, the target gene cannot be translated into a protein due to frameshift mutation, and even if a protein is produced, same is rapidly degraded so that the toxicity of a translated protein with unknown traits is eliminated. The present invention has broad application prospects and can be used in many areas such as gene editing, disease diagnosis and disease treatment.

Description

A gene biological preparation and its preparation method and application technical field

The invention relates to the fields of gene editing and biological cell technology, in particular to a gene biological preparation and its preparation method and application.

Background technique

In recent years, gene editing technology has developed rapidly, especially CRISPR-cas9, CRISPR (Clustered regularly interspaced short palindromicrepeats) regularly clustered interspaced short palindromic repeats; Cas9 (CRISPR associated nuclease) is a CRISPR-related nuclease, and CRISPR-Cas9 is the latest RNA-guided gene editing method using Cas9 nuclease technology. Two articles published in "Science" (Science) on February 15, 2013, proved that the Cas9 system can perform effective targeted enzyme digestion in 293T, K562, iPS and other cells, and non-homologous recombination (NHEJ ) and homologous recombination (HR) each have an efficiency between 3-25%, and the recombination efficiency is equivalent to the cutting effect of TALEN. The article also demonstrates that multiple targets can be targeted for simultaneous shearing. These works will push further targeted gene manipulation to a climax, making multiple gene knockout and knockin easier and more efficient.

After years of research, the technology of editing targeted genes with Cas9 nuclease has matured. Site-directed mutagenesis, gene knockout and gene knockin of target genes can be realized. At present, this technology has been rapidly developed in the fields of animal and plant breeding, directed differentiation of stem cells, and fixed-point repair of genetic diseases.

Under normal circumstances, in the CRISPR-Cas9 gene knockout technology, a gRNA is used to target the target sequence for gene splicing, and the deletion during repair is used to carry out gene splicing, increase bases, cause frameshift mutations, and result in gene silencing; In another case, two gRNAs are used to target the target sequence for gene splicing, resulting in the deletion of gene fragments, resulting in gene silencing. However, there may be a problem in both methods: even if the gene frame shift or gene fragment deletion is caused, the gene can still express the protein normally. At this time, the protein expressed by the gene is not the original wild-type protein, and its function and traits may be altered, and this gene may cause other side effects.

technical problem

Based on this, it is necessary to provide a gene knockout and insertion method to solve the above problems, in which a translatable protein can be produced after the target gene is cleaved in an optimized way, and the knockout gene can be degraded and rendered harmless. The invention also provides the gene biological preparation prepared by using the gene knockout and insertion method and its application.

technical solution

Technical scheme one that the present invention provides is as follows:

A gene knockout insertion method, the method is to use gene editing technology to knock out the gene of the extracellular segment of the expressed protein or the secreted protein in the extracellular segment of the membrane expressed protein or the secreted protein of the target cell sequence and insert a section of IN044 nucleic acid sequence at the knockout gene sequence position; the IN044 nucleic acid sequence is:

AACTGCCGGAATACCGGCCCCTGGCTGAAGAAGGTGCTGAAGTGTAACACACCCGACCCTAGCAAGTTCTTTTCCCAGCTG;

Wherein, the gene editing technology includes one or more of CRISPR, transposon, TALEN and ZFN technologies.

Inserting the IN044 nucleic acid sequence into the extracellular segment gene sequence of the membrane-expressed protein, or inserting the IN044 nucleic acid sequence into the sequence at any position of the secreted protein, is mainly to completely silence the inserted gene in the target cell and complete protein degradation.

In one embodiment, in the gene knockout and insertion method, the amino acid sequence corresponding to the IN044 nucleic acid sequence is: NCRNTGPWLKKVLKCNTPDPSKFFSQL.

The above-mentioned IN044 nucleic acid sequence is inserted into the target cell after synthesizing a DNA sequence. In one embodiment, the DNA sequence is inserted into the gene sequence of the target cell by direct addition.

The above-mentioned CRISPR-cas9 technology inserts the IN044 nucleic acid sequence after gene knockout of target cells by CAS9 protein and gRNA.

The above-mentioned gene editing technology includes one or more of CRISPR, transposon, TALEN and ZFN technology, and the gene or protein and gRNA in the gene editing technology are transferred into the target cell through a delivery vector; wherein, The gene or protein in the gene editing technology includes the CAS9 protein in the CRISPR technology, the TALE and endonuclease FokI coupling element in the TALEN technology, and the zinc finger protein in the ZFN technology. Among the gene editing technologies, the CRISPR-cas9 gene editing technology is preferred.

In one embodiment, in the gene knockout and insertion method, the CAS9 protein and gRNA are transferred into the target cells through a delivery vector; wherein, the delivery system includes lentivirus, retrovirus, common plasmid, additional One or more of body, nano-delivery system, electrotransduction and transposon.

The present invention also provides a gene biological preparation prepared by the above gene knockout and insertion method.

The invention also provides the application of a genetic biological agent in the preparation, prevention and treatment of diseases.

Beneficial effect

The gene knockout and insertion method provided by the present invention and the gene biological preparation prepared therefrom mainly have the following advantages:

1. High gene editing efficiency, up to 100%;

2. After the target gene of the target cell is edited, due to the frameshift mutation, it cannot be translated into protein and the produced protein is rapidly degraded, which also eliminates the side effect problem of toxicity of the translated protein with unknown traits;

3. Gene editing acts on membrane-expressed proteins and secreted proteins, which can cause the extracellular region of membrane-expressed proteins to be ubiquitinated and degraded, making them unable to bind to other proteins, and secreted proteins will be ubiquitinated during secretion Degradation makes it unable to be secreted outside the cell, which can ensure that the edited protein cannot produce the original effect;

4. The present invention provides gene insertion methods and gene biological preparations, which have broad application prospects and can be used in various aspects such as gene editing, disease diagnosis and treatment.

Description of drawings

Fig. 1 is the detection figure of 293T, 293T-CLDN18.2 cells expressing CLDN18.2 of embodiment 1

CT106, CT090, CT032 structural representation of Fig. 2 embodiment 1;

3a, 3b, and 3c are schematic diagrams of CAR expression of CT106 and CT090 in Example 2;

Fig. 4 is the amplification curve of CT106 and CT090 of embodiment 2;

Fig. 5 is the killing effect in vitro of CT106 and CT090 of embodiment 2;

Fig. 6 is the cytokine release effect of CT106 and CT090 of embodiment 2;

Figures 7a, 7b, and 7c show the expression of PD-1 on the surface of CT106 in Example 3 after using gRNA-PD1 to knock in the IN044 sequence;

Figure 8 shows the cell proliferation of CT106 in Example 3 after knocking in the IN044 sequence with gRNA-PD1;

Figure 9 shows the expression of anti-PD1 in the supernatant after the CT032 of Example 4 was knocked into the IN044 sequence using gRNA-antiPD1;

Figure 10 shows the cell proliferation of CT032 in Example 4 after the IN044 sequence was knocked in with gRNA-antiPD1.

BEST MODE FOR CARRYING OUT THE INVENTION

In order to facilitate the understanding of the present invention, the following will describe the present invention more fully.

The present invention provides a gene knockout and insertion method, which uses gene editing technology to knock out the extracellular segment or secreted protein of the membrane-expressed protein of the target cell. The gene sequence of the secreted protein, and insert a piece of IN044 nucleic acid sequence at the position of the knockout gene sequence.

The above-mentioned IN044 nucleic acid sequence is: AACTGCCGGAATACCGGCCCCTGGCTGAAGAAGGTGCTGAAGTGTAACACACCCGACCCTAGCAAGTTCTTTTCCCAGCTG.

Wherein, the gene editing technology includes one or more of CRISPR, transposon, TALEN and ZFN technologies; the gene editing technology includes one or more of CRISPR, transposon, TALEN and ZFN technologies, and the The gene or protein and gRNA in the gene editing technology are transferred into the target cell through a delivery vector; wherein, the gene or protein in the gene editing technology includes the CAS9 protein in the CRISPR technology, the TALE and internal protein in the TALEN technology Nuclease FokI coupling element, zinc finger protein in ZFN technology.

In this embodiment, among the gene editing technologies, the CRISPR-cas9 gene editing technology is preferred.

The gene insertion method of the present invention is to insert the IN044 nucleic acid sequence into the gene sequence of the extracellular segment of the membrane-expressed protein, or to insert the IN044 nucleic acid sequence into any position sequence of the secreted protein, mainly to achieve gene completeness. Function of silencing and complete protein degradation.

The amino acid sequence corresponding to the above-mentioned IN044 nucleic acid sequence is:

NCRNTGPWLKKVLKCNTPDPSKFFSQL.

The above-mentioned IN044 nucleic acid sequence is inserted into the target cell after synthesizing a DNA sequence. In one embodiment, the DNA sequence is inserted into the gene sequence of the target cell by direct addition.

The above-mentioned CRISPR-cas9 technology inserts the IN044 nucleic acid sequence after gene knockout of target cells by CAS9 protein and gRNA.

In one embodiment, in the gene knockout and insertion method, the CAS9 protein and gRNA are transferred into the target cells through a delivery vector; wherein, in the gene knockout and insertion method, the delivery system includes lentivirus, One or more of retroviruses, common plasmids, episomes, nano-delivery systems, electrotransduction and transposons.

The present invention also provides a gene biological preparation prepared by the above gene knockout and insertion method.

The invention also provides the application of a genetic biological agent in the preparation, prevention and treatment of diseases.

The gene knockout and insertion method in the present invention has the following technical advantages:

1. The gene editing efficiency is higher than that of traditional CRISPR-cas9 gene editing.

2. After the target gene is edited, one cannot be translated into a protein due to a frameshift mutation, and the other is that even if the protein is produced, it will be rapidly degraded, which eliminates the toxicity of the translated protein with unknown traits and fully ensures that the gene knockout has no by-products Production.

3. Gene editing acts on membrane-expressed proteins and secreted proteins, which can ensure that the edited proteins cannot produce extracellular effects.

Hereinafter, taking CAR-T cells as target cells as an example, the gene knockout and insertion method of the present invention will be described in detail.

The editable target cells in the present invention, that is, immune cells are not limited to the CAR-T cells mentioned above and below.

In a preferred embodiment, when the CAR used for functional evaluation in the present invention is expressed in T cells, it can perform antigen recognition based on antigen binding specificity or protein receptor binding. The antigen-binding domain is preferably fused to a costimulatory molecule and the intracellular domain of one or more of the CD3ζ chain or partition proteins P2A, T2A, F2A, E2A.

Preferably, the antigen binding domain is fused to one of the combined intracellular domains of CD28, 4-1BB, ICOS signaling domain and CD3ζ signaling domain, respectively.

In the present invention, the intracellular domain used in the functional evaluation CAR includes one of CD28, 4-1BB, the signal transduction domain of ICOS and the signal transduction domain of CD3ζ.

In the present invention, the vector of the CAR expression cassette used for functional evaluation includes one or more of DNA, RNA, plasmid, lentivirus, adenovirus, retrovirus, transposon, and other gene transfer systems. Preferably, the vector is a lentiviral vector.

In the present invention, the Cas9 protein expression cassette vector for gene editing includes one or more of DNA, RNA, plasmid, lentivirus, adenovirus, retrovirus, transposon, purified protein, and other gene transfer systems. Preferably, the carrier is a purified protein carrier.

In the present invention, the physical method for introducing gRNA into host cells (ie, target cells) includes one of calcium phosphate precipitation, lipofection, particle bombardment, microinjection, and electroporation. The physical method of electroporation is preferred.

The gene biological preparation prepared by using CRISPR-cas9 gene knockout and insertion method in the present invention can be applied to drugs for detection, treatment (or prevention) of diseases and the like. The amount and frequency of administration will be determined by such factors as the patient's condition, and the type and severity of the patient's disease, and by the clinical protocol. When an "immunologically effective amount", "inhibitory effective amount" or "therapeutic amount" is indicated, the precise amount of the composition of the invention to be administered can be determined by a physician, taking into account the patient's (subject) age, weight, site size, infection Or individual differences in degree and disease. Genetic biological agents can also be administered at these doses multiple times, but the optimal dose and treatment regimen for a particular patient can be determined by one skilled in the medical art by monitoring the patient for signs of disease and adjusting treatment accordingly.

The administration of the gene biological preparation prepared in the present invention can be carried out in any convenient way, including spraying, injection, swallowing, transfusion, implantation or transplantation. The methods or products described herein can be administered to a patient subcutaneously, intradermally, intraorganically, intranodally, intraspinally, intramuscularly, by intravenous (i.v.) injection or intraperitoneally.

Embodiments of the present invention

The following are descriptions of specific embodiments.

Example 1

The CAR-T cell structure and cell preparation used for functional verification in this example include the following steps:

S100: Isolation of peripheral blood PBMCs and culture of T cells

Mononuclear cells were isolated from donor peripheral blood, density gradient centrifugation was performed using ficol, and T cells were enriched with a T cell sorting kit (CD3 MicroBeads, human-lyophilized, 130-097-043), using conjugated anti-CD3 /anti-CD28 magnetic beads to activate culture and expand T cells; the medium uses TexMACS GMP Medium (Miltenyi Biotec, 170-076-309), containing 10% FBS, 2mM L-glutamine, 100IU/ml rhIL2, all T cells All were cultured in a constant temperature incubator at 37°C with 5% CO ₂ to obtain T cells.

S200: Cell line culture

Cell line overexpressing CLDN18.2: 293T (human embryonic kidney cell line), purchased from ATCC.

Cell line expressing CLDN18.2: 293T-CLDN18.2, constructed by self-produced lentivirus infection.

Cells for packaging: 293T (human embryonic kidney cell line), purchased from ATCC.

Establishment of cell lines overexpressing CLDN18.2: Cloning the base sequence expressing CLDN18.2 into the backbone of the PHBLV lentiviral vector and placing it under the promoter of EF1α (EF-1α) to form PHBLV-EF1α-CLDN18.2 , PHBLV-EF1α-CLDN18.2, lentiviral envelope plasmid pMD2 .G (Addgene, Plasmid #12259) and lentiviral packaging plasmid psPAX2 (Addgene Plasmid#12260) and other three plasmids were transferred into 293T using Lipofectamine3000 to prepare a complete lentiviral expression vector; the virus supernatant was collected at 48h and 72h respectively, and the collected virus supernatant was concentrated by ultracentrifugation (Merck Millipore); the concentrated virus can be used to infect 293T, and finally a 293T cell line overexpressing CLDN18.2 is obtained, named 293T-CLDN18.2.

Figure 1 is the detection diagram of 293T and 293T-CLDN18.2 cells expressing CLDN18.2. In Figure 1, 293T-CLDN18.2 corresponds to the right curve (a curve), indicating that the CLDN18.2 cells on the surface of 293T-CLDN18.2 cells are positive compared to the left curve (b curve) corresponding to the control sample 293T. Culture medium: 293T and 293T-CLDN18.2 were cultured in DMEM medium. All media were supplemented with 10% (v/v) fetal bovine serum.

S300: CAR structure design and lentiviral packaging

CLDN18.2-CAR structure, that is, the CAR structure targeting CLDN18.2:

In the method of the present invention, the second-generation CAR expressed on the membrane of immune cells (CAR-T) is constructed on an expression frame. The core structure of CAR includes secretory signal peptide sequence, CD8 transmembrane region, intracellular segment stimulation signal 4-1BB-CD3ζ, etc. After adding P2A to the CAR structure and synthesizing anti-PD1, it is named CT032; adding P2A to the CAR structure and synthesizing green The fluorescent protein GFP is named CT106; on the basis of the structure of CT106, the IN044 sequence is inserted into the extracellular domain of the CAR structure, named CT090, as shown in Figure 2.

the As shown in Figure 2, CLDN18.2 scFv/TM/4-1BB/CD3ζ/P2A/GFP expresses the chimeric antigen receptor expressed on the cell membrane of immune cells and simultaneously synthesizes green fluorescent protein GFP in the cell, namely CT106; while CLDN18.2 scFv/IN044/TM/4-1BB/CD3ζ/P2A/GFP expresses the expression of antigen on the cell membrane of immune cells. IN044 sequence is inserted into the chimeric receptor and the green fluorescent protein GFP is synthesized in the cell at the same time, namely CT090; CLDN18.2 scFv/TM/4-1BB/CD3ζ/P2A/anti-PD1 represents the chimeric antigen receptor expressed on the cell membrane of immune cells and simultaneously synthesizes anti-PD1 in the cells, namely CT032. the

The expression cassette was cloned into the backbone of the PHBLV lentiviral vector and placed under the promoter of EF1α (EF-1α) to form PHBLV-EF1α-CT032, PHBLV-EF1α-CT106 and PHBLV-EF1α-CT090, and the PHBLV-EF1α-CT032 /CT106/CT090, lentiviral envelope plasmid pMD2 .G (Addgene, Plasmid#12259) and lentiviral packaging plasmid psPAX2 (Addgene Plasmid#12260) and other three viral plasmids, use Lipofectamine3000 to transfer into 293T to prepare lentiviral complete expression vector ; After the cells were cultured for 48h and 72h, they were transferred to centrifuge tubes for centrifugation, and the virus supernatant was collected after the centrifugation stopped, and the supernatant was concentrated by ultracentrifugation (Merck Millipore); the concentrated virus can be used to infect T cells.

S400: Preparation of CAR-T cells.

4.1 Lentivirus infection

After reactivating the primary T cells isolated and purified in step S100 for 1 day, use the three kinds of lentivirus (Lv032/Lv106/Lv107) packaged in step S300 to carry out lentiviral vector infection according to MOI (1-10), and the infected virus T cells were transferred to cell culture flasks and cultured in a constant temperature incubator at 37°C with 5% CO ₂ .

4.2 Detection of cell proliferation and CAR positive rate

On the 6th, 9th, 11th, and 13th day after T cell infection, samples were taken every day to detect the number of cells, and on the 6th day, the CAR positive rate of T cells was detected respectively, and the culture medium was supplemented every 1-2 days.

Using the three kinds of lentiviral vectors in step S300, three kinds of CAR-T cells were successfully constructed, named CT032/CT106/CT090 respectively, and T cells not infected with lentivirus were used as the control (NT).

Example 2

This example is used to illustrate the effect of inserting the IN044 sequence into the extracellular segment of the CAR structure on the expression of CAR. The specific steps are as follows:

S500: expression of CAR positive rate in CT106 and CT090 cells.

Figures 3a, 3b, and 3c respectively represent the CAR positive rate detection charts of NT cells, CT106, and CT090. As shown in Figures 3a, 3b, and 3c, using NT cells as negative controls, CT106 and CT090 cells had normal expression of green fluorescent protein GFP, indicating that the gene expression was normal, and the expression level of GFP could be regarded as the positive rate of gene expression. However, the expression of CAR in CT090 with IN044 insertion sequence was completely silenced, which proved that when the IN044 sequence was inserted into the extracellular region of the membrane-expressed protein, the translated protein of this sequence could mediate protein degradation and realize protein expression silencing.

S600: proliferation of CT106 and CT090 cells.

As shown in Figure 4, the proliferation of NT/CT106/CT090 cells was plotted as a growth curve. Taking NT cells and CT106 cells as controls, the proliferation of CT090 cells was not significantly different from that of NT and CT106 cells, which proved that the insertion of the IN044 sequence had no significant effect on cell proliferation.

S700: Killing status of CT106 and CT090 cells in vitro.

5.1 Comparison of killing efficiency between CT106 and CT090 cells in vitro

CT106 and CT090 were used as effector cells, and 293T and 293T-CLDN18.2 were used as target cells. After mixing and co-culturing for 6 hours according to the effect-to-target ratio of 1:27, 1:9, 1:3, and 1:1, the in vitro killing efficiency was calculated. The results are shown in Figure 5, CT106 cells without IN044 sequence insertion exerted the killing function of normal CAR-T on 293T-CLDN18.2 positive target cells, while CT090 cells with IN044 sequence insertion on 293T-CLDN18.2 positive target cells Same as NT cells, no specific killing effect.

5.2 Detection of cytokine release in supernatants of CT106 and CT090 cells in vitro

The supernatants of CT106 and CT090 in vitro killing experiments were collected and tested for IL-2 and IFN-γ cytokines. The test results are shown in Fig. Positive target cells have killing function, and normally release cytokines, and IFN-γ cytokines are proportional to the number of positive target cells; while CT090 cells with IN044 sequence insertion have the same effect on 293T-CLDN18.2 positive target cells as NT cells , no specific killing effect, and basically no cytokine release, which proves that the insertion of the IN044 sequence leads to the degradation of the CAR structure and the knockout of the gene.

Example 3

This example is used to illustrate that the IN044 sequence can silence the expression of PD-1 protein expressed on the membrane of CAR-T cells using CRISPR-Cas9 knock-in technology. The specific steps are as follows:

S800: Using CRISPR-Cas9 to knock in the IN044 sequence into the PD1 gene in CT106 cells.

6.1. Sequence Synthesis

The IN044 sequence was first synthesized, and amplicons were purified by 40 cycles of PCR (polymerase chain reaction) and Ampure magnetic beads.

6.2. Prepare gRNA-PD1

Design and synthesize gRNA-PD1 targeting the extracellular region of human PD-1 protein, resuspend crRNA and tracrRNA in IDT duplex buffer at a concentration of 200uM. The crRNA and tracrRNA were thawed and dissolved, mixed at a volume ratio of 1:1, incubated at 98°C for 5 min, and cooled at room temperature to form a 100 µM gRNA-PD1 solution.

6.3. Electroporation process

Day 0: Isolate T cells with magnetic beads, wherein, T cells: magnetic beads = 1:3; and add IL-2 cytokine, the amount added is 100 IU/mL;

Day 2: Remove the beads, put the cells in the conical tube and return to the 37°C incubator; in addition, preheat some OPT5+IL2 medium for subsequent use;

Prepare RNP+Cas9

Mix RNP with Cas9 protein (5ug/ul), and the final volume ratio gRNA:PGA:Cas9 is 1:0.8:1.6 in total. Incubate at room temperature for 15 min. During the incubation period, the cells are centrifuged at room temperature at 90 g for 10 min. Resuspend cells with P3 solution (P3: Supplement=9:2), 1.5*10^6~2*10^6 cells/ml per 20ul solution

RNP was incubated with HDR for 1 min, and P3 resuspended cells (16 wells, 20ul) were added to each RNP+HDR tube, and mixed evenly. The RNP mixture was transferred to a dish and electroporated using the EH-115 program. Immediately after electroporation, 80ul of preheated PBS was added, placed in 37°C for another 15min. Transfer the treated cells to an appropriate plate with a 200ul gun and rehydrate with pre-warmed old medium (the cell density should not be less than 1* ¹⁰⁶ cells/ml when knocking in). Place in a 37°C incubator for cultivation.

Day 5: Detection of exogenous gene expression

6.4 Detection of PD1 gene expression in CT106 cells before and after knockout

As shown in Figures 7a, 7b, and 7c, after CT106 inserted the IN044 sequence into the extracellular segment of the PD-1 gene using CRISPR-Cas9 technology, the expression of PD1 on the surface of CT106-IN044 cells was completely silenced, which can prove that this knockout method can effectively silence the cell membrane express protein. As shown in Figure 8, there was no significant difference in proliferation ability between CT106 cells and CT106-IN044 cells.

Example 4

This example is used to illustrate that the IN044 sequence can silence the expression of anti-PD-1 protein secreted by CAR-T cells using CRISPR-Cas9 knock-in technology. The specific steps are as follows:

S900: Knock in the IN044 sequence into the anti-PD1 gene of CT032 cells using CRISPR-Cas9 technology.

Synthesize gRNA-antiPD1, and use the CRISPR-Cas9 technology steps in S800 to knock the IN044 sequence into the anti-PD1 gene of CT032 cells. Results As shown in Figure 9, the antiPD1 recombinant antibody protein was secreted normally in CT032 cells, while the anti-PD1 recombinant antibody protein expression could not be detected in the supernatant of CT032-IN044 cells that had been knocked into IN044 by CRISPR-Cas9. As shown in Figure 10, there was no significant difference in proliferation ability between CT032 cells and CT032-IN044 cells. It is proved that the technology in the present invention can effectively silence the expression of the secreted protein gene, and has no significant effect on the edited cells.

Industrial Applicability

The gene editing method provided by the present invention can effectively silence the edited gene, so that the membrane-expressed protein and secreted protein genes can be completely silenced.

Sequence Listing Free Content

sequence listing

<110> Shenzhen Xiankangda Life Science Co., Ltd.

<120> A gene biological preparation and its preparation method and application

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Claims (10)

A gene knockout and insertion method, characterized in that, inserting a piece of IN044 nucleic acid sequence into the extracellular segment of the membrane-expressed protein or the secreted protein gene by gene editing; the IN044 nucleic acid sequence is: AACTGCCGGAATACCGGCCCCTGGCTGAAGAAGGTGCTGAAGTGTAACACACCCGACCCTAGCAAGTTCTTTTCCCAGCTG. The gene knockout and insertion method according to claim 1, wherein the amino acid sequence corresponding to the IN044 nucleic acid sequence is: NCRNTGPWLKKVLKCNTPDPSKFFSQL. The gene knockout and insertion method according to claim 1, wherein the IN044 nucleic acid sequence is inserted into the target cell after a DNA sequence is synthesized. The gene knockout and insertion method according to claim 3, wherein the DNA sequence is inserted into the gene sequence of the target cell by direct addition. The gene knockout and insertion method according to claim 1, characterized in that gene editing technology is used to knock out gene sequences at any position. The gene knockout and insertion method according to claim 5, wherein the gene editing technology includes one or more of CRISPR, transposon, TALEN and ZFN technologies. The gene knockout and insertion method according to claim 6, characterized in that the gene or protein and gRNA in the gene editing technology are transferred into the target cell through a delivery vector. The gene knockout and insertion method according to claim 7, wherein the delivery vector comprises one or more of DNA, RNA, plasmid, purified protein, lentivirus, adenovirus, retrovirus and transposon Several kinds. A gene biological preparation prepared according to the gene knockout and insertion method according to any one of claims 1 to 8. The application of the gene biological agent described in claim 9 in the preparation, prevention and treatment of diseases.