WO2022012663A1 - Virus insusceptible animal and construction method therefor - Google Patents

Abstract

Provided are a virus insusceptible animal and a construction method therefor. The method comprises knocking out all or part of a gene encoding sialyltransferase in an animal, such as knocking out all or part of a gene encoding a 2,3-galactose sialyloligosaccharide (SAα2, 3Gal) receptor and/or a gene encoding a 2,6-galactose sialyloligosaccharide (SAα2, 6Gal) receptor, so that the animal becomes a virus insusceptible animal.

Description

Virus insensitive animal and construction method thereof technical field

The present application relates to the field of biomedicine, in particular to a virus-insensitive animal and a construction method thereof.

Background technique

Since the infection of animals (such as swine strains) with influenza virus is a relatively complex physiological and pathological process involving multiple receptor signaling pathways, influenza viruses may mutate in the bodies of infected animals (such as swine strains). risk of further infecting people with subsequent influenza viruses.

The CRISPR/Cas9 system can achieve highly flexible and specific genome editing in eukaryotic cells, and is currently the most popular next-generation genome editing technology in the field of genome editing. At present, this technology has been used to construct various gene knockout cell lines and Gene knockout animal model.

Sialyltransferases are involved in cell adhesion, migration, and synapse formation, and are involved in the development and remodeling of the nervous system.

SUMMARY OF THE INVENTION

The present application provides a method for constructing a virus-insensitive animal, and a virus-insensitive animal constructed by the method. Animals (such as pig strains) obtained using the method described in this application have knocked out all or part of the gene encoding sialyltransferase, and become virus-insensitive animals, avoiding infection with human influenza virus and/or avian influenza virus, avoiding the The animal itself serves as an intermediate host for different influenza viruses, and also avoids the risk of the animal serving as a breeding ground for influenza virus recombination and mutation.

In one aspect, the present application provides a method for constructing a virus-insensitive animal, the method comprising knocking out all or part of a gene encoding a sialyltransferase in the animal, thereby making the animal a virus-insensitive animal.

In certain embodiments, the gene encoding a sialyltransferase comprises ST3Gal4 gene, ST6Gal1 gene, ST3Gal3 gene and/or ST3Gal6 gene.

In certain embodiments, the sialyltransferase comprises a 2,3-galactosialo-oligosaccharide (SAα2,3Gal) receptor, and/or a 2,6-galactosialo-oligosaccharide (SAα2,6Gal) receptor receptor.

In certain embodiments, the gene encoding the 2,3-galactosialooligosaccharide (SAα2,3Gal) receptor comprises the ST3Gal4 gene.

In certain embodiments, the gene encoding the 2,3-galactosialyloligosaccharide (SAα2,6Gal) receptor comprises the ST6Gal1 gene.

In certain embodiments, the animal includes livestock.

In certain embodiments, the animal comprises a porcine.

In certain embodiments, the knockout comprises adding, replacing and/or deleting one or more nucleotides in the gene encoding a sialyltransferase, allowing expression of the gene encoding a sialyltransferase The amount is reduced, and/or, the gene encoding the sialyltransferase is substantially not expressed; and/or the amino acid sequence of the sialyltransferase is altered, and/or the sialyltransferase is deactivated live.

In certain embodiments, the method comprises knocking out all or part of a gene encoding a 2,3-galactosialooligosaccharide (SAα2,3Gal) receptor in an animal, and/or knocking out a gene encoding 2,3-galactosialyloligosaccharide (SAα2,3Gal) receptor in an animal All or part of the gene for the 6-galactosialo-oligosaccharide (SAα2,6Gal) receptor, thereby rendering the animal less susceptible to the virus.

In certain embodiments, the knockout comprises adding, replacing and/or deleting one or more nucleotides in the ST3Gal4 gene and/or the ST6Gal1 gene, so that the ST3Gal4 gene and/or the ST6Gal1 gene The expression level of the ST6Gal1 gene is reduced, and/or the ST3Gal4 gene and/or the ST6Gal1 gene is substantially not expressed; and/or the SAα2,3Gal receptor and/or the SAα2,3Gal receptor are reduced The amino acid sequence of , and/or, inactivate the SAα2,3Gal receptor and/or the SAα2,3Gal receptor.

In certain embodiments, the knockout involves knocking out all or part of 2 or more exons of the ST3Gal4 gene.

In certain embodiments, the knockout involves knocking out all or part of one or more exons of the ST6Gal1 gene.

In certain embodiments, the method comprises the step of using one or more deoxyribonucleic acid (DNA) endonucleases to produce a gene within or near the ST3Gal4 gene and/or the ST6Gal1 gene or more single-strand breaks (SSBs) or double-strand breaks (DSBs) resulting in the deletion of all or part of one or more exons of the ST3Gal4 gene and/or the ST6Gal1 gene.

In certain embodiments, the DNA endonuclease comprises a Cas nuclease.

In certain embodiments, the Cas nucleases include Cas9 nucleases, homologues thereof, recombinants of naturally occurring molecules thereof, codon-optimized versions thereof, and/or modified versions thereof.

In certain embodiments, the method further comprises the use of one or more guide RNAs (gRNAs).

In certain embodiments, the gRNA is a single-stranded guide RNA (sgRNA).

In certain embodiments, the gRNA is capable of specifically binding to a target region in the ST3Gal4 gene that may comprise the nucleic acid sequence set forth in any one of SEQ ID NOs: 5-6 and 20.

In certain embodiments, the gRNA capable of specifically binding to the ST3Gal4 gene may comprise the nucleic acid sequence shown in any one of SEQ ID NOs: 9-10 and 21-22.

In certain embodiments, the gRNA is capable of specifically binding to a target region in the ST6Gal1 gene that may comprise the nucleic acid sequence shown in any one of SEQ ID NOs: 7-8.

In certain embodiments, the gRNA capable of specifically binding to the ST6Gal1 gene may comprise the nucleic acid sequence shown in any one of SEQ ID NOs: 11-12 and 23-24.

In certain embodiments, the 5' end of the gRNA includes a nucleic acid sequence shown in (X)n, wherein X is a base selected from any one of A, U, C, and G, and n is Any integer from 0-15. In certain embodiments, the n is 13 or 2.

In certain embodiments, the 3' end of the gRNA includes a backbone sequence.

In certain embodiments, the method comprises: (1) providing a cell that may comprise one or more vectors comprising the gRNA or an in vitro transcription product of the vector; (2) subjecting the The cells are cultured in a culture medium; (3) the cultured cells are transplanted into the oviduct of a recipient female non-human mammal, allowing the cells to develop in the uterus of the female non-human mammal; and (4) ) identify germline transmission in the genetically modified non-human mammals of progeny of the pregnant female of step (3).

In certain embodiments, the virus comprises influenza virus. In certain embodiments, the influenza virus comprises human influenza virus and/or avian influenza virus.

In certain embodiments, the virus-insensitive animal does not substantially express the endogenous SAα2,3Gal receptor and/or the SAα2,6Gal receptor capable of interacting with surface proteins of the virus.

In certain embodiments, the virus-insensitive animal does not substantially express the endogenous SAα2,3Gal receptor and/or the SAα2,6Gal receptor.

In another aspect, the present application provides a gRNA that specifically binds to the ST3Gal4 gene, wherein the gRNA specifically binds to a target region comprising the nucleic acid sequence shown in any one of SEQ ID NOs: 5-6 and 20.

In certain embodiments, the gRNA targets more than 2 different exons in the ST3Gal4 gene.

In certain embodiments, the gRNA may comprise the nucleic acid sequence shown in any one of SEQ ID NOs: 9-10 and 21-22.

In another aspect, the present application provides a gRNA that specifically binds to the ST6Gal1 gene, wherein the gRNA specifically binds to a target region that can comprise the nucleic acid sequence shown in any one of SEQ ID NOs: 7-8.

In certain embodiments, the gRNA targets 1 exon in the ST6Gal1 gene.

In certain embodiments, the gRNA can comprise the nucleic acid sequence set forth in any one of SEQ ID NOs: 11-12 and 23-24.

In certain embodiments, the gRNA is a single-stranded guide RNA (sgRNA).

In certain embodiments, the 5' end of the gRNA includes a nucleic acid sequence shown in (X)n, wherein X is a base selected from any one of A, U, C, and G, and n is Any integer from 0-15.

In certain embodiments, the n is 13 or 2.

In certain embodiments, the 3' end of the gRNA includes a backbone sequence.

In another aspect, the application provides a nucleic acid molecule encoding the gRNA described in the application.

In another aspect, the present application provides vectors that can contain the sequences of the gRNAs described herein.

In another aspect, the application provides a cell that can comprise one or more of the gRNAs described in the application, the nucleic acid molecules described in the application, the gRNA vectors described in the application, and/or the gRNA described in the application. In vitro transcripts of gRNA vectors.

On the other hand, the application provides the gRNA described in the application, the nucleic acid molecule described in the application, the gRNA vector described in the application, the in vitro transcription product of the gRNA vector described in the application, and/or the gRNA vector described in the application. The use of cells in knocking out ST3Gal4 gene and/or ST6Gal1 gene, or the use of constructing virus insensitive to infection.

On the other hand, the application provides a ST3Gal4 gene-deficient cell line, which uses the gRNA described in the application, the nucleic acid molecule described in the application, the gRNA vector described in the application, and the gRNA vector described in the application in vitro. Transcription product, and/or obtained from the cell preparation described herein.

In certain embodiments, the virus-insensitive animal does not substantially express endogenous SAα2,3Gal receptors capable of interacting with viral surface proteins.

In certain embodiments, the virally insensitive animal does not substantially express the endogenous SAα2,3Gal receptor.

On the other hand, the present application provides a ST6Gal1 gene-deficient cell line, which uses the gRNA described in the present application, the nucleic acid molecule described in the present application, the gRNA vector described in the present application, and the gRNA vector described in the present application. Transcription product, and/or obtained from the cell preparation described herein.

In another aspect, the present application provides a virus-insensitive animal prepared according to the method described herein, wherein the virus-insensitive animal does not substantially express endogenous SAα2,6Gal receptors capable of interacting with viral surface proteins.

In certain embodiments, the virally insensitive animal does not substantially express the endogenous SAα2,6Gal receptor.

On the other hand, the application provides a method for preparing a virus-insensitive animal, the method comprising:

(a) providing the virus-insensitive animals described in this application; and

(b) mating the virus-insensitive animal obtained in step (a) with other animals or in vitro fertilization or further gene editing based on the virus-insensitive animal obtained in step (a) or transplanting human tissues and cells into step (a) The obtained virus is not susceptible to animals, and is screened to obtain virus-resistant animals.

In certain embodiments, the method comprises: combining a virus-insensitive animal described herein that substantially does not express endogenous SAα2,3Gal receptors with a virus-insensitive animal described herein that substantially does not express endogenous SAα2,3Gal receptors Viruses for the sexual SAα2,6Gal receptor are not susceptible to animal hybridization.

In certain embodiments, the method comprises: screening for animals that do not substantially express the endogenous SAα2,3Gal receptor and do not substantially express the endogenous SAα2,6Gal receptor after the hybridization, Obtain virus insensitive animals.

In another aspect, the present application provides a virus-insensitive animal prepared according to the method described in the present application.

In certain embodiments, the virus-insensitive animals include livestock. In certain embodiments, the virus-insensitive animals include porcine animals.

In certain embodiments, the virus comprises influenza virus. In certain embodiments, the influenza virus comprises human influenza virus and/or avian influenza virus.

In another aspect, the present application provides a cell or cell line or primary cell culture, wherein the cell or cell line or primary cell culture is derived from the virus-insensitive animal described herein or its progeny.

In another aspect, the present application provides a tissue or organ or a culture thereof, wherein the tissue or organ or a culture thereof is derived from the virus-insensitive animal described in the present application or its progeny.

In another aspect, the present application provides a CRISPR/Cas9 system for specifically targeting the ST3Gal4 gene, which uses a DNA sequence containing the gRNA described in the present application that can specifically target the ST3Gal4 gene.

In another aspect, the present application provides a nucleic acid molecule kit capable of specifically targeting the ST3Gal4 gene, wherein the kit includes the gRNA described in the present application.

In another aspect, the present application provides a set of nucleic acid molecules that can specifically target ST3Gal4 gene, wherein the set of nucleic acid molecules includes the sgRNA described in the present application and the nucleic acid molecules encoding Cas9 protein.

On the other hand, the present application provides a CRISPR/Cas9 system for specifically targeting the ST6Gal1 gene, which is characterized by using a DNA sequence containing the gRNA described in the present application that can specifically target the ST6Gal1 gene.

In another aspect, the present application provides a nucleic acid molecule kit capable of specifically targeting ST6Gal1 gene, wherein the kit includes the gRNA described in the present application.

In another aspect, the present application provides a set of nucleic acid molecules that can specifically target ST6Gal1 gene, wherein the set of nucleic acid molecules includes the sgRNA described in the present application and the nucleic acid molecules encoding Cas9 protein.

In another aspect, the present application provides the use of the virus-insensitive animals described herein in the development of antiviral products, or as a model system for pharmacology, immunology, microbiology and medical research.

On the other hand, the present application provides the application of the virus-susceptible animals described in the present application in screening, validating, evaluating or studying antiviral drugs or combination drugs, and/or pharmacodynamic studies.

Other aspects and advantages of the present application can be readily appreciated by those skilled in the art from the following detailed description. Only exemplary embodiments of the present application are shown and described in the following detailed description. As those skilled in the art will recognize, the content of this application enables those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention to which this application relates. Accordingly, the drawings and descriptions in the specification of the present application are only exemplary and not restrictive.

Description of drawings

The invention to which this application relates is set forth with particularity characteristic of the appended claims. The features and advantages of the inventions involved in this application can be better understood by reference to the exemplary embodiments described in detail hereinafter and the accompanying drawings. A brief description of the drawings is as follows:

Figure 1 shows the results of sequencing and alignment of amplified ST3Gal4 genes in Bama miniature pigs and Landrace pigs. The porcine ST3Gal4 gene can be as described in GenBank Gene ID: 396602.

Figure 2 shows the results of sequencing and alignment of amplified ST6Gal1 genes of Bama miniature pigs and Landrace pigs. The porcine ST6Gal1 gene can be described in GenBank Gene ID: 100302026.

Figure 3 shows the results of the plasmid map of the porcine knockout vector.

Figure 4 shows the results of the plasmid map of the resistance vector.

Figure 5 shows the results of the porcine ST3Gal4 knockout sequencing product. The porcine ST3Gal4 gene can be as described in GenBank Gene ID: 396602.

Figure 6 shows the results of the porcine ST6Gal1 gene knockout sequencing product. The porcine ST6Gal1 gene can be described in GenBank Gene ID: 100302026.

detailed description

The embodiments of the invention of the present application are described below with specific specific examples, and those skilled in the art can easily understand other advantages and effects of the invention of the present application from the contents disclosed in this specification.

Definition of Terms

In this application, the term "sialyltransferase" generally refers to the family of mammalian sialyltransferases. The sialyltransferase may include α2,3-, α2,6-, α2,8- and other subtypes. The sialyltransferase has a type II glycoprotein topology, and has an L-sialic acid modified region and an S-sialic acid modified region on the catalytic region. The sialyltransferases are involved in cell adhesion, migration and synapse formation, and in the development and remodeling of the nervous system. The gene encoding sialyltransferase may include ST3Gal4 gene, ST6Gal1 gene, ST3Gal3 gene and/or ST3Gal6 gene.

In the present application, the term "2,3-galactosialyloligosaccharide (SAα2,3Gal) receptor" generally refers to a sialyltransferase, which may also be referred to as Sialyltransferase 4C (Beta-Galactoside Alpha-2, 3-Sialytransferase. It can be located on the surface of animal cells (such as mammalian cells, such as pig somatic cells). For example, the SAα2,3Gal receptor can be located in the respiratory epithelial cells of animals. In the present application, the sialic acid Can refer to a class of compounds that can act as receptor determinants for viruses such as influenza virus. Said sialic acids can be divided into 5-N-acetylneuraminic acid (Neu5Ac) and 5-N-glycolylneuraminic acid ( Neu5GC), may also include O-acetyl-5-N-acetylneuraminic acid. The sialic acid may be linked to the end of the sugar chain by a glycosidic bond (eg SAα2,3Gal or SAα2,6Gal) through its carbon atom at the second position Different (influenza) viruses can specifically recognize sugar chains of different domains (for example, 3-galactosialic acid or SAα2,3Gal) and use it as a binding receptor.

In this application, the term "2,6-galactosialyloligosaccharide (SAα2,6Gal) receptor" generally refers to a sialyltransferase, which may also be referred to as Sialyltransferase 1 (Beta-Galactoside Alpha-2, 6-Sialyltransferase). Similar to the 2,3-galactosialo-oligosaccharide (SAα2,3Gal) receptor, the 2,6-galactosialo-oligosaccharide (SAα2,6Gal) receptor can also be located in the respiratory epithelial cells of animals. It can also be specifically recognized by (influenza) viruses and use it as a binding receptor.

In the present application, the term "ST3Gal4 gene" generally refers to the gene encoding the 2,3-galactosialooligosaccharide (SAα2,3Gal) receptor. In pigs, the ST3Gal4 gene has a Gene ID of 396602 in GenBank. The porcine ST3Gal4 gene can include 16 exons. The porcine ST3Gal4 gene can have multiple isoforms, for example, can include X3, X6, X2, X5 and X1.

In the present application, the term "ST6Gal1 gene" generally refers to the gene encoding the 2,6-galactosialo-oligosaccharide (SAα2,6Gal) receptor. In pigs, the GeneID of the ST6Gal1 gene in GenBank is 100302026. The porcine ST6Gal1 gene can include 14 exons. The porcine ST6Gal1 gene may have multiple isoforms, which may include, for example, X3, X2, and X1.

In this application, the term "virus insensitive animal" generally refers to an animal that is not or not susceptible to infection by a virus (eg, influenza virus).

In this application, the term "knockout" generally refers to a specific gene in the body (for example, the gene encoding 2,3-galactosialylo-oligosaccharide (SAα2,3Gal) receptor and/or the gene encoding " 2,6-galactosialyloligosaccharide (SAα2,6Gal) receptor gene) inactivation or deletion. In this application, the knockout approach includes DNA homologous recombination, insertion mutation, iRNA, etc., for example, Knockouts can be performed using the CRISPR/Cas9 system.

In this application, the term "viral susceptibility" generally refers to the state in which an organism is susceptible to infection by a virus (eg, influenza virus). In the present application, the infection may refer to a pathological phenomenon in which a virus invades an organism and multiplies in the body. The infection can cause tissue damage and even clinical symptoms. The infection may include a silent infection, that is, only causing the body to produce a specific immune response, causing no or only minor tissue damage, and thus not showing any clinical symptoms.

In this application, the term "livestock" generally refers to animals (eg, mammals) that are domesticated by humans and whose reproduction can be controlled by humans. The livestock may include pigs, cattle, sheep, horses, cats, dogs, camels and/or rabbits.

In this application, the term "suidae" generally refers to Suidae, a family of the suborder Suiformes of the order Artiodactyla. In the present application, the suidae may include animals belonging to the genus Sus. For example, the suidae can include domestic pigs (Sus scrofa domesticus).

In this application, the term "Bama minipig" generally refers to the Bama Xiang pig, which may also be referred to as "two-headed black". The Bama miniature pig has the characteristics of black head and buttocks and white coat color, and is resistant to rough feeding, prolific and precocious.

In this application, the term "Landrace" generally refers to a lean pig breed obtained by crossing a Danish pig with a Yorkshire pig. Its body is long, its coat color is all white, its body is wedge-shaped, light in the front and heavy in the back, fast growth, high feed utilization rate, and high lean meat rate. Landrace pigs are mostly used for breeding lean-meat breeds and preparing hybrid pig breeds. Landrace pigs have defects such as weak physique and poor resistance to stress.

In the present application, the term "reduced expression level" generally refers to the reduced transcription and/or expression level of the ST3Gal4 gene and/or ST6Gal1 gene in an organism compared to the wild type. In the present application, the reduction may refer to a reduction of at least about 30%, at least about 35%, at least about 40%, at least about 45% compared to the wild type of the animal (eg, a pig which may be wild type) , at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% Or more. In the present application, the reduction may be substantially no transcription and/or no expression in the organism.

In the present application, the term "substantially not expressing" generally means that SAα2,3Gal receptors and/or SAα2,6Gal receptors are present in an organism compared to the wild-type of said animal (eg, a pig which may be wild-type). The expression level in the % or less, to about 7% or less, to about 6% or less, to about 5% or less, to about 4% or less, to about 3% or less, to about 2% or less, to about 1% or less, to about 0.5% or less or less.

In this application, the term "endonuclease" generally refers to a class of nucleic acid hydrolases that hydrolyze phosphodiester bonds within DNA molecular chains to generate oligo/oligonucleotides. The endonuclease may have strict restriction sites. In the present application, the endonuclease may have base specificity. In the present application, the endonuclease may include an enzyme that breaks down DNA.

In this application, the term "single-strand break (SSB)" generally refers to DNA damage in which only a single strand of the DNA double-strand is broken. After the single-strand break occurs, the cell may use a variety of DNA damage discovery and repair mechanisms to repair it, for example, may include non-homologous end joining (NHEJ, which can be independent of homologous DNA sequences, through DNA Ligase directly links the broken DNA ends) repair and homologous recombination (HR) repair. In the present application, the repair for the single-strand break may be NHEJ repair. The NHEJ repair can bring about deletion mutations.

In this application, the term "double-strand break (DSB)" generally refers to DNA damage in which both strands of DNA are broken. In the present application, the repair of the single-strand break can also bring about deletion mutations.

In this application, the term "Cas nuclease" generally refers to a class of enzymes that are complementary to CRISPR sequences and are able to use the CRISPR sequence as a guide, thereby recognizing and cleaving a specific DNA strand. Non-limiting examples of Cas proteins include: Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl , Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csf1 , Csf2, Csf3, Csf4, and/or their homologs, or modified forms thereof. In some embodiments, the Cas protein is a Cas9 protein.

In this application, the term "Cas9 nuclease", also known as Csn1 or Csx12, generally refers to a class of proteins in the type II CRISPR/Cas system that are involved in both crRNA biosynthesis and destruction of invading DNA. Cas9 proteins typically include a RuvC nuclease domain and an HNH nuclease domain, which cleave two different strands of a double-stranded DNA molecule, respectively. It has been tested in different bacterial species such as S. thermophiles, Listeria innocua (Gasiunas, Barrangou et al. 2012; Jinek, Chylinski et al. 2012) and Streptococcus pyogenes The Cas9 protein is described in (S. Pyogenes) (Deltcheva, Chylinski et al. 2011). For example, the Cas9 protein of Streptococcus pyogenes, its amino acid sequence can be found in the SwissProt database accession number Q99ZW2; the Neisseria meningitides Cas9 protein, its amino acid sequence can be found in the UniProt database number A1IQ68; Streptococcus thermophilus (Streptococcus thermophilus) Cas9 protein, its amino acid sequence is shown in UniProt database number Q03LF7; Staphylococcus aureus Cas9 protein, its amino acid sequence is shown in UniProt database number J7RUA5.

In this application, the term "guide RNA (gRNA)" generally refers to an RNA component that can be included in CRISPR, also referred to as guide RNA (gRNA). A guide RNA may generally comprise a spacer and a backbone sequence, which may be in the same molecule or in different molecules. The role of the guide RNA can include directing the Cas9 protein to cleave a DNA site complementary to the guide sequence (ie, can direct the Cas9 protein to cleave the target region). In the present application, the guide sequence may be any polynucleotide that is sufficiently complementary to the target region so that the guide sequence hybridizes to the target region and directs the specific binding of the CRISPR complex to the target region sequence. In the present application, the degree of complementarity between the guide sequence and its corresponding target region may be more than about 50% or more. In the present application, the guide sequence can be about 12 or more nucleotides or more in length. In the present application, the target region may be a DNA double-stranded region, which may include a DNA single-strand that may include a nucleotide sequence directly complementary to the guide RNA, or may include another DNA single-strand complementary to the DNA single-strand A single strand of DNA.

In the present application, the backbone sequence may be the remaining sequences other than the guide sequence that are necessary in the guide RNA. For example, the backbone sequence may comprise crRNA sequences (tracr mate sequence, CRISPR RNA) and tracrRNA (transactivating crRNA) sequences, which are generally not altered by changes in the target region. In certain instances, the crRNA sequence can be considered to include the nucleotide sequence of the guide sequence.

In the present application, the guide RNA may be a single-stranded guide RNA (sgRNA), or a double-stranded guide RNA composed of crRNA and tracrRNA. In this application, the structure of the backbone sequence can be derived from any commercially available and/or sequence-known vector/plasmid and/or literature. For example, the backbone vector involved in this application can be A and B in Figure 1 (Figure 1), A, B, C in Figure 3 (Figure 3) in the literature (Nowak et al. Nucleic Acids Research 2016.44:9555-9564), And the parts other than the spacer sequence described in A, B, C, D, and E in Figure 4 (Figure 4).

In this application, the term "single-stranded guide RNA (sgRNA)" is usually a chimeric single-stranded guide RNA, which can generally include a spacer, a crRNA sequence and a tracrRNA sequence. In some cases, the crRNA sequence can be considered to contain the guide sequence. In this case, the single-stranded guide RNA can be considered to include the crRNA sequence and the tracrRNA sequence. Wherein the crRNA sequence and the tracrRNA sequence can be linked into a single molecule through a linker loop. In the present application, the loop forming sequence may be four nucleotides in length, eg, may be GAAA. Longer or shorter loop-forming sequences can also be used, eg, can include triplets (eg, AAA), and additional nucleotides (eg, C or G). In the present application, the loop forming sequence may include CAAA and AAAG. In the present application, the number of the loop-forming sequences may be 2 or more. In the present application, the single-stranded guide RNA may further comprise a transcription termination sequence, such as a Poly-U sequence.

In this application, the term "specifically binds" generally means that a particular nucleotide sequence can bind to another nucleotide sequence. For example, the spacer can specifically bind to the corresponding target region. The specificity can be determined by indicators such as affinity and/or binding strength.

In the present application, the term "5'-N _(17-20) -NGG3'" generally refers to the structure of the target region, which in turn includes, from the 5' end, the nucleotide sequence N _{17 complementary to the spacer. -20} and PAM. Among them, the PAM sequence can be in the form of -NGG, and "N" can be any one of A, T, C, and G. Cas9 can make a double-strand break (Double Strand Break) in front of the PAM sequence. In the present application, N _17-20 may be 17-20 nucleotides in length.

In this application, the term "gRNA vector" generally refers to a vector capable of expressing guide RNAs. In the present application, the gRNA vector can generate gRNA after transcription in vivo, eg, can generate sgRNA (eg, sgRNA directed against porcine ST3Gal4 gene and/or ST6Gal1 gene). In the present application, the gRNA may also express a Cas nuclease, such as a Cas9 nuclease. In the present application, the gRNA vector may be a double-stranded vector or a single-stranded vector.

In this application, the term "in vitro transcription product" generally refers to the product of a transcription process that occurs in vitro. In the present application, the in vitro transcription product may include sgRNA and/or Cas9 mRNA. The in vitro transcription may use reagents such as RNA polymerase, NTP, transcription buffer, PCR Mix and/or de-RNase H _{2 O.} In this application, the in vitro transcription can use sgRNA in vitro transcription kit, and can be operated according to the instructions therein

In this application, the term "germline transmission" generally refers to a genetic trait that can be preserved during reproduction of the germline. In the present application, the germline may be a small group of species from the same or similar origins at the beginning of the lineage. For example, all or a portion of the ST3Gal4 gene and/or ST6Gal1 gene may continue to be in the genome of an animal described herein that is bred (eg, crossed) within the germline and/or between germlines to produce progeny (eg, progeny) knockout form.

In this application, the term "influenza virus" generally refers to a virus capable of causing influenza. The influenza viruses may be referred to as Orthomyxoviridae viruses. The influenza virus may be a negative-strand RNA virus. The influenza virus can infect humans, mammals and/or birds. For example, the influenza viruses can be subdivided into three categories: influenza A viruses (eg, can infect humans and birds); influenza B viruses (eg, can infect humans) and influenza C viruses (eg, can infect humans and mammals) .

In this application, the term "avian influenza virus" generally refers to a virus capable of causing Avian Influenza (AI) in birds. Bird flu viruses can generally infect birds and, in rare cases, pigs. According to the antigenic type of hemagglutinin (16 subtypes, H1-H16) and neuraminidase (9 subtypes, N1-N9) on its mantle. In the present application, the avian influenza virus (eg H5N1) can also infect pigs and/or humans. For example, the avian influenza viruses may include AH5N1, AH7N9, AH7N7, and AH9N2.

In this application, the term "human influenza virus" generally refers to viruses of infectious diseases such as influenza (Influenza). In the present application, the human influenza virus may include influenza A virus; influenza B virus and influenza C virus. Symptoms of the influenza may include high fever, runny nose, sore throat, muscle aches, headache, cough and/or tiredness. For example, the swine flu virus can also infect humans. The swine influenza virus may include A H1N1 and H3N2.

In this application, the term "and/or" should be understood to mean either or both of the alternatives.

In this application, the term "may comprise" generally means including the expressly specified features, but not excluding other elements.

In this application, the term "about" generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.

Detailed description of the invention

In one aspect, the present application provides a method for constructing a virus-insensitive animal, the method comprising knocking out all or part of a gene encoding a sialyltransferase in the animal, thereby making the animal a virus-insensitive animal.

In the present application, for example, the gene encoding sialyltransferase may include ST3Gal4 gene, ST6Gal1 gene, ST3Gal3 gene and/or ST3Gal6 gene. For example, at least one (eg, 1, 2, 3, 4, or more) of the genes encoding sialyltransferases can be knocked out.

In the present application, for example, the sialyltransferase may include a 2,3-galactosialo-oligosaccharide (SAα2,3Gal) receptor, and/or a 2,6-galactosialo-oligosaccharide (SAα2,6Gal) ) receptors.

For example, the gene encoding the 2,3-galactosialooligosaccharide (SAα2,3Gal) receptor may include the ST3Gal4 gene. For example, the gene encoding the 2,3-galactosialooligosaccharide (SAα2,6Gal) receptor may include the ST6Gal1 gene.

In the present application, the animals may include livestock. For example, the animal may include a porcine. For example, the porcine can be a domestic pig, such as a landrace pig.

In the present application, for example, the knockout may include addition, substitution and/or deletion of one or more nucleotides in the gene encoding sialyltransferase such that the Reduced expression (eg, the in vivo transcription and/or expression level of the sialyltransferase-encoding gene is reduced by at least 30%, at least 35%, at least 40%, at least 45% compared to wild-type pigs , at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more), and/or, making The gene encoding the sialyltransferase is substantially not expressed (eg, the transcription and/or expression level of the gene encoding the sialyltransferase in vivo is reduced to less than 15%, to 14%, compared to wild-type pigs % or less, to 13% or less, to 12% or less, to 11% or less, to 10% or less, to 9% or less, to 8% or less, to 7% or less, to 6% or less, to 5% or less, to 4 % or less, to 3% or less, to 2% or less, to 1% or less, to 0.5% or less, to 0.1% or less, or even reaching levels that are difficult to detect by conventional detection methods in the art); and/ Alternatively, the amino acid sequence of the sialyltransferase is altered, and/or the sialyltransferase is inactivated.

In certain embodiments, the method can comprise knocking out all or part of a gene encoding the 2,3-galactosialosialylo oligosaccharide (SAα2,3Gal) receptor in the animal, and/or knocking out in the animal the gene encoding 2 ,6-galactosialo-oligosaccharide (SAα2,6Gal) receptor gene all or part, thereby rendering the animal insensitive to the virus.

In the present application, for example, the knockout may include adding, replacing and/or deleting one or more nucleotides in the ST3Gal4 gene and/or the ST6Gal1 gene, so that the ST3Gal4 gene and/or the ST3Gal4 gene and/or all The expression level of the ST6Gal1 gene is reduced (for example, the transcription and/or expression level of the ST3Gal4 gene and/or the ST6Gal1 gene in vivo is reduced by at least 30%, at least 35%, at least 30% compared to wild-type pigs 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more) , and/or, making the ST3Gal4 gene and/or the ST6Gal1 gene substantially not expressed (e.g., compared with wild-type pigs, the transcription of the ST3Gal4 gene and/or the ST6Gal1 gene in vivo and/or Expression levels decreased to below 15%, to below 14%, to below 13%, to below 12%, to below 11%, to below 10%, to below 9%, to below 8%, to below 7%, to 6% % or less, to 5% or less, to 4% or less, to 3% or less, to 2% or less, to 1% or less, to 0.5% or less, to 0.1% or less, and even using conventional detection in the field levels that are difficult to detect by means);

And/or, the amino acid sequence of the SAα2,3Gal receptor and/or the SAα2,6Gal receptor is changed, and/or, the SAα2,3Gal receptor and/or the SAα2,6Gal receptor are deactivated. live.

The knockout can be performed using any technique known in the art capable of adding, replacing, and/or deleting any base pair and/or nucleotide in the ST3Gal4 gene and/or the ST6Gal1 gene (eg, using CRISPR). /Cas systems, ZFNs, TALENs, etc.), and any combination of these.

In the present application, for example, the knockout may involve knocking out all or part of 2 or more exons of the ST3Gal4 gene.

In the present application, for example, the knockout may involve knocking out all or part of one or more exons of the ST6Gal1 gene.

In the present application, for example, the method may comprise the step of using one or more deoxyribonucleic acid (DNA) endonucleases to produce in or near the ST3Gal4 gene and/or the ST6Gal1 gene One or more single-strand breaks (SSBs) or double-strand breaks (DSBs) resulting in the deletion of all or part of one or more exons of the ST3Gal4 gene and/or the ST6Gal1 gene.

In the present application, for example, the endonuclease may comprise a Cas nuclease.

In the present application, for example, the Cas nuclease can include Cas9 nuclease, homologues thereof, recombinants of naturally occurring molecules thereof, codon-optimized versions thereof, and/or modified versions thereof.

In the present application, Cas9 nuclease sequences and structures may be known to those skilled in the art (see e.g. "Complete genome sequence of an Ml strain of Streptococcus pyogenes, Ferretti JJ, McShan ff.M., Ajdic DJ, Savic DJ, Savic G., Lyon K., Primeaux C., Sezate S., Suvorov AN, Kenton S., Lai HS, Lin SP, Qian Y., Jia HG, Najar FZ, Ren Q., Zhu H., Song L. expand/collapse author list McLaughlin RE, Proc.Natl.Acad.Sci.USA 98:4658-4663 (2001); uCRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Deltcheva E., Chylinski K., Sharma CM, Gonzales K., Chao Y., Pirzada ZA, Eckert MR, Vogel J., Charpentier E., Nature 471:602-607 (2011); and "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity, Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821 (2012).

In the present application, the Cas9 homologues include, but are not limited to, S. pyogenes and S. thermophilus. Other suitable Cas9 nucleases and sequences will also be apparent to those skilled in the art based on the content of this application, and their sequences can be found in Chylinski, Rhun, and Charpentier, "The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems" (2013) RNA Biology 10:5, 726-737.

In the present application, the Cas9 nuclease may include a protein that may comprise a Cas9 protein or a fragment thereof, eg, may comprise at least about 70% identical, at least about 80% identical, at least about 90% identical to wild-type Cas9 Amino acid sequences that are identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical.

In the present application, for example, the method may further comprise the use of one or more guide RNAs (gRNAs).

In the present application, for example, the gRNA may be a single-stranded guide RNA (sgRNA).

In the present application, for example, the gRNA is capable of specifically binding to a target region in the ST3Gal4 gene that may comprise the nucleic acid sequence shown in any one of SEQ ID NOs: 5-6. For example, the gRNA can specifically bind to a target region in the ST3Gal4 gene that can comprise the nucleic acid sequence shown in any one of SEQ ID NOs: 5-6 and 20.

In the present application, the number of the gRNAs capable of specifically binding to the ST3Gal4 gene may be one or more (eg, one, two or more). For example, 2 (1 pair) of the gRNAs that specifically bind to the ST3Gal4 gene can be used. The one or more gRNAs that can specifically bind to the ST3Gal4 gene can target different exons in the ST3Gal4 gene, or can target the same exon in the ST3Gal4 gene.

At this time, the two gRNAs can be located on the same expression vector (for example, on the same knockout vector), or can be located on different expression vectors respectively. The expression vectors respectively containing different said gRNAs can be administered to the test animals (eg, pigs) simultaneously, or they can be administered to the test animals sequentially and/or intermittently.

When the number of the gRNAs is two or more, the target sequences specifically bound by the gRNAs may have a certain distance/interval. For example, the number of bases between two target sequences specifically bound by the gRNA is 3n+1, where n is an integer greater than or equal to 0. For example, in the ST3Gal4 gene, the number of bases spaced between two target sequences to which the gRNAs specifically bind is 4, 7, 10, 13 or more.

In the present application, for example, the gRNA that can specifically bind to the ST3Gal4 gene may comprise the nucleic acid sequence shown in any one of SEQ ID NOs: 9-10. For example, the gRNA capable of specifically binding to the ST3Gal4 gene may comprise the nucleic acid sequence shown in any one of SEQ ID NOs: 9-10 and 21-22.

In the present application, for example, the gRNA is capable of specifically binding to a target region in the ST6Gal1 gene that may comprise the nucleic acid sequence shown in any one of SEQ ID NOs: 7-8.

In the present application, the number of the gRNAs capable of specifically binding to the ST6Gal1 gene may be one or more (eg, one, two or more). For example, 2 (1 pair) of the gRNAs that specifically bind to the ST6Gal1 gene can be used. The one or more gRNAs that can specifically bind to the ST6Gal1 gene can target different exons in the ST3Gal4 gene, or can target the same exon in the ST6Gal1 gene.

At this time, the two gRNAs can be located on the same expression vector (for example, on the same knockout vector), or can be located on different expression vectors respectively. The expression vectors respectively containing different said gRNAs can be administered to the test animals (eg, pigs) simultaneously, or they can be administered to the test animals sequentially and/or intermittently.

When the number of the gRNAs is two or more, the target sequences specifically bound by the gRNAs may have a certain distance/interval. For example, the number of bases between two target sequences specifically bound by the gRNA is 3n+1, where n is an integer greater than or equal to 0. For example, in the ST6Gal1 gene, the number of bases spaced between two target sequences to which the gRNAs specifically bind is 4, 7, 10, 13 or more.

In the present application, for example, the gRNA that can specifically bind to the ST6Gal1 gene may comprise the nucleic acid sequence shown in any one of SEQ ID NOs: 11-12. For example, the gRNA capable of specifically binding to the ST6Gal1 gene may comprise the nucleic acid sequence shown in any one of SEQ ID NOs: 11-12 and 23-24.

In the present application, for example, the 5' end of the gRNA may include a nucleic acid sequence represented by (X)n, wherein X is a base selected from any one of A, U, C, and G, and n is any integer from 0-15. In this application, the n may be 13 or 2, for example.

In the present application, for example, the 3' end of the gRNA may include a backbone sequence.

In the present application, for example, the method may comprise: (1) providing a cell that may comprise one or more of the gRNA vectors or in vitro transcription products of the gRNA vectors; (2) adding the The cells are cultured in a culture medium; (3) the cultured cells are transplanted into the oviduct of a recipient female non-human mammal, allowing the cells to develop in the uterus of the female non-human mammal; and (4) ) identify germline transmission in the genetically modified non-human mammals of progeny of the pregnant female of step (3).

In the present application, specifically, the method may include: the first step, an sgRNA vector capable of expressing the gRNA may be prepared and obtained according to the method described in the present application. In the second step, the obtained in vitro transcription products of the sgRNA vector (for example, Cas9 mRNA may also be included) can be mixed, the mixed solution can be injected into the cytoplasm or nucleus of the animal fertilized egg, and the injected fertilized egg can be transferred to Cultivated in culture medium. In the third step, the well-developed cell-stage embryos are selected and transplanted into the oviduct of the female non-human mammal for continued development to obtain the F0 generation of the non-human mammal. In the fourth step, the extracted genome of the F0 generation is tested by PCR technology to verify whether the ST3Gal4 gene and/or the ST6Gal1 gene in the cells have been successfully knocked out.

In the present application, for example, the virus may include influenza virus. In the present application, for example, the influenza virus may include human influenza virus and/or avian influenza virus.

In the present application, for example, the virus-insensitive animal may substantially not express the endogenous SAα2,3Gal receptor and/or the SAα2,6Gal receptor capable of interacting with surface proteins of the virus (For example, endogenous expression levels of the SAα2,3Gal receptor and/or the SAα2,6Gal receptor are reduced to less than 15%, to less than 14%, to less than 13% compared to wild-type pigs , to 12% or less, to 11% or less, to 10% or less, to 9% or less, to 8% or less, to 7% or less, to 6% or less, to 5% or less, to 4% or less, to 3% or less , to 2% or less, to 1% or less, to 0.5% or less, to 0.1% or less, or even reaching a level that is difficult to detect by conventional detection methods in the art).

In the present application, for example, the virus-insensitive animal may substantially not express the endogenous SAα2,3Gal receptor and/or the SAα2,6Gal receptor (eg, compared to wild-type pigs, The endogenous expression level of the SAα2,3Gal receptor and/or the SAα2,6Gal receptor is reduced to below 15%, to below 14%, to below 13%, to below 12%, to below 11%, To 10% or less, to 9% or less, to 8% or less, to 7% or less, to 6% or less, to 5% or less, to 4% or less, to 3% or less, to 2% or less, to 1% or less, to 0.5% or less, to 0.1% or less, and even to a level that is difficult to detect by conventional detection methods in the art).

In another aspect, the present application provides a gRNA that specifically binds to the ST3Gal4 gene, wherein the gRNA specifically binds to a target region that can comprise the nucleic acid sequence shown in any one of SEQ ID NOs: 5-6. For example, wherein the gRNA-specific binding can comprise the target region of the nucleic acid sequence set forth in any one of SEQ ID NOs: 5-6 and 20.

In the present application, for example, the gRNA can target more than 2 different exons in the ST3Gal4 gene.

In the present application, for example, the gRNA may comprise the nucleic acid sequence shown in any one of SEQ ID NOs: 9-10. For example, the gRNA can comprise the nucleic acid sequence shown in any one of SEQ ID NOs: 9-10 and 21-22.

In another aspect, the present application provides a gRNA that specifically binds to the ST6Gal1 gene, wherein the gRNA specifically binds to a target region comprising the nucleic acid sequence shown in any one of SEQ ID NOs: 7-8.

In the present application, for example, the gRNA can target 1 exon in the ST6Gal1 gene.

In the present application, for example, the gRNA may comprise the nucleic acid sequence shown in any one of SEQ ID NOs: 11-12. For example, the gRNA can comprise the nucleic acid sequence set forth in any of SEQ ID NOs: 11-12 and 23-24.

In the present application, for example, the gRNA may be a single-stranded guide RNA (sgRNA).

In the present application, the gRNA may conform to the sequence arrangement rule of 5'-N(17-20)-NGG3' or 5'-CCN-N(17-20)-3'. For example, the NGG and/or CCN may be PAM sequences.

In the present application, for example, the 5' end of the gRNA may include a nucleic acid sequence represented by (X)n, wherein X is a base selected from any one of A, U, C, and G, and n is any integer from 0-15.

In this application, for example, the n may be 13, and for example, the n may be 2.

In the present application, for example, the 3' end of the gRNA may include a backbone sequence.

In another aspect, the application provides a nucleic acid molecule encoding the gRNA described in the application.

In the present application, the nucleic acid molecule may encompass RNA as well as single- and/or double-stranded DNA. The nucleic acid molecule can be naturally occurring, eg, in the context of a genome, transcript, mRNA, tRNA, rRNA, siRNA, snRNA, plasmid, cosmid, chromosome, chromatid, or other naturally occurring nucleic acid molecule. The nucleic acid molecule can also be a non-naturally occurring molecule, such as recombinant DNA or RNA, artificial chromosomes, engineered genomes, or fragments thereof, or synthetic DNA, RNA, DNA/RNA hybrids, or include non-naturally occurring nucleosides acid or nucleoside. For example, in the case of chemically synthesized molecules, the nucleic acid molecule may comprise nucleoside analogs, such as analogs with chemically modified bases or sugars, and backbone modifications.

In another aspect, the present application provides vectors comprising the sequences of the gRNAs described herein.

In the present application, the vector may also comprise a vector capable of translating the nucleotide sequence of the gRNA sequence. In the present application, the vectors may include plasmids, viral vectors, cosmids, artificial chromosomes and phagemids. The vector may contain one or more marker sequences suitable for use in identifying and/or selecting cells. For example, the markers may include genes encoding proteins that increase or decrease resistance or susceptibility to antibiotics (eg, kanamycin, ampicillin) or other compounds, the activity of which is determined by standards known in the art Genes for detectable enzymes such as galactosidase, alkaline phosphatase, or luciferase, and genes that significantly affect the phenotype of transformed or transfected cells, hosts, colonies, or plaques. For example, the vector may be a vector of PX series, pUC series, pGEM series, pET series, pBAD series, pTET series, or pGEX series.

In another aspect, the application provides a cell comprising one or more gRNAs described in the application, nucleic acid molecules described in the application, gRNA vectors described in the application, and/or gRNAs described in the application In vitro transcription product of the vector.

In the present application, the cells may include eukaryotic cells. For example, the cells can include mammalian cells. For example, the cells may include CHO cells, HEK cells and/or somatic porcine cells. For example, the cells can be fibroblasts, such as porcine fibroblasts.

On the other hand, the application provides the gRNA described in the application, the nucleic acid molecule described in the application, the gRNA vector described in the application, the in vitro transcription product of the gRNA vector described in the application, and/or the gRNA vector described in the application. The use of cells in knocking out ST3Gal4 gene and/or ST6Gal1 gene, or the use of constructing virus insensitive to infection.

In this application, due to the knockout of the endogenous ST3Gal4 gene and/or ST6Gal1 gene in animals (such as pigs), the expression levels of SAα2,3Gal receptors and/or SAα2,6Gal receptors in pigs are significantly down-regulated (even without expression), and/or lost the binding ability of the SAα2,3Gal receptor to the surface protein of avian influenza virus, and the binding ability of the SAα2,6Gal receptor to the surface protein of human influenza virus, respectively, thereby at least avoiding animals (such as pigs) at the same time. Infection with avian and human influenza viruses, avoiding animals (eg, pigs) as mixing vessels and intermediate hosts for recombinant mutation of avian and human influenza viruses. The viruses constructed using the gRNAs described in the present application are not susceptible to animals, and will not act as the above-mentioned intermediate host to simultaneously infect avian influenza viruses and human influenza viruses, so that new mammals (such as pigs and/or human beings) that can be infected will not be produced. ) of the new influenza virus, increasing safety.

On the other hand, the application provides a ST3Gal4 gene-deficient cell line, which uses the gRNA that specifically binds to the target sequence of the ST3Gal4 gene described in the application, the nucleic acid molecule described in the application, the gRNA vector described in the application, The in vitro transcription product of the gRNA vector described in this application, and/or the cell preparation described in this application.

In the present application, for example, the virus-insensitive animal may substantially not express endogenous SAα2,3Gal receptors capable of interacting with viral surface proteins.

In the present application, for example, the virus-insensitive animal may substantially not express the endogenous SAα2,3Gal receptor.

On the other hand, the application provides a ST6Gal1 gene-deficient cell line, which uses the gRNA that specifically binds to the ST6Gal1 gene described in the application, the nucleic acid molecule described in the application, the gRNA vector described in the application, and the gRNA described in the application. The in vitro transcription product of the gRNA vector described above, and/or the cell preparation described in this application.

In another aspect, the present application provides a virus-insensitive animal prepared according to the method described herein, wherein the virus-insensitive animal does not substantially express endogenous SAα2,6Gal receptors capable of interacting with viral surface proteins.

In the present application, for example, the virus-insensitive animal may substantially not express the endogenous SAα2,6Gal receptor.

On the other hand, the application provides a method for preparing a virus-insensitive animal, the method may include:

(a) providing the virus-insensitive animals described in this application; and

(b) mating the virus-insensitive animal obtained in step (a) with other animals or in vitro fertilization or further gene editing based on the virus-insensitive animal obtained in step (a) or transplanting human tissues and cells into step (a) The obtained virus is not susceptible to animals, and is screened to obtain virus-resistant animals.

In the present application, for example, the method may comprise: combining the virus-insensitive animal described herein that does not substantially express the endogenous SAα2,3Gal receptor with the substantially non-expressing animal of any of the present application Viruses derived from SAα2,6Gal receptors are not susceptible to animal hybridization.

In the present application, for example, the method may comprise: screening for animals that do not substantially express the endogenous SAα2,3Gal receptor and do not substantially express the endogenous SAα2,6Gal receptor after the hybridization , to obtain virus-susceptible animals.

For example, pigs in a ST3Gal4 knockout pig line homozygous for the ST3Gal4 gene knockout can be crossed with pigs in a ST6Gal1 knockout pig line homozygous for the ST6Gal1 gene knockout, by further sexual reproduction (e.g. After further selfing), the population was expanded, and a stable ST6Gal1 and ST3Gal4 double gene knockout pig line was established.

On the other hand, the present application provides a virus-insensitive animal prepared according to the method described in the present application.

In the present application, for example, the virus-insensitive animals may include livestock. In the present application, for example, the virus-insensitive animals may include porcine animals. For example, the virus insensitive animal can include pigs.

In the present application, for example, the virus may include influenza virus. In the present application, for example, the influenza virus may include human influenza virus and/or avian influenza virus.

In another aspect, the present application provides a cell or cell line or primary cell culture, wherein the cell or cell line or primary cell culture is derived from the virus-insensitive animal described herein or its progeny.

In another aspect, the present application provides a tissue or organ or a culture thereof, wherein the tissue or organ or a culture thereof is derived from the virus-insensitive animal described in the present application or its progeny.

In the cells or cell lines or primary cell cultures described herein, or in the tissues or organs described herein, or cultures thereof, the expression of genes encoding sialyltransferases may be reduced, such as The expression level of the ST3Gal4 gene and/or the ST6Gal1 gene can be reduced (for example, the transcription and/or expression level of the ST3Gal4 gene and/or the ST6Gal1 gene in vivo is reduced compared to wild-type pigs. at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% %, at least 95% or more), and/or, the gene encoding a sialyltransferase is not substantially expressed, for example, the ST3Gal4 gene and/or the ST6Gal1 gene is substantially not expressed (for example, compared to wild-type pigs In comparison, the transcription and/or expression levels of the ST3Gal4 gene and/or the ST6Gal1 gene in vivo are reduced to less than 15%, to less than 14%, to less than 13%, to less than 12%, to less than 11%, to 10% or less, to 9% or less, to 8% or less, to 7% or less, to 6% or less, to 5% or less, to 4% or less, to 3% or less, to 2% or less, to 1% or less, to 0.5% or less, to 0.1% or less, and can even reach a level that is difficult to detect by conventional detection methods in the field);

And/or, in the cells or cell lines or primary cell cultures described in the present application, or in the tissues or organs or cultures thereof described in the present application, the SAα2,3Gal receptor and/or all The amino acid sequence of the SAα2,6Gal receptor is altered, and/or the SAα2,3Gal receptor and/or the SAα2,6Gal receptor loses activity (for example, loses the ability to specifically bind to viral proteins) .

In another aspect, the present application provides a CRISPR/Cas9 system for specifically targeting the ST3Gal4 gene, which uses a DNA sequence containing the gRNA described in the present application that can specifically target the ST3Gal4 gene.

In another aspect, the present application provides a nucleic acid molecule kit capable of specifically targeting ST3Gal4 gene, wherein the kit can include the gRNA described in the present application.

On the other hand, the present application provides a set of nucleic acid molecules that can specifically target ST3Gal4 gene, wherein the set of nucleic acid molecules may include the sgRNA described in the present application and the nucleic acid molecules encoding Cas9 protein.

On the other hand, the present application provides a CRISPR/Cas9 system for specifically targeting the ST6Gal1 gene, which is characterized by using a DNA sequence containing the gRNA described in the present application that can specifically target the ST6Gal1 gene.

In another aspect, the present application provides a nucleic acid molecule kit capable of specifically targeting ST6Gal1 gene, wherein the kit may include the gRNA described in the present application.

On the other hand, the present application provides a set of nucleic acid molecules that can specifically target ST6Gal1 gene, wherein the set of nucleic acid molecules may include the sgRNA described in the present application and the nucleic acid molecules encoding Cas9 protein.

In the present application, the kit may include Cas protein, and/or nucleic acid molecules encoding Cas protein. The kit may include a targeting vector.

In the present application, in the set of nucleic acid molecules, the sgRNA and the nucleic acid molecule encoding the Cas9 protein may be located in the same vector, or may be located in different vectors.

In another aspect, the present application provides the use of the virus-insensitive animals described herein in the development of antiviral products, or as a model system for pharmacology, immunology, microbiology and medical research.

In another aspect, the present application provides the application of the virus-insensitive animals described in the present application in screening, validating, evaluating or researching antiviral drugs or combination drugs, and/or pharmacodynamic studies.

In this application, the conventional methods of pharmacology, immunology, microbiology and medical research, as well as methods for screening, validating, evaluating or researching antiviral drugs and methods for studying the efficacy of antiviral drugs are routine in the art Those skilled in the art can learn about the method by referring to the literatures and textbooks disclosed in the art.

Without intending to be limited by any theory, the following examples are only used to illustrate the virus insensitive animals, preparation methods and uses of the present application, and are not intended to limit the scope of the invention of the present application.

Example

Example 1 Amplification of target gene

The genomic DNAs of Bama miniature pigs and Landrace pigs were obtained respectively, and the genomic DNAs were used as amplification templates, and the primers in Table 1 were used to amplify the ST3Gal4 gene (about 537bp or about 664bp in length) and the ST6Gal1 gene (about 698bp in length or about 664bp in length) respectively. about 958bp). For example, the sequence of the forward primer corresponding to ST3Gal4 can be as shown in SEQ ID: 1, the sequence of the reverse primer corresponding to ST3Gal4 can be as shown in SEQ ID: 2, for example, the sequence of the forward primer corresponding to ST6Gal1 can be as shown in SEQ ID : 3, the sequence of the reverse primer corresponding to ST6Gal1 can be as shown in SEQ ID: 4.

Table 1 Primers for amplifying ST3Gal4 gene and ST6Gal1 gene

The PCR reaction system is as follows:

Genomic DNA 2μL

Forward primer (10pM) 1μL; Reverse primer (10pM) 1μL

2X Taq Enzyme Master Mix 25μL

_{ddH 2} O 21 μL

Total 50μL

PCR reaction conditions are as follows:

The PCR reaction product was subjected to agarose gel electrophoresis (1%, that is, 1 g of agarose gel was added to 100 mL of electrophoresis buffer). After electrophoresis, the target band was cut under ultraviolet light, and then recovered using a gel recovery kit (QIAGEN). The band of interest was sequenced (see Figures 1 and 2 for results).

The results showed that the sequences of ST3Gal4 gene and ST6Gal1 gene of Bama miniature pigs and Landrace pigs were completely consistent with the nucleotide sequences recorded in the database (see Table 2). It can be seen that the ST3Gal4 and ST6Gal1 genes of Bama miniature pigs and Landrace pigs have been correctly amplified.

Table 2 Target sequences

species gene name GenBank Gene ID pig ST3Gal4 396602 pig ST6Gal1 100302026

Example 2 Knock out (Knock out) sgRNA design

Using the online design tool Cas-Designer (http://www.rgenome.net/cas-designer/), the nucleotide sequences of the porcine ST3Gal4 gene and ST6Gal1 gene were input for sgRNA design.

sgRNA sequences were selected for subsequent knockout experiments. To further improve the knockout efficiency, a pair of sgRNAs are designed for each gene, so that they can be knocked out simultaneously.

See Table 3 for the nucleotide sequence of the target sequence corresponding to the sgRNA. Wherein the nucleotides underlined (-) are PAM regions. Among them, the target sequences of sgRNA1 and sgRNA2 that specifically bind to ST3Gal4 gene are located in two different exons of ST3Gal4 gene, respectively. The target sequences of sgRNA1 and sgRNA2 that specifically bind to ST6Gal1 gene are located in the same exon of ST6Gal1 gene.

Table 3 sgRNAs knocking out ST3Gal4 gene and ST6Gal1 gene

Example 3 Construction of knockout (Knock out) vector

1. The vector adopts pX330-U6-Chimeric_BB-CBh-hSpCas9 purchased from addgene company, the specific sequence of the vector is available at http://www.addgene.org/42230/sequences/;

2. f1ori is the single-stranded DNA replication origin, Cas9 is the Cas9 protein coding sequence in the CRISPR-Cas9 system, AmpR promoter is the AmpR gene promoter, AmpR is the Amp resistance coding sequence, ori is the replication origin, U6 promoter is U6 promoter, chickenβ-actin promoter is chicken β-globin promoter, CMV enhancer is CMV enhancer, Bbs I is the restriction endonuclease Bbs I restriction site, and sgRNA represents the corresponding sgRNA that can be produced after transcription DNA sequence (see figure for plasmid map). The specific sgRNA sequences involved are shown in Table 4, and the ST3Gal4 gene or ST6Gal1 knockout vectors of pigs, cats and dogs were obtained respectively. The sequences contained in the vector are shown in Table 5.

Table 4 Sequences of targeting vectors for knocking out ST3Gal4 gene

Table 5 Sequences of targeting vectors for knocking out ST6Gal1 gene

3. Resistance vector (pHY58_Puro): In pHY58_Puro, PGK promoter is the eukaryotic gene expression promoter, PuroR is the puro resistance coding sequence, ori is the origin of replication, AmpR promoter is the AmpR gene promoter, and AmpR is the Amp resistance. sex coding sequence, (see Figure 4 for the plasmid map).

Example 4 Construction of ST3Gal4 and ST6Gal1 gene knockout animal models

Construction of porcine ST3Gal4 knockout fibroblasts using CRISPR/Cas9 technology. Pig secondary oocytes were taken and cultured to maturity in vitro. The egg cell nucleus was removed by a microinjector, and the constructed ST3Gal4 knockout cells were transplanted into enucleated oocytes by somatic cell nuclear transfer (SCNT) technology, and then activated by electrofusion technology. Cell. The cells were briefly cultured in vitro and then transplanted into the oviducts of recipient sows for development. The obtained knockout pigs were crossed and selfed to expand the population and establish a stable ST3Gal4 knockout pig line.

Construction of porcine ST6Gal1 knockout fibroblasts using CRISPR/Cas9 technology. Pig secondary oocytes were taken and cultured to maturity in vitro. The egg cell nucleus was removed by a microinjector, and the constructed ST6Gal1 knockout cells were transplanted into enucleated oocytes by somatic cell nuclear transfer (SCNT) technology, and then activated by electrofusion technology. Cell. The cells were briefly cultured in vitro and then transplanted into the oviducts of recipient sows for development. The obtained knockout pigs were crossed and selfed to expand the population and establish a stable ST6Gal1 knockout pig line.

The pigs in the ST3Gal4 knockout pig line homozygous for the ST3Gal4 gene were crossed with the pigs in the ST6Gal1 gene knockout pig line homozygous for the ST6Gal1 gene. ST6Gal1 and ST3Gal4 double knockout pig lines.

For example, porcine ST3Gal4 and ST6Gal1 knockout fibroblasts can be constructed using CRISPR/Cas9 technology. Pig secondary oocytes were taken and cultured to maturity in vitro. The egg cell nucleus was removed by a microinjector, and the constructed ST3Gal4/ST6Gal1 knockout cells were transplanted into enucleated oocytes by somatic cell nuclear transfer (SCNT) technology, and then activated by electrofusion technology. transplanted cells. The cells were briefly cultured in vitro and then transplanted into the oviducts of recipient sows for development. The obtained knockout pigs were crossed and selfed to expand the population and establish a stable ST3Gal4/ST6Gal1 knockout pig line. in particular,

1 Construction of porcine ST3Gal4/ST6Gal1 knockout fibroblast cell line

(1) The pig fetus (with intact fetal membranes) at 35-40 days of pregnancy was dissected out from the maternal uterus, washed in 75% ethanol, and the alcohol on the surface of the fetal membranes of the pig fetus was washed and removed with DPBS, and the fetal membrane was cut. membrane, remove the intact pig fetus, and cut the skin tissue of the abdomen, back and limbs of the pig fetus;

(2) Cut the skin tissue into pieces as much as possible, add 4-5mL of 200U/mL collagenase, pipette and mix well, put it into a 37°C cell incubator for digestion for about 30min;

(3) Observe under the microscope, when the tissue becomes loose and transparent, the digestion is terminated immediately, centrifuged, the supernatant digestion solution is aspirated, and then the tissue pellet at the bottom of the tube is resuspended with 4 ml of DMEM complete medium containing 16% FBS;

(4) Discard the supernatant, resuspend the cells in DMEM complete medium containing 16% FBS, and culture at 38.5°C, 5% CO ₂ ;

(5) After the cells grow to the logarithmic growth phase, digest the cells with 0.05% trypsin to collect the cells, adjust the ^{total amount of cells in a cell suspension to about 1.5×10 6} cells, and use the nucleofection instrument of Lonza company and Mammalian Fibroblast Transfection Kit, follow the steps below for nucleofection:

a) One reaction requires 100 μl of nuclear transfer reaction solution, and each reaction solution needs to be mixed in advance with 82 μl of NucleofectorTM Basic Solution plus 18 μl of Supplement 1, that is, according to the ratio of 4.5:1 to mix and return to room temperature;

b) Add to a 100 μl reaction solution according to the ratio of ST3Gal4-Cas9 targeting vector plasmid: ST6Gal1-Cas9 targeting vector plasmid: resistance plasmid = 2 μg: 2 μg: 1 μg (the total amount is less than 8ug);

c) Wash a part of the cell suspension with DPBS, centrifuge at 1500g for 5 min and remove the supernatant as much as possible, and resuspend the pellet with a nucleotransfer reaction solution containing Cas9 plasmid and resistance plasmid;

d) The nuclear transfer system is carefully added to the electroporation cup provided with the kit. The electroporation cup is placed in the cup groove of the Lonza nucleator, and the optimal nuclear transfer program (such as program U023) is selected. Immediately after electroporation transfection, the liquid in the electroporation cup is gently aspirated in the ultra-clean bench, and then transferred to a 2ml 16% FBS in DMEM complete medium, mix gently;

(6) Plate the cell suspension in 20 10cm dishes with a suitable volume;

(7) Put it into a cell incubator, 38.5°C, 5% CO _{2 to} cultivate. After nuclear transfer, the medium was changed to complete medium containing puromycin (puromycin) after 24 hours, and the concentration of the first drug screening was 0.8 μg/mL, and the medium was changed every 2-3 days after that;

(8) Decrease the concentration of the drug according to the state of the cells under the microscope. When single cell clones begin to appear in the culture dish (usually 10-14 days), the final concentration of puromycin needs to be maintained at 0.2 μg/mL;

(9) Select single-cell clones that cover the entire field of view under the 4x microscope field of view, mark the clones with an oily pen on the outside of the bottom of the dish where the clones are located, carefully pick them with a clone ring, and culture them in a 24-well plate;

(10) When the cells grow to the logarithmic growth phase, digest the cells with trypsin, take 4/5 of the cells and inoculate them in a 12-well plate or a 6-well plate, and continue to culture the remaining 1/5 in the original well for subsequent genes. type identification;

(11) When the cells in the 12-well plate or 6-well plate are overgrown, trypsin digestion and centrifugation to collect the cells, then resuspend them in cell freezing solution, and freeze 2-5 tubes according to the cell volume and cell status, label the number, and freeze Put the storage tube into the programmed cooling box, and then slowly cool down in the -80℃ refrigerator for 12 hours, then take out the cryovial and store it in liquid nitrogen for later use;

(12) When the cells in the 24-well plate are full, after digesting and collecting the cells, add an appropriate amount of NP-40 lysis buffer (about 10-25 μl) according to the amount of cells to resuspend the cell pellet, and then lyse and extract genomic DNA. The lysis procedure is as follows:

55℃ 1 hour

95℃ 5min

4℃ +∞

(13) The genomic DNA obtained by cleavage can be stored at -20°C after corresponding marker number until use.

2Construction of a pig model with two-gene knockout of ST3Gal4/ST6Gal1 from pigs

(1) Resuscitate ST3Gal4/ST6Gal1 knockout monoclonal cells;

(2) Obtain immature eggs from fresh sow ovaries. After washing the egg wash, select eggs with better quality in the mature solution under a microscope, and culture them in a 38.5°C, 5% CO2 cell incubator for 42 hours. -44h until the oocyte matures;

(3) The granulosa cells located outside the zona pellucida were removed from the mature oocytes. Then, select the oocytes whose granulosa cells have been removed and the first polar body has been discharged, and then remove their nucleus and put them in a 38.5°C incubator for later use;

(4) The ST3Gal4/ST6Gal1 knockout monoclonal cells were injected into the enucleated oocytes, and one cell was injected into each oocyte;

(5) Electric shock fusion, reconstituted embryos are activated, transferred to embryo maturation medium, and cultured in a 38.5°C, 5% CO2 cell incubator until blastocysts are formed;

(6) Embryo transfer: transfer the well-developed embryos into the uterus of the surrogate sow that is in heat at the same time, take care of the surrogate sow, and use B-ultrasound to detect the pregnancy of the recipient pig 1 month later. If the recipient pig is pregnant, monitor closely until farrowing.

Example 5 Phenotypic identification of knockout animal models

Whether the ST3Gal4 gene is knocked out in the somatic cells of the ST3Gal4 knockout pig lines and the ST6Gal1 and ST3Gal4 double gene knockout pig lines constructed in Example 4 was examined by using gene sequencing technology. The resulting pigs were found to be positive pigs. And the experiment confirmed that the pig line constructed in Example 4 had good growth and development despite the knockout of the ST3Gal4 gene.

Gene sequencing technology was used to test whether the ST6Gal1 gene was knocked out in the somatic cells of the ST6Gal1 knockout pig lines and the ST6Gal1 and ST3Gal4 double gene knockout pig lines constructed in Example 4. The resulting pigs were found to be positive pigs. And the experiment confirmed that the pig line constructed in Example 4 had good growth and development despite the knockout of the ST6Gal1 gene.

For example, specifically,

1 Identification of porcine ST3Gal4/ST6Gal1 knockout fibroblast cell line

(1) Sequencing primers were designed for the CDS region sequences of pig ST3Gal4 and ST6Gal1 genes respectively, and the primer sequences are shown in Table 1.

(2) PCR reaction:

Select the appropriate annealing temperature for PCR according to the Tm value of the primers. After PCR, the PCR products are sent to Beijing Qingke Xinye Biotechnology Co., Ltd. and Sangon Bioengineering (Shanghai) Co., Ltd. for sequencing, and the sequencing results correspond to align the genome sequences.

The PCR reaction system is as follows:

PCR reaction conditions are as follows:

The PCR reaction product was subjected to agarose gel electrophoresis (1%, that is, 1 g of agarose gel was added to 100 mL of electrophoresis buffer). After electrophoresis, the target band was cut under ultraviolet light, and then recovered using a gel recovery kit (QIAGEN). target band and sequenced.

The identification results are shown in Figure 5 and Figure 6, indicating that the PCR product sequencing results show that the pig ST3Gal4 and ST6Gal1 genes have been successfully knocked out.

Due to the knockout of the endogenous ST3Gal4 gene and/or ST6Gal1 gene in animals (eg pigs), the expression of SAα2,3Gal receptors and/or SAα2,6Gal receptors in pigs is significantly down-regulated (or even not expressed), and/or Or lose the binding ability of SAα2,3Gal receptor to the surface protein of avian influenza virus, and the binding ability of SAα2,6Gal receptor to the surface protein of human influenza virus, so as to avoid at least the simultaneous infection of animals (such as pigs) with avian influenza virus and Human influenza viruses, avoiding animals (eg, pigs) as mixing vessels and intermediate hosts for recombinant variation of avian and human influenza viruses. The viruses constructed using the gRNAs described in the present application are not susceptible to animals, and will not act as the above-mentioned intermediate host to simultaneously infect avian influenza viruses and human influenza viruses, so that new mammals (such as pigs and/or human beings) that can be infected will not be produced. ) of the new influenza virus, increasing safety. The pig strain obtained by using the method described in the present application can avoid simultaneous infection of human influenza virus and avian influenza virus, thereby avoiding using pigs as an intermediate host for different influenza viruses, and avoiding providing a breeding ground for influenza viruses to recombine and mutate.

The embodiments of the present application are described in detail above. However, the present application is not limited to the specific details of the above-mentioned embodiments. Within the scope of the technical concept of the present application, various simple modifications can be made to the technical solutions of the present application. It belongs to the protection scope of this application. In addition, it should be noted that the specific technical features described in the above-mentioned specific embodiments can be combined in any suitable manner unless they are inconsistent. The combination method will not be specified otherwise. In addition, the various embodiments of the present application can also be combined arbitrarily, as long as they do not violate the idea of the present application, they should also be regarded as the content disclosed in the present application.

Claims (66)

A method for constructing a virus-insensitive animal, the method comprising knocking out all or part of a gene encoding a sialyltransferase in the animal, thereby making the animal a virus-insensitive animal. The method according to claim 1, wherein the gene encoding sialyltransferase comprises ST3Gal4 gene, ST6Gal1 gene, ST3Gal3 gene and/or ST3Gal6 gene. The method of any one of claims 1-2, wherein the sialyltransferase comprises a 2,3-galactosialo-oligosaccharide (SAα2,3Gal) receptor, and/or 2,6-galactose Sialyloligosaccharide (SAα2,6Gal) receptor. The method of claim 3, wherein the gene encoding the 2,3-galactosialooligosaccharide (SAα2,3Gal) receptor comprises the ST3Gal4 gene. The method of any one of claims 3-4, wherein the gene encoding the 2,6-galactosialooligosaccharide (SAα2,6Gal) receptor comprises the ST6Gal1 gene. The method of any one of claims 1-5, wherein the animal comprises a livestock. The method of any one of claims 1-6, wherein the animal comprises a porcine. The method of any one of claims 1-7, wherein the knockout comprises adding, replacing and/or deleting one or more nucleotides in the gene encoding a sialyltransferase such that the The expression level of the gene encoding a sialyltransferase is reduced, and/or, the gene encoding a sialyltransferase is substantially not expressed; and/or, The amino acid sequence of the sialyltransferase is altered, and/or the sialyltransferase is inactivated. The method of any one of claims 5-8, wherein the knockout comprises adding, replacing and/or deleting one or more nucleotides in the ST3Gal4 gene and/or the ST6Gal1 gene such that The expression level of the ST3Gal4 gene and/or the ST6Gal1 gene is reduced, and/or, the ST3Gal4 gene and/or the ST6Gal1 gene is substantially not expressed; and/or, altering the amino acid sequence of the SAα2,3Gal receptor and/or the SAα2,3Gal receptor, and/or inactivating the SAα2,3Gal receptor and/or the SAα2,3Gal receptor. The method of any one of claims 4-9, wherein the knockout involves knocking out all or part of 2 or more exons of the ST3Gal4 gene. The method of any one of claims 5-10, wherein the knockout involves knocking out all or part of 1 or more exons of the ST6Gal1 gene. The method according to any one of claims 4-11, wherein the method comprises the steps of: The use of one or more deoxyribonucleic acid (DNA) endonucleases to generate one or more single-strand breaks (SSBs) or double-strand breaks (SSBs) or double-strand breaks ( DSB) such that one or more exons of the ST3Gal4 gene and/or the ST6Gal1 gene are deleted in whole or in part. 13. The method of claim 12, wherein the DNA endonuclease comprises a Cas nuclease. The method of claim 13, wherein the Cas nuclease comprises a Cas9 nuclease, a homologue thereof, a recombinant of a naturally occurring molecule thereof, a codon-optimized version thereof, and/or a modified version thereof. The method of any one of claims 1-14, wherein the method further comprises the use of one or more guide RNAs (gRNAs). The method of claim 15, wherein the gRNA is a single-stranded guide RNA (sgRNA). The method according to any one of claims 15-16, wherein the gRNA is capable of specifically binding to a target region in the ST3Gal4 gene comprising the nucleic acid sequence shown in any one of SEQ ID NOs: 5-6 and 20. The method according to any one of claims 15-17, wherein the gRNA capable of specifically binding to the ST3Gal4 gene comprises a nucleic acid sequence shown in any one of SEQ ID NOs: 9-10 and 21-22. The method according to any one of claims 15-18, wherein the gRNA is capable of specifically binding to a target region in the ST6Gal1 gene comprising the nucleic acid sequence shown in any one of SEQ ID NOs: 7-8. The method according to any one of claims 15-19, wherein the gRNA capable of specifically binding to the ST6Gal1 gene comprises a nucleic acid sequence shown in any one of SEQ ID NOs: 11-12 and 23-24. The method according to any one of claims 15-20, wherein the 5' end of the gRNA comprises a nucleic acid sequence shown in (X)n, wherein X is selected from A, U, C and G any base, and n is any integer from 0-15. 21. The method of claim 21, wherein the n is 13 or 2. The method of any one of claims 15-22, wherein the 3' end of the gRNA comprises a backbone sequence. The method of any of claims 1-23, wherein the method comprises: (1) providing a cell comprising one or more vectors comprising the gRNA or an in vitro transcription product of the vector; (2) culturing the cells in a culture medium; (3) transplanting the cultured cells into the oviduct of the recipient female non-human mammal, allowing the cells to develop in the uterus of the female non-human mammal; and (4) identifying germline transmission in the genetically modified non-human mammal of the progeny of the pregnant female of step (3). The method of any one of claims 1-24, wherein the virus comprises influenza virus. The method of claim 25, wherein the influenza virus comprises human influenza virus and/or avian influenza virus. The method of any one of claims 3-26, wherein the virus-insensitive animal does not substantially express the endogenous SAα2,3Gal receptor and/or the endogenous SAα2,3Gal receptor capable of interacting with surface proteins of the virus or the SAα2,6Gal receptor. The method of any one of claims 3-27, wherein the virus insensitive animal does not substantially express the endogenous SAα2,3Gal receptor and/or the SAα2,6Gal receptor. A gRNA that specifically binds to the ST3Gal4 gene, wherein the gRNA specifically binds to a target region comprising a nucleic acid sequence shown in any one of SEQ ID NOs: 5-6 and 20. The gRNA of claim 29, which targets more than 2 different exons in the ST3Gal4 gene. The gRNA of any one of claims 29-30, comprising the nucleic acid sequence shown in any one of SEQ ID NOs: 9-10 and 21-22. A gRNA that specifically binds to the ST6Gal1 gene, wherein the gRNA specifically binds to the target region comprising the nucleic acid sequence shown in any one of SEQ ID NOs: 7-8. The gRNA of claim 32, which targets 1 exon in the ST6Gal1 gene. The gRNA of any one of claims 32-33, comprising the nucleic acid sequence shown in any one of SEQ ID NOs: 11-12 and 23-24. The gRNA of any one of claims 29-34, wherein the gRNA is a single-stranded guide RNA (sgRNA). The gRNA according to any one of claims 29-35, wherein the 5' end of the gRNA comprises a nucleic acid sequence shown in (X)n, wherein X is selected from among A, U, C and G any base, and n is any integer from 0-15. The gRNA of claim 36, wherein the n is 13 or 2. The gRNA of any one of claims 29-37, wherein the 3' end of the gRNA comprises a backbone sequence. A nucleic acid molecule encoding the gRNA of any one of claims 29-38. A vector comprising the sequence of the gRNA of any one of claims 29-38. A kind of cell, it comprises the gRNA described in any one of claim 29-38, the nucleic acid molecule described in claim 39, the gRNA carrier described in claim 40, and/or claim 40 The in vitro transcription product of the gRNA vector described above. The gRNA of any one of claims 29-38, the nucleic acid molecule of claim 39, the gRNA carrier of claim 40, and/or the in vitro transcription product of the gRNA carrier of claim 40, and/ Or the use of the cell according to claim 41 in knocking out the ST3Gal4 gene and/or the ST6Gal1 gene, or the use in constructing a virus that is not susceptible to infection. A kind of ST3Gal4 gene deletion cell line, it is to use the gRNA described in any one of claim 29-31, the nucleic acid molecule described in claim 39, the gRNA carrier described in claim 40, and/or claim 40 The in vitro transcription product of the gRNA vector, and/or the cell preparation according to claim 41. The virus-insensitive animal prepared by the method of any one of claims 1-28, wherein the virus-insensitive animal does not substantially express endogenous SAα2,3Gal receptors capable of interacting with viral surface proteins. The virus-insensitive animal prepared by the method of any one of claims 1-28, wherein the virus-insensitive animal does not substantially express endogenous SAα2,3Gal receptors. A kind of ST6Gal1 gene deletion cell line, it is to use the gRNA described in any one of claim 32-34, the nucleic acid molecule described in claim 39, the gRNA carrier described in claim 40, the gRNA described in claim 40 The in vitro transcription product of the vector, and/or obtained from the cell according to claim 41. The virus-insensitive animal prepared by the method of any one of claims 1-28, wherein the virus-insensitive animal does not substantially express endogenous SAα2,6Gal receptors capable of interacting with viral surface proteins. The virus-insensitive animal prepared by the method of any one of claims 1-28, wherein the virus-insensitive animal does not substantially express the endogenous SAα2,6Gal receptor. A method for preparing a virus-insensitive animal, the method comprising: (a) providing a virus-insensitive animal prepared by the method of any one of claims 1-28; and (b) mating the virus-insensitive animal obtained in step (a) with other animals or in vitro fertilization or further gene editing based on the virus-insensitive animal obtained in step (a) or transplanting human tissues and cells into step (a) The obtained virus is not susceptible to animals, and is screened to obtain virus-resistant animals. The method of claim 49, wherein the method comprises combining the virus-insensitive animal of any one of claims 44-45 that does not substantially express endogenous SAα2,3Gal receptors with claims 47- The virus of any one of 48 that does not substantially express the endogenous SAα2,6Gal receptor is not susceptible to sexual reproduction in animals. The method of claim 50, wherein the method comprises: the post-hybridization screening for substantially no expression of the endogenous SAα2,3Gal receptor and no substantial expression of the endogenous SAα2,6Gal receptor animals that are not susceptible to the virus. The virus obtained by the method according to any one of claims 49-51 is not susceptible to animals. The virus-insensitive animal of claim 52, which includes livestock. The virus-insensitive animal of any one of claims 52-53, which includes a porcine. The virus-insensitive animal of any one of claims 52-54, wherein the virus comprises an influenza virus. The virus-insensitive animal of claim 55, wherein the influenza virus comprises human influenza virus and/or avian influenza virus. A cell or cell line or primary cell culture, wherein said cell or cell line or primary cell culture is derived from the virus-insensitive animal of any one of claims 52-56 or its progeny. A tissue or organ or a culture thereof, wherein the tissue or organ or a culture thereof is derived from the virus-insensitive animal of any one of claims 52-56 or a progeny thereof. A CRISPR/Cas9 system for specifically targeting and knocking out ST3Gal4 gene, which uses a DNA sequence containing the gRNA capable of specifically targeting ST3Gal4 gene according to any one of claims 29-31. A nucleic acid molecule kit capable of specifically targeting ST3Gal4 gene, wherein the kit comprises the gRNA according to any one of claims 29-31. A complete set of nucleic acid molecules capable of specifically targeting ST3Gal4 gene, wherein the complete set of nucleic acid molecules comprises the gRNA according to any one of claims 29-31 and a nucleic acid molecule encoding a Cas9 protein. A CRISPR/Cas9 system for specifically targeting and knocking out ST6Gal1 gene, characterized in that a DNA sequence containing the gRNA capable of specifically targeting ST6Gal1 gene according to any one of claims 32-34 is used. A nucleic acid molecule kit capable of specifically targeting ST6Gal1 gene, wherein the kit comprises the gRNA according to any one of claims 32-34. A complete set of nucleic acid molecules capable of specifically targeting ST6Gal1 gene, wherein the complete set of nucleic acid molecules comprises the gRNA according to any one of claims 32-34 and a nucleic acid molecule encoding a Cas9 protein. Use of the virus-insensitive animal of any one of claims 44-45, 47-48 and 52-56 in the development of antiviral products, or as a model system for pharmacology, immunology, microbiology and medical research. Use of the virus-insensitive animal described in any one of claims 44-45, 47-48 and 52-56 in screening, validating, evaluating or researching antiviral drugs or combination drugs, and/or pharmacodynamic studies.

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