Patent Description:
With the continuous progress of modern medicine, blood transfusion has been widely used in clinical practice, but it is also facing more and more problems, including serious risk of blood transfusion caused by high incidence of infectious diseases such as Acquired Immune Deficiency Syndrome, Hepatitis B, Hepatitis C, etc. Blood demand is increasing and the amount of blood donation is relatively reduced, and blood resources are becoming increasingly scarce. Animal red blood cells (RBCs) can be used to develop alternatives to human RBCs for blood transfusion. Porcine red blood cells (pRBCs) have been widely used in the development of heterogeneous blood transfusion red blood cells, and there are a lot of similarities between pRBCs and human RBCs. Meanwhile, when raised under conditions free from specific pathogens and biosafety, pRBCs do not carry human pathogenic microorganisms. The pRBCs do not express MHC antigens, i.e., swine leukocyte antigens (SLA), therefore the immunogenicity is reduced. There are no nuclei in pRBCs, so it is impossible for pRBCs to carry porcine endogenous retrovirus.

However, the direct use of wild-type pRBCs in clinical blood transfusion may cause many problems. The infusion of wild-type pRBCs into a primate may result in a consistent hyperacute rejection. For example, since wild-type pRBCs express Gal and non-Gal antigens in human with natural hemolytic antibodies, so the lysis of blood cells can be directly caused by antibody-antigen binding and/or complement activation.

Therefore, it is urgently needed at present to acquire modified pRBCs which can be directly used for human blood transfusion.

The application provides for a method of preparing a blood product according to claim <NUM>. The blood product has at least one of the following properties: <NUM>) the binding to immunoglobulin in human serum is significantly reduced; <NUM>) having an significant effect on overcoming hyperacute rejection; <NUM>) effectively solving the problem of blood shortage in clinical practice; <NUM>) providing valuable material resources for clinical blood transfusion; <NUM>) effectively knocking out the GGTA1 gene in pigs; <NUM>) effectively knocking out the CMAH gene in pigs; <NUM>) effectively knocking out the β4GalNT2 gene in pigs; <NUM>) being suitable for mass production; <NUM>) effective quality control; and/or <NUM>) safe and reliable, without carrying pathogenic microorganisms and/or viruses.

It is surprisingly found in the application that, by knocking out appropriate exons in the genes to be knocked out, for example, by designing a suitable targeting SgRNA sequence against specific exons in the genes to be knocked out, the knockout efficiency of the genes to be knocked out (for example, comprising the GGTA1 gene, the CMAH gene and/or the β4GalNT2 gene) can be significantly improved by means of a CRISPR/Cas9 vector combination. A blood product derived from the gene knockout pig is then obtained, thus effectively reducing the hyperacute rejection caused by xenotransplantation, allowing it to be an alternative for human blood and play a variety of experimental and clinical functions.

A blood product may derive from a gene knockout pig, a GGTA1 gene, a CMAH gene and a β4GalNT2 gene of the gene knockout pig are knocked out, wherein one or more nucleotides in the β4GalNT2 gene encoding one or more amino acids in exon <NUM> are deleted such that the β4GalNT2 gene is knocked out.

One or more nucleotides in the GGTA1 gene encoding one or more amino acids in exon <NUM> are deleted such that the GGTA1 gene is knocked out.

One or more nucleotides in the CMAH gene encoding one or more amino acids in exon <NUM> are deleted such that the CMAH gene is knocked out.

The gene knockout pig is prepared by using a CRISPR/Cas9 vector combination.

According to the invention , the exon <NUM> of the GGTA1 gene, the exon <NUM> of the CMAH gene and the exon <NUM> of the β4GalNT2 gene serve as the parts targeted by CRISPR/Cas9.

The CRISPR/Cas9 vector combination comprises a GGTA1-CRISPR/Cas9 vector, a CMAH-CRISPR/Cas9 vector and a β4GalNT2-CRISPR/Cas9 vector, the GGTA1-CRISPR/Cas9 vector contains the SgRNA nucleotide sequence specifically targeting the GGTA1 gene of SEQ ID No: <NUM>, the CMAH-CRISPR/Cas9 vector contains the SgRNA nucleotide sequence specifically targeting the CMAH gene of SEQ ID No: <NUM>, and the β4GalNT2-CRISPR/Cas9 vector contains the SgRNA nucleotide sequence specifically targeting the β4GalNT2 gene of SEQ ID No: <NUM>.

In some embodiments, the GGTA1-CRISPR/Cas9 vector comprises the nucleotide sequence of SEQ ID No: <NUM>, the CMAH-CRISPR/Cas9 vector comprises the nucleotide sequence of SEQ ID No: <NUM>, and the β4GalNT2-CRISPR/Cas9 vector comprises the nucleotide sequence of SEQ ID No: <NUM>.

In some embodiments, the blood product comprises red blood cells of the gene knockout pig. In some embodiments, the blood product comprises peripheral blood mononuclear cells (PBMC) of the gene knockout pig.

In some embodiments, the red blood cells have a reduced aGal antigen level, a reduced Neu5Gc antigen level and a reduced Sda-like antigen level.

In some embodiments, the PBMC have a reduced aGal antigen level, a reduced Neu5Gc antigen level and a reduced Sda-like antigen level.

In some embodiments, the binding level of the red blood cells of the gene knockout pig to human immunoglobulin is reduced compared to red blood cells derived from a wild-type pig. In some embodiments, the binding level of the PBMC of the gene knockout pig to human immunoglobulin is reduced compared to PBMC derived from a wild-type pig.

In some embodiments, the red blood cells of the gene knockout pig have a comparable level of binding to human immunoglobulin compared to human-derived red blood cells. In some embodiments, the PBMC of the gene knockout pig have a comparable level of binding to human immunoglobulin compared to human-derived PBMC.

In some embodiments, the human immunoglobulin comprises human IgG and/or human IgM.

In some embodiments, the agglutination reaction of the red blood cells of the gene knockout pig to human serum is reduced compared to red blood cells derived from a wild-type pig. In some embodiments, the agglutination reaction is caused by an IgM antibody against a blood group antigen and/or an IgG antibody against a blood group antigen.

In some embodiments, the likelihood of a hemolytic transfusion reaction occurring after the red blood cells of the gene knockout pig are introduced into a human body is reduced compared to red blood cells derived from a wild-type pig.

The blood product may be used in the preparation of blood products for infusion into human.

The blood products for infusion into human do not substantially cause and/or are capable of ameliorating hyperacute rejection.

In this application, the term "gene knockout" generally refers to a genetic engineering means for silencing a gene and/or failing to express its encoded protein. For example, the gene knockout can use the CRISPR/Cas system.

In this application, the term "GGTA1 gene" generally refers to a gene encoding α-<NUM>,<NUM>-galactosyl transferase (GGTA1). In this application, the accession number of the GGTA1 gene of the pig (Sus scrofa) in Ensemble is ENSSSCG00000005518. The accession number of the GGTA1 pseudogene of the pig in GenBank is <NUM>.

In this application, the term "CMAH gene" generally refers to a gene encoding cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH). In this application, the accession number of the CMAH gene of the pig (Sus scrofa) in Ensemble is ENSSSCG00000001099. The accession number of the CMAH gene of the pig in GenBank is <NUM>.

In this application, the term "β4GalNT2 gene" generally refers to a gene encoding β-<NUM>,<NUM>-N-acetyl galactosaminyl transferase <NUM> (β4GalNT2). In this application, the accession number of the β4GalNT2 gene of the pig (Sus scrofa) in Ensemble is ENSSSCG00000040942. The accession number of the β4GalNT2 gene of the pig in GenBank is <NUM>. The β4GalNT2 gene of the pig and its products may be important non-Gal antigens that cause xenotransplantation rejection.

In this application, the term "exon" generally refers to a part of an eukaryotic gene. In this application, the exon may be a coding gene which can be still preserved after splicing and can be expressed as a protein during the protein biosynthesis. The exon can also be known as an expression sequence. A linearly expressed eukaryotic gene may have multiple exons. For example, the exon may be blocked with an intron. In this application, exon X (X is a positive integer) may represent the Xth exon of the gene.

In this application, the term "blood product" generally refers to blood and products prepared from blood. For example, the blood product may comprise blood, red blood cells and their constituent products, platelets and their constituent products, plasma, plasma protein products and/or coagulation factor products.

In this application, the term "CRISPR/Cas9 vector combination" generally refers to a combination of vectors comprising CRISPR (i.e., Clustered regularly interspaced short palindromic repeats, CRISPR) and the Cas gene. In this application, the CRISPR/Cas9 vector combination may be utilized to realize the gene knockout (see Deveau et al. , <NUM>; Horvath and Barrangou, <NUM>).

In this application, the term "SgRNA" generally refers to a single-stranded chimeric antibody RNA (Single guide RNA, sgRNA) in an artificial CRISPR/Cas9 system (see Deltcheva et al. , <NUM>; Bikard and Marraffini, <NUM>). The SgRNAmay be about 20bp in length. In this application, the SgRNA can bind to a target sequence, and then bind to the Cas9 protein to form a complex.

In this application, the term "red blood cells" generally refers to cells in the blood which deliver oxygen to each tissue, also known as "erythrocyte"or "red blood cells". The primary functional molecules of red blood cells may be hemoglobin, which can bind to oxygen molecules in lung, then release the bound oxygen molecules in tissues. In this application, the red blood cells can also deliver carbon dioxide. In this application, the red blood cells in mammals (for example, pigs) may have no nuclei. The red blood cells may also have no mitochondria.

In this application, the term "peripheral blood mononuclear cells (PBMC)" generally refers to peripheral blood mononuclear cell with round nuclei. PBMC may comprise lymphocytes (for example, T cells, B cells or NK cells) and monocytes. PBMC may be extracted from whole blood of mammals (for example, pigs) (for example, may be obtained by gradient centrifugation).

In this application, the term "αGal antigen" generally refers to enzymes (GT, αGal or α1,<NUM> galactosyl transferase) encoded by genes of α1,3galactosyl transferase (aGal, GGTA, GGT1, GT, αGT, GGTA1 or GGTA-<NUM>). The αGal antigen may be an epitope or antigen recognized by human immune system. Removal of αGal antigens from transgenic organ materials cannot eliminate the human immune response caused by the materials.

In this application, the term "Neu5Gc antigen" generally refers to N-glycolylneuraminic acid (Neu5Gc). The Neu5Gc antigen may be a sialic acid generated by the catalysis of the CMAH. In this application, the CMAH can catalyze the conversion of sialic acid N-acetyl neuraminic acid (Neu5Ac) to Neu5Gc. The Neu5Gc antigen may be an epitope or antigen recognized by human immune system.

In this application, the term "Sda-like antigen" generally refers to a glycosyl transferase of a pig. The Sda-like antigen may be synthesized by the catalysis of β <NUM>,4N-acetyl galactosaminyl transferase. In this application, the Sda-like antigen may comprise Sda and similar glycans associated with blood group or blood tests.

In this application, the term "a wild-type pig" generally refers to any known pig (Sus scrofa) without any modifications at genetic and/or protein level (for example, deletion, insertion, substitution and/or modification of one or more nucleotides and/or one or more amino acids). For example, the wild-type pig may be Sus scrofa domestica, for example may be Berkshire, Chester White, Duroc, Hampshire, Hereford Pig, Landrace, Poland-China swine, Spotted pig or Yorkshire. For example, the wild-type pig may be a wild boar. In this application, the wild-type pig may be a complete individual pig, or may be organs, tissues, body liquid and/or cells of a pig.

In this application, the term "human immunoglobulin" generally refers to an immunologic substance produced by the human immune system after antigen stimulation. For example, the human immunoglobulin may comprise IgA, IgD, IgE, IgG and IgM subtypes. The human immunoglobulin may be known as an antibody (for example, IgG subtype), its monomer may be a Y-shaped molecule, and the antibody may be composed of <NUM> polypeptide chains, comprising two identical heavy chains and two identical light chains, wherein the light chains and the heavy chains may comprise variable portions -V regions (also known as variable regions), and constant portions - C regions (also known as constant regions).

In this application, the term "blood group antigen" generally refers to an antigen of blood types A, B, AB and O in the AB antigen standard contained in human red blood cells.

In this application, the term "agglutination reaction" generally refers to a serological reaction caused by the binding of an antigen to an antibody. In this application, the antigen (for example, the red blood cells of the gene knockout pig) may be known as agglutinogen. The antibody (for example, an IgM antibody against a blood group antigen and/or an IgG antibody against a blood group antigen) may be known as agglutinin. The agglutination reaction may be characterized by the appearance of small clumps of agglutination visible to the naked eye.

In this application, the term "hemolytic transfusion reaction" generally refers to immune hemolytic transfusion reaction. For example, when the blood recipient is injected incompatible red blood cells or donor plasma with the presence of alloantibodies, the incompatible red blood cells and/or the donor plasma may result in damages to the red blood cells. For example, the hemolytic transfusion reaction may comprise acute (for example, reaction occurs within <NUM> hours after the blood transfusion) hemolytic transfusion reaction (AHTR) and delayed (for example, reaction occurs several days or weeks after the blood transfusion) hemolytic transfusion reaction (DHTR). For example, the hemolytic transfusion reaction may comprise intravascular hemolysis and extravascular hemolysis.

In this application, the term "hyperacute rejection" (HAR) generally refers to a failure occurred rapidly (for example, within several minutes after the transplantation) after the transplantation of exogenous organs, tissues and/or cells into a recipient. The HAR may occur in heart and/or kidney. The HAR may be associated with thymic T cells.

In this application, the term "Gal" generally refers to a terminal oligosaccharide generated from α1,<NUM>-galactosyl transferase (GGTA1). In mammals (e.g., human), the Gal may be the primary binding antigen for antibodies naturally produced in vivo. In this application, all antibodies or antigen-binding fragments thereof that do not bind to the Gal may be considered to be non-Gal antibodies.

In one aspect, the application provides for a method of preparing a blood product as claimed in claim <NUM>.

For example, one or more (for example, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or more) nucleotides in the β4GalNT2 gene encoding one or more (for example, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or more) amino acids in exon <NUM> may be deleted or added such that the β4GalNT2 gene is knocked out and/or does not express functional β4GalNT2 encoded products. In this application, the knockout efficiency of the β4GalNT2 gene may be more than about <NUM>%, for example, may be more than about <NUM>%, more than about <NUM>%, more than about <NUM>%, more than about <NUM>%, more than about <NUM>%, more than about <NUM>%, more than about <NUM>%, more than about <NUM>%, more than about <NUM>% or more than about <NUM>%.

In this application, one or more (for example, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or more) nucleotides in the GGTA1 gene encoding one or more (for example, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or more) amino acids in exon <NUM> may be deleted or added such that the GGTA1 gene is knocked out and/or does not express functional GGTA1 encoded products. In this application, the knockout efficiency of the GGTA1 gene may be more than about <NUM>%, for example, may be more than about <NUM>%, more than about <NUM>%, more than about <NUM>%, more than about <NUM>%, more than about <NUM>%, more than about <NUM>%, more than about <NUM>%, more than about <NUM>%, more than about <NUM>% or more than about <NUM>%.

In this application, one or more (for example, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or more) nucleotides in the CMAH gene encoding one or more (for example, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or more) amino acids in exon <NUM> may be deleted or added such that the CMAH gene is knocked out and/or does not express functional CMAH encoded products. In this application, the knockout efficiency of the CMAH gene may be more than about <NUM>%, for example, may be more than about <NUM>%, more than about <NUM>%, more than about <NUM>%, more than about <NUM>%, more than about <NUM>%, more than about <NUM>%, more than about <NUM>%, more than about <NUM>% or more than about <NUM>%.

In this application, the gene knockout pig is prepared by using a CRISPR/Cas9 vector combination.

The exon <NUM> of the GGTA1 gene, the exon <NUM> of the CMAH gene and the exon <NUM> of the β4GalNT2 gene serve as the parts targeted by CRISPR/Cas9.

For example, CRISPR target sequence of the GGTA1 gene may be located in exon <NUM> of the gene, near the initiation codon. Knockout of the GGTA1 gene may further comprise inserting one or more (for example, may insert <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or more) bases (for example, may insert one base) in exon <NUM> of the gene. For example, T can be inserted between T and C of exon <NUM> of the GGTA1 gene.

For example, CRISPR target sequence of the CMAH gene may be located in exon <NUM> of the gene, near the initiation codon. Knockout of the CMAH gene may further comprise inserting one or more (for example, may insert <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or more) bases (for example, may insert one base) in exon <NUM> of the gene. For example, A can be inserted between A and G of exon <NUM> of the GGTA1 gene.

For example, CRISPR target sequence of the β4GalNT2 gene may be located in exon <NUM> of the gene. For example, knockout of the β4GalNT2 gene may comprise deleting one or more (for example, may delete <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or more) bases in exon <NUM> of the gene. For example, <NUM> bases may be deleted. For example, bases of ACTCACGAAC may be deleted.

In some instances, CRISPR target sequence of the p4GalNT2 gene may not be located in exon <NUM> of the gene.

It is surprisingly found in the application that gene knockout in respect of exon <NUM> of the β4GalNT2 gene can significantly improve the knockout efficiency of the β4GalNT2 gene. Moreover, the knockout efficiency of the β4GalNT2 gene caused by the knockout in respect of exon <NUM> of the β4GalNT2 gene may be significantly higher than the knockout efficiency of knockout in respect of exon <NUM> of the β4GalNT2 gene.

In this application, the CRISPR/Cas9 vector combination comprises a GGTA1-CRISPR/Cas9 vector, a CMAH-CRISPR/Cas9 vector and a β4GalNT2-CRISPR/Cas9 vector. The GGTA1-CRISPR/Cas9 vector contains the SgRNA nucleotide sequence specifically targeting the GGTA1 gene of SEQ ID No: <NUM>. The CMAH-CRISPR/Cas9 vector contains the SgRNA nucleotide sequence specifically targeting the CMAH gene of SEQ ID No: <NUM>. And, the β4GalNT2-CRISPR/Cas9 vector contains the SgRNA nucleotide sequence specifically targeting the β4GalNT2 gene of SEQ ID No: <NUM>.

In some instances, the GGTA1-CRISPR/Cas9 vector may comprise the nucleotide sequence of SEQ ID No: <NUM>. In some instances, the CMAH-CRISPR/Cas9 vector may comprise the nucleotide sequence of SEQ ID No: <NUM>. And/or, in some instances, the β4GalNT2-CRISPR/Cas9 vector may comprise the nucleotide sequence of SEQ ID No: <NUM>.

For example, the blood product may comprise red blood cells of the gene knockout pig. Further for example, the blood product may comprise peripheral blood mononuclear cells (PBMC) of the gene knockout pig.

In this application, the red blood cells may have a reduced aGal antigen level, a reduced Neu5Gc antigen level and a reduced Sda-like antigen level.

In this application, the PBMC may have a reduced aGal antigen level, a reduced Neu5Gc antigen level and a reduced Sda-like antigen level.

In this application, the GGTA1 gene can encode α1,<NUM> galactosyl transferase. The functional α1,<NUM> galactosyl transferase may catalyze the formation of galactose α1,<NUM>-galactose (aGal) residues on glycoproteins. The aGal antigen may be an antigen or an epitope thereof recognized by human immune system.

In this application, the CMAH gene may be responsible to the synthesis of N-glycolyl neuraminic acid (Neu5Gc). For example, the CMAH gene may encode cytidine monophospho-N-acetylneuraminic acid hydroxylase, which can catalyze the conversion of sialic acid N-acetylneuraminic acid to the Neu5Gc antigen. The Neu5Gc antigen may be an antigen or an epitope thereof recognized by human immune system.

In this application, the β4GalNT2 gene can encode β1,<NUM> N-acetylgalactosaminyl transferase <NUM> glycosyl transferase (β4GalNT2). The functional β4GalNT2 may produce Sda-like glycans (for example, the Sda-like antigen). The Sda-like antigen may be an antigen or an epitope thereof recognized by human immune system.

In this application, for example, the binding level of the red blood cells of the gene knockout pig to human immunoglobulin may be reduced (for example, may be reduced by at least about <NUM> times, at least about <NUM> times, at least about <NUM> times, at least about <NUM> times or more) compared to red blood cells derived from a wild-type pig. Further for example, the binding level of the PBMC of the gene knockout pig to human immunoglobulin may be reduced (for example, may be reduced by at least about <NUM> times, at least about <NUM> times, at least about <NUM> times, at least about <NUM> times, at least about <NUM> times or more) compared to PBMC derived from a wild-type pig.

In this application, for example, the red blood cells of the gene knockout pig may have a comparable level of binding to human immunoglobulin compared to human-derived red blood cells (for example, may be about <NUM>%~<NUM>%, about <NUM>%~<NUM>%, about <NUM>%~<NUM>%, about <NUM>%~<NUM>%, about <NUM>%~<NUM>% or about <NUM>%~<NUM>% of the binding level of human-derived red blood cells to human immunoglobulin). Further for example, the PBMC of the gene knockout pig may have a comparable level of binding to human immunoglobulin compared to human-derived PBMC (for example, may be about <NUM>%~<NUM>%, about <NUM>%~<NUM>%, about <NUM>%~<NUM>%, about <NUM>%~<NUM>%, about <NUM>%~<NUM>% or about <NUM>%~<NUM>% of the binding level of human-derived PBMC to human immunoglobulin).

In this application, the human immunoglobulin may comprise human IgG and/or human IgM.

In this application, the agglutination reaction of the red blood cells of the gene knockout pig in human serum may be reduced compared to red blood cells derived from a wild-type pig (for example, may be reduced by at least about <NUM> times, at least about <NUM> times, at least about <NUM> times, at least about <NUM> times or more). In this application, the agglutination reaction may be caused by an IgM antibody against a blood group antigen and/or an IgG antibody against a blood group antigen.

In this application, the likelihood of a hemolytic transfusion reaction occurring after the red blood cells of the gene knockout pig are introduced into a human body may be reduced compared to red blood cells derived from a wild-type pig (for example, may be reduced by at least about <NUM> times, at least about <NUM> times, at least about <NUM> times, at least about <NUM> times or more).

The blood products which may be used for infusion into human may not substantially cause and/or be capable of ameliorating hyperacute rejection. The amelioration aims to make any advance or progress towards the treatment and/or relief of hyperacute rejection. For example, the blood product may be used to ameliorate at least one symptom associated with the hyperacute rejection selected from the group consisting of: thrombotic occlusion, graft vasculature bleeding, neutrophil influx, ischemia, plaque, edema, cyanosis, edema, organ failure, organ dysfunction and/or necrosis, glomerular capillary thrombosis, hemolysis, fever, coagulation, reducedbile production, hypotension, elevated serum transaminase level, elevated alkaline phosphatase level, jaundice, lethargy, acidosis, hyperbilirubinemia, and/or thrombocytopenia.

The blood product may comprise plasma, serum albumin, placental serum albumin, intravenous immunoglobulin, intramuscular immunoglobulin, histamine immunoglobulin, specific immunoglobulin, hepatitis B immunoglobulin, rabies immunoglobulin, tetanus immunoglobulin, blood coagulation factor VIII, prothrombin complex, fibrinogen, antilymphocyte immunoglobulin, antithrombin III, topical lyophilized fibrin adhesive, lyophilized thrombin, and/or S/D-FFP.

To address the immunological rejection present in existing clinical transfusion of xenogeneic red blood cells a CRISPR/Cas9 vector combination may be used in the preparation of blood products for gene knockout pigs.

Technical Solution: The use of the CRISPR/Cas9 vector combination of the application in the preparation of blood products for gene knockout pigs, wherein the gene knockout pigs are pigs with their GGTA1 gene, CMAH gene and β4GalNT2 gene being knocked-out. The CRISPR/Cas9 vector combination comprises a GGTA1-CRISPR/Cas9 vector, a CMAH-CRISPR/Cas9 vector and a β4GalNT2-CRISPR/Cas9 vector; the GGTA1-CRISPR/Cas9 vector contains the SgRNA nucleotide sequence specifically targeting the GGTA1 gene of SEQ ID No: <NUM>, the CMAH-CRISPR/Cas9 vector contains the SgRNA nucleotide sequence specifically targeting the CMAH gene of SEQ ID No: <NUM>, the β4GalNT2-CRISPR/Cas9 vector contains the SgRNA nucleotide sequence specifically targeting the β4GalNT2 gene of SEQ ID No: <NUM>.

For example, the blood products may be red blood cells.

For example, the nucleotide sequence of the GGTA1-CRISPR/Cas9 vector is SEQ ID No: <NUM>; the nucleotide sequence of the CMAH-CRISPR/Cas9 vector is SEQ ID No: <NUM>; the nucleotide sequence of the β4GalNT2-CRISPR/Cas9 vector is SEQ ID No: <NUM>.

Wherein, the CRISPR/Cas9 vector combination can be constructed as follows:.

When the CRISPR/Cas9 vector is a GGTA1-CRISPR/Cas9 vector, the SgRNA nucleotide sequence in the step (<NUM>) is SEQ ID No: <NUM>; when the CRISPR/Cas9 vector is CMAH-CRISPR/Cas9, the SgRNA nucleotide sequence in the step (<NUM>) is SEQ ID No: <NUM>; when the CRISPR/Cas9 vector is a β4GalNT2-CRISPR/Cas9 vector, the SgRNA nucleotide sequence in the step (<NUM>) is SEQ ID No: <NUM>.

The use of the CRISPR/Cas9 vector combination in the preparation of blood products for gene knockout pigs may comprise the following steps:.

The separation step of red blood cells may be as follows: The blood stored in the anticoagulation tube was added into a centrifuge tube, and diluted with a PBS solution. AFicoll-paque separation liquid was added to form a separation system, which was centrifuged to obtain a four-layer solution, including successively, from top to bottom, a plasma layer, a monocyte layer, a Ficoll-paque layer and a red blood cell layer. The top three layers were discarded, and the red blood cells were rinsed with a PBS solution to get a solution of red blood cells. For example, the volume ratio of the blood, the PBS solution and the Ficoll-paque separation liquid in the separation system may be <NUM>:<NUM>:<NUM>.

For example, the conditions for centrifugation may be: at <NUM>, centrifugation at <NUM> for <NUM>.

In this application, it should be understood that the use of the terms "a/an" and "the" and "at least one" as well as similar indications comprises singular and plural referents. Unless otherwise stated herein or clearly contradicted in the context, when the term "at least one" is used followed by one or more of the listed items (for example, "at least one of A and B"), it should be understood to be selected from one of the listed items (A or B) or any combination of two or more of the listed items (A and B).

In this application, unless otherwise noted, the term " comprise ", "have", "include" as well as "contain" should all be understood as non-exclusive terms (i.e., mean "comprise, but not limited to").

Not wishing to be bound by any theory, the following embodiments are only intended to illustrate the working modes of the device, method and system of the application and are not intended to limit the scope of the invention.

The specific characteristics of the invention referred in the application are set forth in the appended claims. The features and advantages of the invention referred in the application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:.

Firstly, based on the DNA sequences of GGTA1/CMAH/β4GalNT2 genes, sgRNA (single guide RNAs) targeting GGTA1, CMAH and β4GalNT2 genes were synthesized, thus constructing a GGTA1-CRISPR/Cas9 vector, a CMAH-CRISPR/Cas9 vector and a β4GalNT2-CRISPR/Cas9 vector respectively, with pX330 as the skeleton plasmid.

Firstly, based on the porcine GGTA1 gene sequence published in Genbank, the exon <NUM> of GGTA1 gene was selected as the CRISPR/Cas9 target. According to the design principle of cas9 target, the <NUM>' end was G, and the <NUM>' end was PAM sequence (NGG). The SgRNA sequence was designed to be GAAAATAATGAATGTCAA, as shown in <FIG>. Its nucleotide sequence is SEQ ID No: <NUM>.

The GGTA1-CRISPR/Cas9 vector was prepared as follows:.

The SgRNA sequence was cloned onto the pX330 skeleton vector, the specific steps of which were as follows:.

<NUM>µg pX330 plasmid was digested with a restriction endonuclease BbsI;
<NUM>. The digested pX330 plasmid was separated using an agarose gel (an agarose gel at a concentration of <NUM>%, i.e., <NUM> agarose gel being added into <NUM> electrophoresis buffer), the digestion product was then purified and recovered by a gel extraction kit (QIAGEN);
<NUM>. The forward and reverse oligonucleotide sequences synthesized in step III were annealed as follows:.

A ligation reaction was initiated following the system below: reaction at room temperature for <NUM>.

The ligation system was treated with a plasmid-safe exonuclease to eliminate the incorrect-ligated plasmid:.

Reaction at <NUM> for <NUM>
<NUM>. Transformation.

Mini-extraction of plasmid, sequencing, identification of the successful construction of the target plasmid.

The constructed CRSAPR/Cas9 vector is named as GGTA1-CRISPR/Cas9, its nucleotide sequence is shown in SEQ ID No: <NUM>.

Firstly, based on the porcine CMAH gene sequence published in Genbank, the exon <NUM> of CMAH gene was selected as the CRISPR/Cas9 target. According to the design principle of cas9 target, the <NUM>' end was G, and the <NUM>' end was PAM sequence (NGG). The SgRNA guide sequence was designed to be GAGTAAGGTACGTGATCTGT, as shown in <FIG>. Its nucleotide sequence is SEQ ID No: <NUM>.

The CMAH-CRISPR/Cas9 vector was prepared as follows:.

The constructed CRSAPR/Cas9 vector is named as CMAH-CRISPR/Cas9, its nucleotide sequence is shown in SEQ ID No: <NUM>.

Firstly, based on the porcine β4GalNT2 gene sequence published in Genbank, the exon <NUM> of β4GalNT2 gene was selected as the CRISPR/Cas9 target. According to the design principle of cas9 target, the <NUM>' end was G, and the <NUM>' end was PAM sequence (NGG). The guide sequence was designed to be GGTAGTACTCACGAACACTC as shown in <FIG>. Its nucleotide sequence is SEQ ID No: <NUM>.

The β4GalNT2-CRISPR/Cas9 vector was prepared as follows:.

The constructed CRSAPR/Cas9 vector is named as β4GalNT2-CRISPR/Cas9, its nucleotide sequence is SEQ ID No: <NUM>.

The GGTA1-CRISPR/Cas9 vector, the CMAH-CRISPR/Cas9 vector and the β4GalNT2-CRISPR/Cas9 vector (their profiles can be seen in <FIG>, <FIG> sequentially) widely present in mammals, which express GGTA1/CMAH/β4GalNT2 genes respectively, comprise a U6 promoter, an enhancer of a CMV-chicken-β-actin gene (CMV-chicken-β-actin enhancer), and contain a resistant gene for screening in mammal cells - Neomycin gene and a resistant gene for screening in prokaryotic cells - ampicillin gene. The U6 promoter of β-skeletal muscle actin gene (CMV-chicken-β-actin promoter) which can be extensively expressed can ensure the extensive expression of downstream genes.

The GGTA1-CRISPR/Cas9 vector, CMAH-CRISPR/Cas9 vector and β4GalNT2-CRISPR/Cas9 vector constructed in Embodiment <NUM> were co-transfected into porcine fetal fibroblasts together with tdTomato plasmid. Single-cell clones were obtained by G418 screening and identified by sequencing to obtain GGTA1/CMAH/β4GalNT2 triple knockout porcine fetal fibroblasts. GGTAl/CMAH/β4GalNT2 triple knockout Landrace pigs were prepared by somatic cell nuclear transfer (SCNT). The genome of a newborn piglet was extracted, amplified by PCR primers, and ligated with a T vector for genotyping.

The nucleofection experiments were performed by using a mammalian fibroblast nucleofection kit (Lonza) and a Lonza Nucleofactor™ 2b nucleofection instrument.

Formulation of a nucleofection reaction liquid, the system was as follows:.

The three constructed plasmids and the Tdtomato plasmid were added into the <NUM>µL nucleofection reaction liquid obtained in the previous step <NUM> at a mass ratio of <NUM>:<NUM> respectively and mixed evenly, being careful not to generate bubbles during the process;
<NUM>. The cell suspension prepared in step I was rinsed twice with Dulbecco's Phosphate Buffered Saline (DPBS, Gibco), digested at <NUM> for <NUM>. A DMEM complete medium containing fetal bovine serum at a volume percent of <NUM>% was then used to terminate the digestion. After then, the suspension was centrigued at <NUM> rpm for <NUM>. The supernatant was discarded. The cells were resuspended with the nucleofection reaction liquid containing plasmids in the previous step <NUM>, being careful to avoid the generation of bubbles during the process of resuspension;
<NUM>. The nucleofection system was added into the electroporation cuvettes contained in the kit carefully, being careful to prevent the bubbles. The electroporation cuvettes containing <NUM>µL PBS were firstly placed in the cuvette troughs of the Lonza nucleofector. After the program was adjusted by selecting U023 nucleofection procedures, the electroporation cuvettes containing cells were electroporated, and the liquid in the electroporation cuvettes was then immediately sucked out in a Clean Bench gently and transferred into <NUM> DMEM complete medium containing fetal bovine serum of <NUM>% by volume, and mixed gently;
<NUM>. Preparing several culture dishes (<NUM>) each containing <NUM> of complete medium. The cell suspension after nucleofection was pipetted and added into one culture dish containing the complete medium, and mixed evenly. The number of cells was observed under a microscope and counted such that the culture dish contained about <NUM> to <NUM> cells in a single field of view under the microscope. The remaining dishes were all added with the cell suspension following this final amount, mixed evenly and then placed in a constant temperature incubator at <NUM> and <NUM>% of CO<NUM> for culture.

When the cells were overgrown at the bottom of the wells in the <NUM>-well plate, they were digested with <NUM>% (<NUM>/<NUM>) of trypsin and collected. Then <NUM> NP-<NUM> lysis buffer was added into the cells to lyse the cells and extract genomic DNA from the cells. The lysis procedures were: at <NUM> for <NUM> - at <NUM> for <NUM> - at <NUM>. Upon the completion of the reaction, the genomic DNA was kept at -<NUM>;
<NUM>. Corresponding PCR primers were designed according to GGTA1/CMAH/β4GalNT2 gene target information. The PCR primer sequences were respectively:.

The GGTA1/CMAH/β4GalNT2 target gene was amplied by PCR reaction. The PCR reaction system was as follows:.

The reaction conditions were as follows:.

The amplification of CMAH target gene was performed the same as in the above steps; and the amplification of β4GalNT2 target gene was performed the same as in the above steps. The PCR reaction products were subjected to agarose gel electrophoresis (<NUM>%, i.e., <NUM> agarose gel being added into <NUM> electrophoresis buffer). At the end of electrophoresis, the target band was cut under ultraviolet light, and then recovered by a gel extraction kit (QIAGEN), and the concentration of the recovered PCR products was determined by using NanoDrop <NUM>;
<NUM>. The recovered PCR products were ligated with T vectors by using a TAKARA pMD™<NUM>-T Vector Cloning Kit. The T vector reaction system was as follows:.

The reaction condition of T vector ligation was reaction at <NUM> for <NUM>;
<NUM>. The T vector ligation products obtained in the above step <NUM> were transformed with competent cells (TIANGEN). After the transformation, the competent cells were coated onto an Amp-resistant LB agar solid medium, and cultured in a constant temperature incubator at <NUM> overnight;
<NUM> to <NUM> monoclonal colonies were sorted from the medium cultured overnight and sent to a sequencing company for sequencing. The sequencing results were then compared with the target GGTA1/CMAH/β4GalNT2 information to determine whether the cell line was GGTA1/CMAH/β4GalNT2 gene knockout cell line;.

A total of <NUM> monoclonal cell lines were sorted, wherein there was one biallelic knockout cell line in which three genes were knocked-out simultaneously, being numbered <NUM>#. The genotype of the clone is shown in Table <NUM>:.

It was found from the results that, the knockout efficiencies of knocking-out GGTA1 (the nucleotide sequence of GGTA1 fragment of WT in Table <NUM> is as shown in SEQ ID No: <NUM>), CMAH (the nucleotide sequence of CMAH fragment of WT in Table <NUM> is as shown in SEQ ID No: <NUM>), β4GalNT2 (the nucleotide sequence of β4GalNT2 fragment of WT in Table <NUM> is as shown in SEQ ID No: <NUM>) genes were <NUM>%, <NUM>% and <NUM>%, respectively.

Since compared with GGTA1/CMAH double knockout, the binding to IgM, IgG of human is significantly reduced in GGTA1/CMAH/β4GalNT2 triple knockout, so triple knockout is necessary.

A total of <NUM> GGTA1/CMAH/β4GalNT2 knockout pigs (TKO) were born, being numbered from <NUM> to <NUM> (as shown in <FIG>). The <NUM> born boars were consistent with the results of cell genotyping.

Following the steps in Embodiment <NUM>, the GGTA1-CRISPR/Cas9 vector constructed in Embodiment <NUM> was used alone to get GGTA1 single gene knockout (GGTA1-KO) pigs.

After the GGTA1/CMAH/β4GalNT2 knockout pigs prepared in Embodiment <NUM> were weaned, blood was drawn and peripheral blood mononuclear cells (PBMC) were isolated, and the gene knockout profile of the piglets was determined by flow cytometer.

PBMC were isolated as follows: To <NUM>µL anticoagulant blood was added <NUM> times volume of red blood cell lysis buffer (BD, diluted with deionized water by <NUM> times), and the lysis was performed at room temperature for <NUM> to <NUM>. After centrifugation, the supernatant was discarded. The remainings were rinsed with a pre-cooled washing liquid <NUM>% FBS (the solvent was PBS, <NUM>% means <NUM> FBS/<NUM> PBS)(promote cell sedimentation), and centrifuged to obtain PBMC precipitates.

The commercialized human serum was inactivated in a water bath kettle at <NUM> for <NUM> and then used to incubate the obtained PBMC on ice for <NUM>, which were then centrifuged at <NUM> rpm for <NUM>, washed with PBS for three times, blocked with goat serum with a volume ratio of <NUM>% at <NUM> for <NUM>, and washed with PBS for additional three times. After incubation with antibodies specifically binding to GGTA1, CMAH and β4GalNT2, the antibodies were washed away with PBS, resuspended and the mean fluorescence intensity was determined on a machine.

The results were shown in <FIG>, which, from top to bottom, showed the expression profiles of GGTA1, CMAH and β4GalNT2 in sequence. Wherein, PBS control group was the blank control, the isotype control group was chick IgY, WT was wild-type pig. The results showed that, the three antigens (α-<NUM>,<NUM>-galactosyl transferase (GGTA1), CMP-N-acetylneuraminic acid hydroxylase (CMAH) and β-<NUM>,<NUM>-N-acetylgalactosaminyl transferase <NUM> (β4GalNT2)) were not expressed in TKO pigs. In other words, GGTA1 gene, CMAH gene and β4GalNT2 gene in TKO have all been knocked out successfully.

PBMC were separated from GGTA1/CMAH/β4GalNT2 knockout pigs (TKO), GGTA1-KO pigs prepared in Embodiment <NUM>, as well as human and wild-type pigs following the process as described in <NUM>.

The commercialized human serum was inactivated in a water bath kettle at <NUM> for <NUM> and then used to incubate the obtained PBMC on ice for <NUM>, which were then centrifuged at <NUM> rpm for <NUM>, washed with PBS for three times, blocked with goat serum with a volume ratio of <NUM>% at <NUM> for <NUM>, and washed with PBS for additional three times. After incubation with human specific immunoglobulin antibodies (i.e., anti-human IgM antibody and anti-human IgG antibody), the antibodies were washed away with PBS, resuspended and the mean fluorescence intensity was determined on a machine.

The results were shown in <FIG>, which showed the level of binding to immunoglobulins IgM and IgG in human serum in sequence. The results showed that, compared with wild-type pigs, the binding level of PBMC of TKO to human immunoglobulins IgM and IgG was greatly reduced, with little difference from the level of binding to human PBMC in normal circumstances. However, although GGTA1-KO pigs are a little superior to wild-type pigs, the level of binding to human immunoglobulins IgM and IgG was still significantly different from that to human PBMC. It can be seen that PBMCs of TKO were capable of overcoming human hyperacute rejection.

The GGTA1/CMAH/β4GalNT2 knockout pigs (TKO) prepared in Embodiment <NUM> were immobilized, from the anterior vena cava of which <NUM> blood was drawn with a sterile syringe, and placed in an anticoagulation tube and preserved at <NUM> for one week. <NUM> of the above anticoagulant blood was taken and added into a <NUM> centrifuge tube, and then <NUM> PBS solution was added for dilution and mixed evenly. The diluted blood was slowly added into a <NUM> centrifuge tube containing <NUM> Ficoll-paque separation liquid (GE Company), at which there were two layers, wherein the upper layer was blood, and the lower layer was Ficoll-paque separation liquid. After centrifugation at <NUM> and at <NUM> for <NUM>, the liquid phase was divided into four layers, which were successively, from top to bottom, a plasma layer, a monocyte layer, a Ficoll-paque layer and a red blood cell layer. The supernatant was discarded, and the red blood cells were remained and resuspended with <NUM> PBS solution and mixed evenly. After centrifugation at <NUM> and at <NUM> for <NUM>, the supernatant was discarded, and <NUM> PBS solution was added for resuspension and mixed evenly. After centrifugation at <NUM> and at <NUM> for <NUM>, the supernatant was discarded, and <NUM> PBS was added for resuspension, ready for use.

Following this process, human RBCs and RBCs of wild-type pigs (WT) can be obtained respectively.

IB4 agglutinin interacted with carbohydrates ligated to α galactose generated from the expression products of GGTA1; and DBA agglutinin interacted with the structures of carbohydrates generated from the expression products of β4GalNT2.

<NUM>×<NUM><NUM> red blood cells prepared in step (<NUM>) were placed in a <NUM> EP tube and centrifuged at <NUM> rpm for <NUM>, discarding the supernatant. The cell precipitates were resuspended with <NUM>µL of IB4 agglutinin (purchased from Invitrogen) or DBA agglutinin (purchased from Invitrogen) dilution (at a dilution ratio of <NUM>:<NUM>) diluted with PBS, and incubated at <NUM> in dark for <NUM>. The samples which have not been incubated with agglutinin were used as blank control. They were washed twice with a PBS solution, the centrifuged precipitates were resuspended with <NUM>µL of PBS solution, detected with BD FACSCalibur flow cytometry, and analyzed using FlowJo <NUM> software, with the results being shown in <FIG>.

Columns <NUM> and <NUM> in <FIG> successively showed the results after incubation with IB4 agglutinin and DBA agglutinin respectively. Wherein, WT means wild-type pigs. It was indicated from the results in <FIG> that, unlike WT, the antigen flow results of RBC of TKO and human (RBC of human type O) against IB4 agglutinin and DBA agglutinin were all negative.

CMAH gene was capable of synthesizing saccharide molecule Neu5Gc.

<NUM>×<NUM><NUM> red blood cells prepared in step (<NUM>) were placed in a <NUM> EP tube and centrifuged at <NUM> rpm for <NUM>, discarding the supernatant. The cells were resuspended with <NUM>µL of <NUM>% diluted blocking liquid (free from mammal serum) and incubated at <NUM> in dark for <NUM>. They were washed twice with a PBS solution, the centrifuged precipitates were then resuspended with <NUM>µL of Neu5Gc antibody (Purified anti-Neu5Gc Antibody (biolegend, <NUM>)) dilution (at a dilution ratio of <NUM>: <NUM>) diluted with a PBS solution, and incubated at <NUM> for <NUM>. The samples which have not been incubated with antibodies were used as blank control. They were washed twice with a PBS solution, and the cell precipitates were then resuspended with <NUM>µL of goat-anti-chick IgY antibody (invitrogen, A11039) dilution (at a dilution ratio of <NUM>:<NUM>) diluted with a PBS solution, incubated at <NUM> in dark for <NUM>, and centrifuged at <NUM> rpm for <NUM>, discarding the supernatant. They were washed twice with a PBS solution, the centrifuged precipitates were then resuspended with <NUM>µL of PBS solution, detected with BD FACSCalibur flow cytometry, and analyzed using FlowJo <NUM> software, with the results being shown in <FIG>.

Column <NUM> in <FIG> showed the results after incubation with Neu5Gc antibody. Wherein, WT means wild-type pigs. It was indicated from the results in <FIG> that, unlike WT, the antigen flow results of RBC of TKO and human (RBC of human type O) against Neu5Gc antibody were all negative.

Human type AB serum was inactivated in advance by incubation at <NUM> for <NUM>. <NUM>×<NUM><NUM> red blood cells prepared in step (<NUM>) were placed in a <NUM> EP tube and centrifuged at <NUM> rpm for <NUM>, discarding the supernatant. The cell precipitates were resuspended with <NUM>µL of <NUM>% (v/v) human AB serum dilution diluted with a PBS solution and incubated at <NUM> for <NUM>. The samples which have not been incubated with human AB serum were used as blank control. They were washed twice with a PBS solution, and the cells were then resuspended with <NUM>µL of <NUM>% (v/v) ready-to-use normal goat serum and incubated at <NUM> for <NUM>. They were washed twice with a PBS solution, and the cell precipitates were then resuspended with <NUM>µL of goat-anti-human IgG or IgM antibody (anti-human IgM (invitrogen, A18842); anti-human IgG (invitrogen, A18830) dilution (at a dilution ratio of <NUM>:<NUM>) diluted with a PBS solution, incubated at <NUM> in dark for <NUM>, and centrifuged at <NUM> rpm for min, discarding the supernatant. They were washed twice with a PBS solution, the centrifuged precipitates were then resuspended with <NUM>µL of PBS, detected with BD FACSCalibur flow cytometry, and analyzed using FlowJo <NUM> software, with the results being shown in <FIG>.

The results in <FIG> showed that WT means wild-type pigs. It was indicated from the results in <FIG> that, the capabilities of human red blood cells and red blood cells of TKO to bind to human IgG and IgM were all significantly lower than that of red blood cells of wild-type pigs. It can be seen that RBCs of TKO are capable of overcoming human hyperacute rejection.

See Embodiment <NUM> for the acquisition process of RBC.

The collected porcine blood, after being washed for three times, was formulated to a <NUM>% red blood cell suspension. <NUM>µL of <NUM>% (v/v) RBCs (WT pigs and TKO pigs) suspension and <NUM>µL of normal human (types A, B, AB and O) serum were respectively added into glass test tubes, incubated at <NUM> for <NUM>, centrifuged at <NUM> for <NUM>, discarding the supernatant. They were washed with normal saline for <NUM> times, into which were respectively added <NUM>µL of anti-human IgG antibodies (Shanghai Blood Biomedicine Co. LTD), centrifuged at <NUM> for <NUM> at ambient temperature, and shaked gently. After then, the agglutination degrees were observed, with the results being shown in <FIG>.

It was indicated from the results in <FIG> that, regardless of gender, the agglutination intensities between RBCs of TKO pigs and human type A, B, AB and O sera were significantly lower than the agglutination intensities between RBCs of wild-type pig and human type A, B, AB and O sera. The agglutination degrees were determined following the vertical coordinates in <FIG>: <NUM>: No agglutination or hemolysis; ±: turbid background, small scattered incompact agglutination blocks, after shaking, the agglutination blocks became invisible; <NUM>+: turbid background, small scattered incompact agglutination blocks, after shaking, the agglutination blocks were still visible; <NUM>+: incompact agglutination blocks, clear background, after shaking, the background became turbid; <NUM>+: several compact agglutination blocks, clear background; <NUM>+: one compact agglutination block. With the increase of the number, the agglutination degree increased continuously.

Each <NUM> drops of anti-A human polyclonal antibody, anti-B human polyclonal antibody and anti-D human polyclonal antibody (all purchased from The Institute of Blood Transfusion, Chinese Academy of Medical Sciences) were added into <NUM> drop of <NUM>% RBCs (WT pigs, TKO pigs and human) suspension to be detected after being washed for three times. They were added into a test tube together, mixed evenly, and centrifuged at <NUM> for <NUM> seconds. The agglutination degrees were observed by naked eyes, with the results being shown in <FIG>.

It was indicated from the results in <FIG> that compared with red blood cells of wild-type pigs, the agglutination degree of red blood cells of TKO pigs to human serum was significantly weakened.

<FIG> shows the agglutination titers of red blood cells of WT pigs and TKO pigs to human AB serum respectively. The maximum dilution of RBC at which obvious agglutination phenomenon appeared was used as the agglutination titer. In <FIG>, the agglutination degrees were determined as follows: <NUM>: No agglutination or hemolysis; ±: turbid background, small scattered incompact agglutination blocks, after shaking, the agglutination blocks became invisible; <NUM>+: turbid background, small scattered incompact agglutination blocks, after shaking, the agglutination blocks were still visible; <NUM>+: incompact agglutination blocks, clear background, after shaking, the background became turbid; <NUM>+: several compact agglutination blocks, clear background; <NUM>+: one compact agglutination block. With the increase of the number, the agglutination degree increased continuously. +S means strong, i.e., strengthened; +W means weak, i.e., weakened. <NUM> (type A) and <NUM> (type O) blood samples were taken from WT pigs, <NUM> (type A) and <NUM> (type O) blood samples were taken from TKO pigs.

See Embodiment <NUM> for the acquisition process of RBCs (WT pigs, TKO pigs and human). These RBCs were used as donor red blood cells.

Human type A, type B, type AB, and type O sera (all taken from healthy blood donors) were used as recipient sera.

Two tiny test tubes were taken and marked as the main tube and the self control tube respectively. The main tube was added with <NUM> drops of recipient serum and <NUM> drop of donor red blood cell suspension; the self control tube was added with <NUM> drops of recipient serum and <NUM> drop of recipient red blood cell suspension. They were mixed evenly by shaking and centrifuged at <NUM> for <NUM> seconds. The agglutination degrees were observed by naked eyes, with the results being shown in <FIG>.

It was indicated from the results in <FIG> that during the determination of red blood cell agglutination caused by an IgM antibody against a blood group antigen, the agglutinations of red blood cells of TKO pigs in various blood group sera of human were significantly reduced compared to those of red blood cells of wild-type pigs.

The loading of the main tube and the self control tube was achieved following the operational steps of saline method as described in Embodiment <NUM>. The mixture was mixed evenly and incubated at <NUM> for <NUM> minutes. Red blood cells were washed for three times, and the tubes were dried by patting after the last washing. Each tube was added with <NUM> drop of antihuman IgG antibody (purchased from Shanghai Blood Biomedicine Co. LTD), mixed, and centrifuged at <NUM> for <NUM> seconds. The results were observed and shown in <FIG>.

It was indicated from the results in <FIG> that during the determination of red blood cell agglutination caused by an IgG antibody against a blood group antigen, the agglutinations of red blood cells of TKO pigs in various blood group sera of human were significantly reduced compared to those of red blood cells of wild-type pigs.

Human blood samples were taken from blood donors, who meet the National standards for blood donors' health examination. <NUM> of whole blood was draw from each person, and preserved at <NUM>, ready for use. <NUM> (type A), <NUM> (type O) blood samples were taken from WT pigs, <NUM># (type A), <NUM># (type O) blood samples were taken from TKO pigs.

Lymphocyte separation liquid (AS1114545, Axis-Shield, Norway), RPMI <NUM> basic medium (gibco, US), fetal bovine serum (FBS, <NUM>, Sciencell, US), Wright-Giemsa Stain (DN0007, Leagene Biotech. , Ltd), Methanol (Sinopharm). Chamber system (154534PK, Thermo Fisher, US), Upright Microscope (BX53, Olympus, Japan).

<NUM> of the whole blood was transferred into a <NUM> centrifuge tube, into which was added an equal amount of PBS for dilution and mixed evenly. Into a <NUM> centrifuge tube was added <NUM> of the lymphocyte separation liquid, and the diluted blood was added gently to the top of the lymphocyte separation liquid in the centrifuge tube, and centrifuged at <NUM> rpm at room temperature for <NUM>, during which the centrifugal speed rose and fell slowly. After centrifugation, the cell layer at which PBMC were located was white. This layer of cells were pipetted into another <NUM> centrifuge tube, which were washed twice by adding PBS, resuspended in RPMI <NUM> +<NUM>% FBS medium, and cultivated in the chamber system (<NUM>µL/well, <NUM> cells), and incubated in an incubator at <NUM> and <NUM>% of CO<NUM> for <NUM> hour to make them adhere to the wall.

Porcine red blood cells pRBC (WT pigs and TKO pigs) were drawn, washed twice with PBS, and counted. Sera were drawn from human whole blood. <NUM>×<NUM><NUM> red blood cells were mixed with <NUM>µL of sera evenly, incubated in an incubator at <NUM> and <NUM>% of CO<NUM> for <NUM> hour. A positive control group (Incubation of human-derived anti-D antibody and human red blood cells) and a negative control group (Incubation of type AB serum and human type O red blood cells) were set. They were washed with PBS for <NUM> times, resuspended in <NUM>µL RPMI <NUM> +<NUM>% FBS medium.

The adherent PBMCs were sucked out of the medium, then <NUM>µL of pRBCs which have been reacted with the serum were added, incubated in an incubator at <NUM> and <NUM>% of CO<NUM> for <NUM> hours, rinsed with PBS twice, immobilized with methanol at room temperature for <NUM>, and stained with Wright-Giemsa Stain at room temperature for <NUM> minute; an equal amount of phosphate dilution was added and left at room temperature for <NUM>. The stain was then flushed away with water. It was dried and then photographed.

The mean phagocytic index was determined as follows: observation using a microscope, photographing, and counting more than <NUM> cells. The mean phagocytic index = Number of adherent or phagocytic red blood cells/Total number of cells × <NUM>%.

Claim 1:
A method of preparing a blood product, comprising preparing a gene knockout pig and isolating a blood product from said gene knockout pig;
wherein said gene knockout pig is prepared through somatic cell nuclear transfer by using a CRISPR/Cas9 vector combination; the exon <NUM> of said GGTA1 gene, the exon <NUM> of said CMAH gene and the exon <NUM> of said β4GalNT2 gene serve as the parts targeted by CRISPR/Cas9;
wherein the CRISPR/Cas9 vector combination comprises a GGTA1-CRISPR/Cas9 vector, a CMAH-CRISPR/Cas9 vector and a β4GalNT2-CRISPR/Cas9 vector, said GGTA1-CRISPR/Cas9 vector contains the sgRNA nucleotide sequence specifically targeting the GGTA1 gene of SEQ ID No: <NUM>, said CMAH-CRISPR/Cas9 vector contains the sgRNA nucleotide sequence specifically targeting the CMAH gene of SEQ ID No: <NUM>, and said β4GalNT2-CRISPR/Cas9 vector contains the sgRNA nucleotide sequence specifically targeting the β4GalNT2 gene of SEQ ID No: <NUM>.