Method for constructing genetically-engineered animal used for xenotransfusion

Abstract

Provided is a method for constructing a genetically-engineered animal used for xenotransfusion, belonging to the technical field of genetic engineering. Provided is a method for performing gene modification on a porcine red blood cell, which successfully realizes expressing a human protective protein in the porcine red blood cell, improves the compatibility of the porcine red blood cell in a human immune system, reduces immune rejection and reduces the phagocytic effect of macrophages. The present invention is based on a porcine fibroblast which has undergone triple knockout by three cultured antigens α-gal, Sda and Neu5Gc, and uses a CRISPR-cas9 system to design an efficient sgRNA so as to complete site-directed integration of an exogenous DNA sequence at a porcine safe harbor; and using an EF1α promoter for driving the expression of the human protective protein achieves the expression of the protein on the surface of the porcine red blood cell. The provided transgenic strategy enables an exogenous gene to be stably expressed on the surface of the porcine red blood cell, thereby further improving the survival ability of the porcine red blood cell in human bodies. Therefore, the porcine red blood cell can be used as a xeno-sourced red blood cell for reservation, thus alleviating the problem of insufficient blood reservation.

Description

A method for constructing genetically engineered animals for xenotransfusion Technical Field

[0001] The present invention belongs to the field of genetic engineering technology, and in particular relates to a method for constructing genetically engineered animals for xenotransfusion. Background Art

[0002] With rapid advances in technology, the dynamics of local warfare and the types of weapons used in modern warfare have undergone dramatic changes. The widespread use of high-explosive and high-energy weapons has significantly increased their lethality and destructive power, expanding the area of ​​casualty. This has led to a significant increase in the incidence of limb injuries, which accounted for over 80% of combat injuries during the Gulf War. Severe penetrating or blast injuries during wartime often cause life-threatening hemorrhage. These casualties are critically ill and urgently needed, and treatment is limited by battlefield conditions. Due to insufficient blood product supplies, they can quickly develop hemorrhagic shock. Although limb replantation technology has met clinical needs, limbs are prone to avascular necrosis during transport from the front lines, ultimately resulting in extremely high disability rates. Effectively treating these limb-related combat injuries, improving survival rates on the battlefield and in post-traumatic care, and reducing mortality and disability rates remain critical challenges in military medicine worldwide. This invention adapts to the needs of modern warfare, explores the application of humanized gene-modified pig blood in the treatment of limb combat injuries, improves the level of treatment for limb combat injuries, and reduces the amputation rate and mortality rate of limb combat injuries, which is of great significance.

[0003] Blood transfusion is also a crucial life-saving measure, especially in cases of acute hemorrhage, severe anemia, and traumatic surgery, when patients urgently need new blood to sustain life. In recent years, the global pandemic has led to a severe shortage of blood, with blood products in short supply and blood banks in many regions experiencing critical shortages.

[0004] Transfusing blood from non-human animals to humans is called xenotransfusion. Research has found that pig red blood cells (pRBCs) share similar characteristics to human red blood cells, including size and lifespan. pRBCs can survive normally in the human body. Humanized, genetically modified pig-derived red blood cells are not only easily accessible, but also facilitate the mass production of blood products and can be stored directly in hospitals for future use.

[0005] However, there are three major obstacles to xenotransfusion: blood type incompatibility, immune rejection, and the risk of infection by pathogenic microorganisms. The AO blood type of pigs is similar to the ABO blood type system of humans, and the compatibility of the blood type system can be ensured by screening O-type pigs. After pig red blood cells enter the human body, immune rejection and agglutination reactions lead to hemolysis of the red blood cells. Depending on the location of the hemolytic reaction, it can be divided into intravascular hemolysis and extravascular hemolysis. The former is mainly caused by the specific binding of xenoantigens on the surface of pig red blood cells to natural antibodies in human serum, activating the complement system to form a membrane attack complex, resulting in red blood cell lysis. The latter is because the SIPα (Signal regulatory protein-α) protein on the surface of human macrophages cannot recognize the "self" cell signal CD47 protein on the pig red blood cell membrane, triggering the phagocytosis of macrophages.

[0006] Transgenic pigs have been used in many fields, but they remain subject to certain limitations, primarily due to limited efficiency and uncertainty regarding integration sites. The CRISPR / Cas system, widely found in eubacteria and archaea, is an adaptive immune system that protects the host from secondary viral invasion. This system combines RNA transcribed by the CRISPR system with proteins translated by the Cas system to cleave DNA. Currently, the most widely used CRISPR / Cas system in genome editing is the type II SpCas9 system. This system uses a chimeric guide RNA, formed by crRNAs and tracrRNAs, in conjunction with the nuclease Cas9 to recognize and cleave DNA, creating double-strand breaks at specific sites in the genome. Compared to ZFNs and TALENs, the CRISPR / Cas9 system, with its extreme simplicity and low construction cost, has rapidly become a mainstream genome editing technology and has become an essential research tool in modern life science laboratories. CD55 (Cluster of differentiation 55), also known as decay-accelerating factor (DAF), is a complement regulatory protein encoded by the CD55 gene. The human CD55 protein has a molecular weight of 70 kDa and can protect cells from complement attack by inhibiting the formation of the membrane attack complex (MAC). Because mature red blood cells lack nuclei, current research reports have not yet achieved the expression of the human complement regulatory protein CD55 on the surface of porcine red blood cells.

[0007] Knocking out the major xenoantigens in pigs can reduce antibody binding in human serum by over 90%, but they still face complement- and immune cell-mediated rejection. The construction of transgenic pigs that express human complement regulatory proteins on the surface of red blood cells has important implications for xenotransfusions.

[0008] Summary of the Invention

[0009] One object of the present invention is to provide an sgRNA for constructing a genetically engineered animal. Another object of the present invention is to provide a method for constructing a genetically engineered animal for xenotransfusion, as well as the constructed genetically engineered animal and its application.

[0010] The present invention provides an sgRNA, whose nucleotide sequence is shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5.

[0011] Furthermore, the nucleotide sequence of the sgRNA is shown in SEQ ID NO: 3.

[0012] The present invention also provides another sgRNA, whose nucleotide sequence is shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10.

[0013] Furthermore, the nucleotide sequence of the sgRNA is shown in SEQ ID NO:8.

[0014] The present invention also provides a method for constructing a genetically engineered animal, the method comprising the following steps:

[0015] (1) Connecting the sgRNA described in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 to a vector to construct a knockout vector;

[0016] (2) constructing a target DNA sequence expressing human protective protein;

[0017] (3) The knockout vector and the vector containing the target DNA sequence expressing the human protective protein are co-transfected into animal fibroblasts, and the positive clone cells with site-specific integration of the human protective protein are obtained by sequencing and identification;

[0018] (4) Using positive cloned cells as nuclear donors and enucleated animal oocytes as recipients, somatic cell nuclear transplantation is performed to obtain reconstructed embryos;

[0019] (5) The reconstructed embryo is transferred into the body of a female animal, and the female animal is raised until it gives birth, and the offspring is obtained.

[0020] Furthermore, in step (1), the vector is a pX458 vector.

[0021] Furthermore, in step (3), the vector containing the target DNA sequence for expressing human-derived protective protein is a pUC57 cloning vector containing the target DNA sequence for expressing human-derived protective protein.

[0022] In the present invention, the human protective protein refers to a protein that can enhance the biocompatibility of heterologous cells in the host body and improve the survival ability of heterologous cells.

[0023] Furthermore, in step (2), the human protective protein includes one or two of human complement regulatory protein and human anti-cellular rejection protein; preferably, the human anti-cellular rejection protein is human anti-macrophage phagocytosis protein or human anti-T cell activation protein; more preferably, the human complement regulatory protein is CD55, CD46 or CD59, the human anti-macrophage phagocytosis protein is CD47, and the human anti-T cell activation protein is CTLA4.

[0024] Furthermore, in step (2), the target DNA sequence for expressing human protective protein includes a left homology arm, an EF1α promoter, a CDS sequence of a human protective protein gene, a polyA sequence, a right homology arm, and an HSV-TK sequence connected in sequence; preferably, the target DNA sequence for expressing human protective protein is as shown in SEQ ID NO: 6 or SEQ ID NO: 11.

[0025] Furthermore, in step (3), the fibroblasts are fibroblasts in which heterologous antigens are knocked out, preferably fibroblasts in which three antigens, α-gal, Sda and Neu5Gc, are knocked out.

[0026] Furthermore, the animal is a pig.

[0027] Furthermore, the method comprises the following steps:

[0028] (1) Connect the sgRNA to the vector to construct knockout vector 1;

[0029] (2) constructing a target DNA sequence 1 for expressing human protective protein;

[0030] (3) The knockout vector 1 and the vector containing the target DNA sequence 1 expressing the human protective protein were co-transfected into animal fibroblasts, and positive clone cells with site-specific integration of the human protective protein were obtained by sequencing and identification;

[0031] (4) using the positive cloned cells obtained in step (3) as nuclear donors and the enucleated animal oocytes as recipients to perform somatic cell nuclear transplantation to obtain reconstructed embryos;

[0032] (5) The reconstructed embryo obtained in step (4) is transferred into a female animal, the female animal is raised until it gives birth, and the offspring are obtained to obtain a transgenic animal;

[0033] (6) Connecting the sgRNA to the vector to construct knockout vector 2;

[0034] (7) Constructing target DNA sequence 2 for expressing human protective protein;

[0035] (8) co-transfecting the knockout vector 2 and the vector containing the target DNA sequence 2 expressing the human-derived protective protein into the transgenic animal fibroblasts of step (5), and sequencing and identifying the positive clone cells with site-specific integration of the human-derived protective protein;

[0036] (9) using the positive cloned cells obtained in step (8) as nuclear donors and the enucleated animal oocytes as recipients to perform somatic cell nuclear transplantation to obtain reconstructed embryos;

[0037] (10) taking the reconstructed embryo obtained in step (9) and transferring it into the body of a female animal, raising the female animal until giving birth, and then taking the offspring;

[0038] Wherein, the sgRNA in step (1) is as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, the target DNA sequence 1 for expressing human-derived protective protein in step (2) is as shown in SEQ ID NO: 6, the sgRNA in step (6) is as shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10, and the target DNA sequence 2 for expressing human-derived protective protein in step (7) is as shown in SEQ ID NO: 11; or, the sgRNA in step (1) is as shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10, the target DNA sequence 1 for expressing human-derived protective protein in step (2) is as shown in SEQ ID NO: 11, the sgRNA in step (6) is as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, and the target DNA sequence 2 for expressing human-derived protective protein in step (7) is as shown in SEQ ID NO: As shown in ID NO:6.

[0039] The present invention also provides a genetically engineered animal constructed using the above method.

[0040] The present invention also provides the use of the genetically engineered animal in xenotransfusion.

[0041] Compared with the existing technology, the present invention has achieved the following beneficial effects: The present invention provides a method for genetically modifying pig red blood cells, successfully achieving the expression of human protective proteins in pig red blood cells, improving the compatibility of pig red blood cells in the human immune system, reducing immune rejection, and reducing the phagocytic effect of macrophages. Relying on the fibroblasts of three antigen knockout pigs (Triple-Knockout; TKO) of α-gal (GGTA1 gene knockout), Sda (B4GalNT2 gene knockout) and Neu5Gc (CMAH gene knockout) that have been cultivated, the CRISPR-cas9 system is used to design sgRNA to complete the site-specific integration of exogenous DNA sequences in the safe harbor of the pig genome, and the EF1α promoter is used to drive the expression of human protective proteins, thereby achieving the expression of such proteins on the surface of pig red blood cells. The transgenic strategy provided by the present invention can allow exogenous genes to be stably expressed on the surface of pig red blood cells, further improving the viability of pig red blood cells in the human body, and can be stored as red blood cells of heterologous origin, alleviating the problem of insufficient blood reserves.

[0042] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0043] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 is a schematic diagram of the target DNA sequence structure.

[0045] FIG2 is the identification of site-specific integration of CD55 transgene in TKO / CD55 transgenic pigs at the DNA level.

[0046] Figure 3 shows the DNA sequencing results of GGTA1, B4GalNT2 and CMAH gene knockout in TKO / CD55 transgenic pigs.

[0047] FIG4 shows the expression of human CD55 protein in TKO / CD55 transgenic pig pRBCs as determined by flow cytometry.

[0048] Figure 5 is a comparison of the agglutination effects of pig pRBCs with different genotypes.

[0049] FIG6 is a comparison of the binding effects of different genotype pig pRBCs with IgG and IgM antibodies in human serum.

[0050] FIG7 shows the CDC test results of porcine pRBCs of different genotypes and human sera of different blood types.

[0051] FIG8 is a comparison of the complement-mediated immune killing effects of pig pRBCs of different genotypes.

[0052] FIG9 shows the DNA level identification results of TKO / CD55 / CD47 transgenic pigs.

[0053] FIG10 shows the expression of human CD47 protein in TKO / CD55 / CD47 transgenic pig pRBCs as determined by flow cytometry.

[0054] FIG11 shows the results of phagocytosis of different genotype pig pRBCs by monkey macrophages. DETAILED DESCRIPTION

[0055] The raw materials and equipment used in the present invention are all known products and are obtained by purchasing commercially available products.

[0056] The α-gal (GGTA1 gene knockout), Sda (B4GalNT2 gene knockout), and Neu5Gc (CMAH gene knockout) antigen knockout pigs (Triple-Knockout; TKO) were obtained from Chengdu Zhongke Oger Biotechnology Co., Ltd.

[0057] Example 1: Construction of GGTA1KO / B4GALNT2KO / CMAHKO / hCD55 transgenic pigs (abbreviated as TKO / CD55 transgenic pigs)

[0058] Step 1: Design an sgRNA that specifically recognizes the target sequence DNA at the ROSA26 site in the porcine genome and ligate it into the pX458 vector recovered by BbsⅠ digestion to construct a knockout vector. Verify the knockout efficiency and select a highly efficient sgRNA (Table 1) for subsequent experiments.

[0059] Table 1. sgRNA sequences and their knockout efficiency

[0060] The knockout efficiency verification method is as follows:

[0061] The sgRNA knockout plasmid was transfected into the pig ear fibroblast cell line. After culturing for 48-72 hours after transfection, the GFP-positive cell population was recovered by flow cytometry, the DNA of the positive cell population was extracted, the knockout target sequence was amplified by PCR, the target sequence was recovered by gel and then cloned into the T vector by TA, and 20-30 monoclonal colonies were picked for sequencing.

[0062] The verification results are shown in Table 1. It can be seen that pX458-Rosa26-sgRNA-3 has the highest knockout efficiency, and pX458-Rosa26-sgRNA-3 was selected for subsequent experiments.

[0063] Step 2: Use homologous recombination technology to sequentially connect the left homology arm, EF1α promoter, CDS sequence of human CD55 gene, polyA sequence, right homology arm, HSV-TK sequence (including promoter and polyA), add restriction endonuclease sites NheI and HindIII sequences at the left and right ends, respectively, and connect the target DNA sequence to the pUC57 cloning vector. Figure 1 is a schematic diagram of the target DNA sequence structure.

[0064] The target DNA sequence is:

[0065] Step 3: Transfect the constructed cloning vector into competent cells, screen the positive bacteria and expand them, extract the plasmid and perform double digestion with NheI and HindⅢ restriction endonucleases, recover the target DNA sequence by gel, purify it and co-transfect it with the Rosa26-sgRNA-3 knockout vector into fibroblasts of α-gal, Sda and Neu5Gc antigen knockout pigs (Table 2), recover the GFP-positive cell population by flow cytometry, screen the monoclonal cells by limiting dilution, culture for about 10 days, pick the monoclonal cells for DNA level identification, and identify them by PCR technology to obtain CD55 site-specific integration positive clones.

[0066] Step 4: Through somatic cell cloning technology, the positive clone point cells are injected into the enucleated oocyte for fusion, and the reconstructed embryo is transferred into the uterus of the recipient sow for pregnancy.

[0067] Step 5: The recipient sows gave birth to four healthy piglets and two weak piglets (dead) through natural delivery, and the constructed TKO / CD55 transgenic pigs were obtained.

[0068] The following are the DNA level identification, protein level identification and protection effect verification of the born piglets:

[0069] (1) DNA level identification

[0070] After the piglets were born, a small amount of ear tissue was cut, and the piglet ear sample DNA was extracted using a DNA extraction kit according to the instructions. After DNA extraction, primers were designed according to the CD55 transgenic target DNA sequence for identification. There are two pairs of primers in total, one pair of primers near the left and right homologous arms to identify the CD55 site-specific integration. After PCR amplification, the product was run on a gel to identify the site-specific integration of the left and right arms. All piglets had CD55 site-specific integration (Figure 2). At the same time, PCR amplified the GGTA1, B4GALNT2, and CMAH gene editing target sequences. The target sequences were recovered by a gel recovery kit and cloned into a T vector. Ten monoclonal colonies were picked for sequencing and identification. After TA cloning and sequencing, the GGTA1, B4GALNT2, and CMAH genes of all piglets had been knocked out (Figure 3).

[0071] (2) Protein level identification

[0072] When the piglets were 1 month old, 5 mL of pig whole blood was collected and centrifuged at 900 g for 5 min. The blood was washed twice with PBS and the red blood cells were separated. 10 6 Erythrocytes were treated with 5 μL of PE-mouse anti-human CD55 antibody and incubated at 4°C for 30 minutes. The cells were washed three times with PBS and resuspended. Flow cytometry was used to analyze the expression of exogenous proteins on the erythrocyte surface. Mean fluorescence intensity (MFI) was used to represent CD55 expression. Wild-type porcine erythrocytes and human erythrocytes were used as controls.

[0073] The results are shown in Figure 4. While wild-type porcine erythrocytes do not express human CD55 (orange), human CD55 expression is detectable on the surface of TKO / CD55 transgenic pig erythrocytes. Furthermore, compared to human erythrocytes (red), the expression level of human CD55 on TKO / CD55 transgenic pig erythrocytes (blue) is approximately four times that of human erythrocytes.

[0074] (3) Protection effect verification

[0075] When piglets reached one month of age, 5 mL of whole blood was collected and centrifuged at 900 g for 5 minutes. The blood was washed twice with PBS, and red blood cells were isolated. The protective effect was verified by in vitro hemagglutination, IgG / IgM antibody binding, and CDC (complement-dependent cytotoxicity) assays. Wild-type pigs (WT) and pigs with α-gal, Sda, and Neu5Gc knockouts (TKO) were used as controls. Homo type A serum was inactivated from humans with blood type A, Homo type B serum was inactivated from humans with blood type B, Homo type AB serum was inactivated from humans with blood type AB, and Homo type O serum was inactivated from humans with blood type O.

[0076] The in vitro hemagglutination test method is as follows: take 10 6 Red blood cells were incubated with 25% heat-inactivated mixed human serum at room temperature for 30 minutes and then the red blood cell agglutination was observed under a microscope.

[0077] The IgG / IgM antibody binding test method is as follows: take 10 6 Red blood cells were incubated with 25% heat-inactivated mixed human serum at 4°C for 30 minutes, washed twice with PBS, and then fluorescently labeled anti-human IgG antibodies and fluorescently labeled anti-human IgM antibodies were added, incubated at 4°C for 30 minutes, washed twice with PBS, and resuspended in PBS to detect the fluorescence intensity on the red blood cell surface by flow cytometry.

[0078] The CDC test method is as follows: take 10 6 Red blood cells were incubated with 25% heat-inactivated human serum at 4°C for 30 minutes, washed once with PBS, and supplemented with 25% rabbit complement. The cells were incubated at 37°C for 30 minutes, centrifuged at 900 g for 5 minutes, and 100 μL of the supernatant was transferred to a 96-well plate. The OD560 of the sample was measured using a microplate reader to calculate the red blood cell mortality rate. Red blood cell mortality rate = ([AC] / [BC]) × 100%, where A represents the experimental value (OD), B represents the maximum mortality group (red blood cells lysed by red blood cell lysis buffer), and C represents the minimum mortality group (negative control, without complement and serum). Each experiment was repeated three times.

[0079] The results show:

[0080] 1. Compared with wild-type pigs, depletion of the three pig antigens reduced the degree of agglutination (Figure 5);

[0081] 2. Removal of the three porcine antigens significantly reduced the binding of porcine erythrocytes to IgG and IgM antibodies in human serum. The antibody binding levels of TKO and TKO / CD55 porcine erythrocytes were essentially the same, with no significant difference (Figure 6).

[0082] 3. CDC tests were performed on porcine erythrocytes with human sera of different blood types. There was no significant difference in the results between different blood types and the same genotype of porcine erythrocytes (Figure 7).

[0083] 4. CDC tests were performed on WT, TKO / CD55, and TKO pig red blood cells with mixed human serum. The results showed that removal of the three pig antigens significantly inhibited complement-mediated immune killing, and the introduction of the human CD55 gene could further inhibit complement-mediated killing (Figure 8).

[0084] Example 2: Construction of GGTA1KO / B4GALNT2KO / CMAHKO / hCD55 / hCD47 transgenic pigs (abbreviated as TKO / CD55 / CD47 transgenic pigs)

[0085] After successfully obtaining TKO / CD55 transgenic pigs in Example 1, it was verified that the EF1α promoter can be used to express exogenous proteins on red blood cells, and the human protective protein CD55 was successfully expressed in pig red blood cells, which improved the compatibility of pig red blood cells in the human immune system and reduced immune rejection.

[0086] On this basis, this example further introduces human anti-macrophage phagocytic protein CD47 into the H11 locus of TKO / CD55 transgenic pigs to reduce the phagocytosis of pig red blood cells by macrophages. The specific steps are as follows:

[0087] Step 1. Referring to the method of Example 1, an sgRNA that specifically recognizes the target sequence DNA was designed at the H11 site of the porcine genome and ligated into the pX458 vector recovered by BbsⅠ digestion to construct a knockout vector. The knockout efficiency was verified and an efficient sgRNA (Table 2) was selected for subsequent experiments.

[0088] Table 2. sgRNA sequences and their knockout efficiency

[0089] The verification results are shown in Table 2. It can be seen that H11-sgRNA-2 has the highest knockout efficiency, and H11-sgRNA-2 was selected for subsequent experiments.

[0090] Step 2: Referring to the method of Example 1, an integration vector of the human CD47 gene was constructed by homologous recombination technology.

[0091] The target DNA sequence is:

[0092] Step 3. Referring to the method of Example 1, the constructed cloning vector was transfected into competent cells, positive bacteria were screened and expanded, the plasmid was extracted and double-digested with NheI and HindⅢ restriction endonucleases, the target DNA sequence was recovered by gel, and after purification, it was co-transfected with the H11-sgRNA-2 knockout vector into TKO / CD55 transgenic pig fibroblasts, and the GFP-positive cell population was recovered by flow cytometry. Monoclonal cells were screened by limiting dilution, and monoclonal cells were picked for DNA level identification after culture for about 10 days. They were identified by PCR technology to obtain CD47 site-specific integration positive clones.

[0093] Step 4: Referring to the method of Example 1, the positive clone point cells are injected into the enucleated oocytes through somatic cell cloning technology for fusion, and the reconstructed embryos are transferred into the uterus of the recipient sow for pregnancy.

[0094] Step 5: The recipient sow gave birth to 7 healthy piglets through natural delivery, and the constructed TKO / CD55 / CD47 transgenic pigs were obtained.

[0095] The following are the DNA level identification, protein level identification and protection effect verification of the newborn piglets:

[0096] (1) DNA identification

[0097] After the piglets were born, a small amount of ear tissue was removed and DNA was extracted from the piglet ears using a DNA extraction kit according to the instructions. After DNA extraction, primers were designed based on the CD47 transgenic target DNA sequence for identification. Three pairs of primers were used: the first pair identified the integrity of the promoter and CD47 cDNA region, and a pair of primers near each of the left and right homologous arms identified site-specific integration of CD47. After PCR amplification, the products were run on a gel to identify site-specific integration of the left and right arms. All piglets showed site-specific integration of CD47 (Figure 9).

[0098] (2) Protein expression identification

[0099] When the piglets were 1 month old, 5 mL of pig whole blood was collected and centrifuged at 900 g for 5 min. The blood was washed twice with PBS and the red blood cells were separated. 10 6 Erythrocytes were treated with 5 μL of FITC-mouse anti-human CD47 antibody and incubated at 4°C for 30 minutes. The cells were washed three times with PBS and resuspended. Flow cytometry was used to analyze the expression of CD47 on the erythrocyte surface. Mean fluorescence intensity was used to represent CD47 expression. Wild-type porcine erythrocytes and human erythrocytes were used as controls.

[0100] The results are shown in Figure 10. While wild-type porcine erythrocytes do not express human CD47 (orange), human CD47 expression is detectable on the surface of TKO / CD55 / CD47 transgenic pig erythrocytes. Furthermore, compared to human erythrocytes (red), the expression level of human CD47 on TKO / CD55 / CD47 transgenic pig erythrocytes (blue) is approximately 10 times higher.

[0101] (3) Protection effect verification

[0102] The CD47 function was verified by using monkey macrophages to phagocytize pig red blood cells as follows:

[0103] Monkey macrophage isolation: Obtain fresh monkey lungs. Clamp the larynx below the larynx with two hemostats (or double ligate with string). Ligate the artery leading to the heart with string, carefully avoiding damage to the lung's integrity. Wash debris from the outside of the lung with a wash solution (1× PBS + 10% RPMI-1640 + 3× P / S). Place the lung on a tray and move it to a biosafety cabinet. Cut the middle of the two hemostats. Heat the tracheal opening with an alcohol burner and wash with wash solution. Instill a small amount of wash solution into the lung through the trachea. Ligate the larynx with a hemostats. Gently massage the lung surface for 2-3 minutes. Collect the first lung wash in a sterile bottle. Occasionally irrigate the lung surface with PBS to prevent drying. Use a small amount of wash solution for the first wash, and gradually increase the amount of wash solution. Collect the second, third, and fourth lung washes in the same manner (the number of washes depends on the turbidity of the wash solution). Filter the resulting lung lavage fluid through a disposable cell sieve (70 mesh) into sterile bottles, then aliquot into sterile 50 mL centrifuge tubes and centrifuge at 1,000 rpm for 4 minutes. Discard the supernatant from the centrifuged cells, resuspend the cell pellet in washing solution, and centrifuge at 1,000 rpm for 4 minutes. Remove the red blood cells using red blood cell lysis buffer according to the manufacturer's instructions. Repeat the red blood cell lysis as needed based on the lysis results. After lysis is complete, resuspend the cells in DMEM medium containing 10% serum.

[0104] Pig red blood cell staining: After collecting 5 mL of pig whole blood, centrifuge at 900 g for 5 minutes, wash twice with PBS, separate the red blood cells, and take 10 6 For red blood cells, add 1 mL of PBS solution containing 5 uM CFSE and incubate at 37°C in the dark for 20 min. Then add 5 mL of DMEM medium containing 1% serum and incubate at 37°C in the dark for 5 min. Centrifuge at 1500 rpm for 5 min, remove the supernatant, and resuspend the cells in DMEM medium containing 10% serum.

[0105] Phagocytosis: Monkey macrophages and CFSE-labeled porcine red blood cells were incubated at a ratio of 1:1. After mixing the cells, they were placed in an incubator and incubated for 4 hours. After the incubation was completed, red blood cell lysis buffer was used to remove the non-phagocytic porcine red blood cells. The cells were then analyzed by flow cytometry to detect the CFSE-positive ratio of monkey macrophages, which was the phagocytic red blood cells.

[0106] The results are shown in Figure 11: Compared with TKO / CD55 pig erythrocytes, the number of TKO / CD55 / CD47 pig erythrocytes phagocytosed by monkey macrophages decreased by about 50%, indicating that the expression of CD47 significantly reduced the phagocytosis of pig erythrocytes by monkey macrophages.

[0107] In summary, the present invention provides a method for genetically modifying pig red blood cells, successfully achieving the expression of human protective proteins in pig red blood cells, improving the compatibility of pig red blood cells in the human immune system, reducing immune rejection, and reducing the phagocytic effect of macrophages. Relying on the fibroblasts of three antigen-knockout pigs of α-gal, Sda and Neu5Gc that have been cultivated, the CRISPR-cas9 system is used to design efficient sgRNA to complete the site-specific integration of exogenous DNA sequences in the pig safe harbor, and the EF1α promoter is used to drive the expression of human protective proteins to achieve the expression of such proteins on the surface of pig red blood cells. The transgenic strategy provided by the present invention can allow exogenous genes to be stably expressed on the surface of pig red blood cells, further improving the viability of pig red blood cells in the human body, and can be stored as red blood cells of heterologous origin to alleviate the problem of insufficient blood reserves.

Claims

1. A sgRNA, characterized in that Its nucleotide sequence is shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:

5.

2. The sgRNA according to claim 1, characterized in that Its nucleotide sequence is shown in SEQ ID NO:

3.

3. A sgRNA, characterized in that Its nucleotide sequence is shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:

10.

4. The sgRNA according to claim 1, characterized in that Its nucleotide sequence is shown in SEQ ID NO:

8.

5. A method for constructing a genetically engineered animal, characterized in that: The method comprises the following steps: (1) connecting the sgRNA according to any one of claims 1 to 4 to a vector to construct a knockout vector; (2) constructing a target DNA sequence for expressing human protective protein; (3) The knockout vector and the vector containing the target DNA sequence expressing the human protective protein are co-transfected into animal fibroblasts, and the positive clone cells with site-specific integration of the human protective protein are obtained by sequencing and identification; (4) Using positive cloned cells as nuclear donors and enucleated animal oocytes as recipients for somatic cell nuclear transplantation to obtain reconstructed embryos; (5) The reconstructed embryo is transferred into the body of a female animal, and the female animal is reared until it gives birth, and the offspring is obtained.

6. The method according to claim 5, characterized in that In step (1), the vector is a pX458 vector.

7. The method according to claim 5, characterized in that In step (2), the human protective protein includes one or two of human complement regulatory protein and human anti-cellular rejection protein; preferably, the human anti-cellular rejection protein is human anti-macrophage phagocytosis protein or human anti-T cell activation protein; more preferably, the human complement regulatory protein is CD55, CD46 or CD59, the human anti-macrophage phagocytosis protein is CD47, and the human anti-T cell activation protein is CTLA4.

8. The method according to claim 5, characterized in that In step (2), the target DNA sequence for expressing human protective protein includes a left homologous arm, an EF1α promoter, a CDS sequence of a human protective protein gene, a polyA sequence, a right homologous arm, and an HSV-TK sequence connected in sequence; preferably, the target DNA sequence for expressing human protective protein is as shown in SEQ ID NO: 6 or SEQ ID NO:

11.

9. The method according to claim 5, characterized in that In step (3), the fibroblasts are fibroblasts in which heterologous antigens are knocked out, preferably fibroblasts in which three antigens, α-gal, Sda and Neu5Gc, are knocked out.

10. The method according to any one of claims 5 to 9, characterized in that: The animal is a pig.

11. The method according to any one of claims 5 to 10, characterized in that: The method comprises the following steps: (1) Connecting sgRNA to the vector to construct knockout vector 1; (2) constructing a target DNA sequence 1 for expressing human protective protein; (3) The knockout vector 1 and the vector containing the target DNA sequence 1 expressing the human protective protein are co-transfected into animal fibroblasts, and the positive clone cells with site-specific integration of the human protective protein are obtained by sequencing and identification; (4) using the positive cloned cells obtained in step (3) as nuclear donors and enucleated animal oocytes as recipients to perform somatic cell nuclear transplantation to obtain reconstructed embryos; (5) taking the reconstructed embryo obtained in step (4) and transferring it into a female animal, raising the female animal until it gives birth, and taking the offspring to obtain a transgenic animal; (6) Connecting the sgRNA to the vector to construct knockout vector 2; (7) constructing a target DNA sequence 2 for expressing human protective protein; (8) co-transfecting the knockout vector 2 and the vector containing the target DNA sequence 2 expressing the human protective protein into the transgenic animal fibroblasts of step (5), and sequencing and identifying the positive clone cells with site-specific integration of the human protective protein; (9) using the positive cloned cells obtained in step (8) as nuclear donors and enucleated animal oocytes as recipients to perform somatic cell nuclear transplantation to obtain reconstructed embryos; (10) taking the reconstructed embryo obtained in step (9) and transferring it into a female animal, raising the female animal until giving birth, and then taking the offspring; Wherein, the sgRNA in step (1) is as described in any one of claims 1-2, the target DNA sequence 1 for expressing human-derived protection protein in step (2) is as shown in SEQ ID NO: 6, the sgRNA in step (6) is as described in any one of claims 3-4, and the target DNA sequence 2 for expressing human-derived protection protein in step (7) is as shown in SEQ ID NO: 11; or, the sgRNA in step (1) is as described in any one of claims 3-4, the target DNA sequence 1 for expressing human-derived protection protein in step (2) is as shown in SEQ ID NO: 11, the sgRNA in step (6) is as described in any one of claims 1-2, and the target DNA sequence 2 for expressing human-derived protection protein in step (7) is as shown in SEQ ID NO:

6.

12. A genetically engineered animal constructed by the method according to any one of claims 5 to 11.

13. Use of the genetically engineered animal according to claim 12 in xenotransfusion.

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