WO2022012512A1 - 敲除猪异种抗原的基因的gRNA及其应用 - Google Patents

敲除猪异种抗原的基因的gRNA及其应用 Download PDF

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WO2022012512A1
WO2022012512A1 PCT/CN2021/105973 CN2021105973W WO2022012512A1 WO 2022012512 A1 WO2022012512 A1 WO 2022012512A1 CN 2021105973 W CN2021105973 W CN 2021105973W WO 2022012512 A1 WO2022012512 A1 WO 2022012512A1
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grna
gene
β4galnt2
nucleic acid
seq
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PCT/CN2021/105973
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French (fr)
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戴一凡
杨海元
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金佩奇生物科技(南京)有限公司
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Publication of WO2022012512A1 publication Critical patent/WO2022012512A1/zh

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Definitions

  • the present application relates to the field of biomedicine, in particular to a gRNA that specifically targets the ⁇ 4GalNT2 gene and its application.
  • genes encoding certain proteins that elicit immune system responses can be knocked out by gene editing.
  • the present application provides a gRNA that specifically targets the ⁇ 4GalNT2 gene, the gRNA comprising the nucleotide sequence shown in any one of SEQ ID NO. 6-7.
  • the gRNA described in this application significantly improves the gene knockout efficiency.
  • the knockout efficiency of the ⁇ 4GalNT2 gene can reach at least 20% or more (for example, at least 21% or more, 22% or more, 23% or more, 24% or more, or more than 25%. , 26% or more, 27% or more, 28% or more, 29% or more or higher).
  • Using two of the sgRNAs can increase the knockout efficiency, for example, by at least 5% or more (eg, at least 10% or more, 15% or more, 20% or more, 25% or more, or at least 10% or more, 15% or more, 20% or more, 25% or more, or at least 5% or more, compared to using one type of gRNA for knockout. 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, or higher).
  • the gRNA that specifically targets the ⁇ 4GalNT2 gene described in this application can also be used for gene knockout together with the gRNA that specifically targets the GGTA1 and/or CMAH gene, so that the knockout efficiency of the simultaneous knockout of the three genes can reach at least 10 % or more (for example, at least 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 16% or more, 17% or more, 18% or more, 19% or more, 20% or more, 25% or more or higher).
  • the sgRNA described in this application can have high knockout efficiency and/or stable knockout efficiency.
  • the present application also provides nucleic acid molecules encoding the gRNAs, cells, vectors, vector transcripts, kits and/or systems comprising the nucleic acid molecules or sgRNAs, and animal models, cells, tissues, and animal models prepared according to the gRNAs. /or organs, and their applications.
  • the application provides a gRNA that specifically targets the ⁇ 4GalNT2 gene, wherein the gRNA specifically binds to the nucleotide sequence shown in any one of SEQ ID NO.1-2.
  • the gRNA comprises the nucleotide sequence shown in SEQ ID NO.16.
  • the gRNA comprises the nucleotide sequence set forth in any one of SEQ ID NOs. 6-7.
  • the sgRNA comprises the nucleotide sequence-backbone sequence-3' shown in 5'-(X)n-SEQ ID NO. 16, wherein X is selected from A, U, C and G The base of any one, and n is any integer from 0-15.
  • the gRNA is a single-stranded guide RNA (sgRNA).
  • the application provides a gRNA combination, which includes the gRNA specifically targeting the ⁇ 4GalNT2 gene, the gRNA specifically targeting the GGTA1 gene and the gRNA specifically targeting the CMAH gene described in the application.
  • the gRNA that specifically targets the GGTA1 gene comprises the nucleotide sequence shown in SEQ ID NO.9.
  • the gRNA specifically targeting the CMAH gene comprises the nucleotide sequence shown in SEQ ID NO.10.
  • the application provides one or more isolated nucleic acid molecules encoding the gRNAs described herein that specifically target the ⁇ 4GalNT2 gene.
  • the present application provides a vector comprising the nucleic acid molecule described herein.
  • the present application provides cells comprising the gRNA specifically targeting the ⁇ 4GalNT2 gene, the nucleic acid molecule, the vector, and/or an in vitro transcription product of the vector.
  • the present application provides the gRNA that specifically targets the ⁇ 4GalNT2 gene, the nucleic acid molecule, the vector, the in vitro transcription product of the vector and/or the cell in which the ⁇ 4GalNT2 gene is knocked out use in or in the construction of animal models.
  • the application provides a ⁇ 4GalNT2 gene-deficient cell line, which uses the sgRNA specifically targeting the ⁇ 4GalNT2 gene, the nucleic acid molecule, the vector, the in vitro transcription product of the vector and / or obtained from the cell preparation.
  • the present application provides a method for constructing an animal model, the method comprising administering to cells of the animal at least two gRNAs that specifically target the ⁇ 4GalNT2 gene, thereby knocking out all or part of the ⁇ 4GalNT2 gene, wherein the The gRNA specifically binds to the nucleotide sequence shown in any one of SEQ ID NO. 1-2.
  • the gRNA that specifically targets the ⁇ 4GalNT2 gene comprises the nucleotide sequence shown in SEQ ID NO.16.
  • the gRNA specifically targeting the ⁇ 4GalNT2 gene comprises the nucleotide sequence shown in any one of SEQ ID NO. 6-7.
  • the gRNA specifically targeting the ⁇ 4GalNT2 gene comprises the nucleotide sequence-backbone sequence-3' shown in 5'-(X)n-SEQ ID NO. 16, wherein X is selected from A base of any of A, U, C, and G, and n is any integer from 0-15.
  • the gRNA is a single-stranded guide RNA (sgRNA).
  • the method comprises the step of using one or more deoxyribonucleic acid (DNA) endonucleases to generate one or more single-strand breaks in or near the ⁇ 4GalNT2 gene (SSB) or double-strand break (DSB), resulting in the deletion of all or part of one or more exons of the ⁇ 4GalNT2 gene.
  • DNA deoxyribonucleic acid
  • the method further comprises administering to the cells of the animal a gRNA that specifically targets the GGTA1 gene, thereby knocking out all or part of the GGTA1 gene.
  • the gRNA that specifically targets the GGTA1 gene comprises the nucleotide sequence shown in SEQ ID NO.9.
  • the method further comprises administering to the cells of the animal a gRNA that specifically targets the CMAH gene, thereby knocking out all or part of the CMAH gene.
  • the gRNA specifically targeting the CMAH gene comprises the nucleotide sequence shown in SEQ ID NO.10.
  • the method comprises the step of using one or more deoxyribonucleic acid (DNA) endonucleases to produce an 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 GGTA1 gene and/or the CMAH gene.
  • DNA deoxyribonucleic acid
  • the DNA endonuclease comprises a Cas nuclease.
  • the Cas nucleases include Cas9 nucleases, homologues thereof, recombinants of naturally occurring molecules thereof, codon-optimized versions thereof, and/or modified versions thereof.
  • the method comprises: a) providing a cell comprising one or more in vitro transcription products comprising a vector described herein or the gRNA vector; b) subjecting the The cells are cultured in a culture medium; c) the cultured cells are transplanted into the fallopian tubes of a recipient female non-human mammal, allowing the cells to develop in the uterus of the female non-human mammal; and d) an identification step c) Germline transmission in genetically modified non-human mammals in progeny of pregnant females.
  • the animal comprises a pig.
  • the present application provides an animal model prepared according to the method for constructing an animal model described in the present application, wherein the animal does not express the ⁇ 4GalNT2 gene and/or ⁇ -1,4-N-acetylgalactosaminyltransferase 2 .
  • the animal does not express the GGTA1 gene and/or ⁇ Gal.
  • the animal does not express the CMAH gene and/or Neu5Gc.
  • the present application provides a method for preparing an animal model, the method comprising: a) providing the animal model described in the present application; b) mating the animal model obtained in step a) with other animals or in vitro fertilization or The animal model obtained based on step a) is further subjected to gene editing or the human tissue and cells are transplanted into the animal model obtained in step a) and screened to obtain an animal model.
  • the present application provides an animal model prepared according to the method for preparing an animal model.
  • the animal comprises a pig.
  • 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 animal model described herein or its progeny.
  • 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 animal model or progeny thereof.
  • the present application provides a CRISPR/Cas9 system for specifically targeting the ⁇ 4GalNT2 gene, comprising using a DNA sequence containing the gRNA specifically targeting the ⁇ 4GalNT2 gene described in the present application.
  • the present application provides a nucleic acid molecule kit that can specifically target the ⁇ 4GalNT2 gene, wherein the kit includes the gRNA that specifically targets the ⁇ 4GalNT2 gene.
  • the present application provides a set of nucleic acid molecules that can specifically target the ⁇ 4GalNT2 gene, wherein the set of nucleic acid molecules includes the sgRNA and Cas9 mRNA that specifically target the ⁇ 4GalNT2 gene.
  • the present application provides said cells or cell lines or primary cell cultures, said tissues or organs or cultures thereof in organ and/or tissue transplantation product development, or as pharmacological, immunological and Model systems for medical research, or applications in the validation, evaluation or study of immune rejection.
  • the present application provides the use of the animal model in the development of organ and/or tissue transplantation products, or as a model system for pharmacological, immunological and medical research.
  • the present application provides the application of the animal model in verifying, evaluating or studying immune rejection.
  • Figure 1 shows a schematic diagram of the GGTA1-CRISPR/Cas9 targeting vector.
  • Figure 2 shows a schematic diagram of the CMAH-CRISPR/Cas9 targeting vector.
  • Figure 3 shows a schematic diagram of the dual sgRNA ⁇ 4GalNT2-CRISPR/Cas targeting vector.
  • single-strand break generally refers to a phenomenon in which one strand of a DNA molecule is cleaved and broken. When only one of the two strands of the DNA double helix is defective, the other strand can be used as a template to direct the correction of the damaged strand. DNA endonucleases can cause single-strand breaks.
  • double-strand break generally refers to a phenomenon that occurs when two single strands of a double-stranded DNA molecule are cleaved at the same location. Double-strand breaks can induce DNA repair, possibly causing genetic recombination, and cells also have systems that act on double-strand breaks that occur at other times. Double-strand breaks can occur periodically during the normal cell replication cycle or can be enhanced under certain circumstances, such as ultraviolet light, inducers of DNA breaks (eg, various chemical inducers). Many inducers can cause DSBs to occur indiscriminately across the genome, and DSBs can be regularly induced and repaired in normal cells.
  • the original sequence can be reconstructed with full fidelity, but, in some cases, small insertions or deletions (called “indels”) are introduced at DSB sites.
  • double-strand breaks can also be induced specifically at specific locations, which can be used to cause targeted or preferential genetic modification at selected chromosomal locations.
  • This homology-directed repair is used to insert the sequence of interest provided by the use of a "donor" polynucleotide into the desired chromosomal location.
  • in vitro transcription product generally refers to a product that is synthesized by in vitro transcription of DNA, or that becomes messenger RNA (mRNA) after processing.
  • In vitro transcription products may include precursor messenger RNA (pre-mRNA) and processed mRNA itself. After DNA strands are transcribed into transcripts, newly synthesized primary transcripts can be modified in several ways to convert to their mature functional forms to produce different proteins and RNAs (e.g. mRNA, tRNA, rRNA, lncRNA, miRNA, etc. ).
  • the term “in vitro transcript” may include exons, introns, 5'UTRs and 3'UTRs.
  • the "vector” generally refers to a nucleic acid molecule capable of self-replication in a suitable host for transfer of the inserted nucleic acid molecule into and/or between host cells.
  • the vectors may include vectors primarily for the insertion of DNA or RNA into cells, vectors primarily for replication of DNA or RNA, and vectors primarily for expression of transcription and/or translation of DNA or RNA.
  • the carrier also includes a carrier having a variety of the above-mentioned functions.
  • the vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell.
  • the vector can produce the desired expression product by culturing a suitable host cell containing the vector.
  • the vector may contain additional features in addition to the transgene insert and backbone: promoter, genetic marker, antibiotic resistance, reporter gene, targeting sequence, protein purification tag.
  • Vectors called expression vectors are used in particular to express transgenes in target cells, and usually have control sequences.
  • the vectors described in this application may be expression vectors, which may include viral vectors (lentiviral vectors and/or retroviral vectors), phage vectors, phagemids, cosmids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), Bacterial artificial chromosomes (BACs) or P1 derived artificial chromosomes (PACs) and/or plasmids.
  • viral vectors lentiviral vectors and/or retroviral vectors
  • phage vectors phagemids
  • cosmids cosmids
  • cosmids cosmids
  • artificial chromosomes such as yeast artificial chromosomes (YAC),
  • the term "pig” generally refers to any pig known in the art, including but not limited to: wild pig, domestic pig, mini pig, wild pig (Sus scrofa pig), domestic pig (Sus scrofa domesticus pig) ) and inbred pigs.
  • the pig may be selected from the group comprising: Landrace (also known as Landrace), Hampshire, Duroc ), Chinese Meishan, Chester White, Berkshire Goettingen, Landrace/Yorkshire/Chester White, Yucatan ( Yueatan, Barna, Wuzhishan, Xi Shuang Banna and Pietrain.
  • DNA endonucleic acid (DNA) endonuclease generally refers to an enzyme that can hydrolyze phosphodiester bonds inside DNA molecular chains, thereby producing oligonucleotides.
  • DNA endonucleases can include enzymes with no base specificity and enzymes capable of recognizing and cleaving specific bases or base sequences.
  • Cas nuclease generally refers to CRISPR-associated nucleases, a type of DNA endonuclease that can form double-strand breaks at specific DNA sequences. Cas nucleases are often complementary to CRISPR sequences and can use the CRISPR sequence as a guide to recognize and cut specific DNA strands.
  • Cas nucleases may include, but are not limited to, the following group: 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 homologues, or modified forms thereof.
  • Cas9 nuclease may also be referred to as, Csn1 or Csx12, generally referring to the RNA-guided DNA nucleic acid associated with type II CRISPR (Regularly Interspaced Short Palindromic Repeats) adaptive immune system Endonuclease.
  • Cas9 nucleases can also include wild-type proteins, orthologs, and functional and non-functional mutants thereof.
  • the Cas9 nuclease can be derived from any suitable bacteria.
  • Cas9 nucleases typically include a RuvC nuclease domain and an HNH nuclease domain, which cleave two different strands of a double-stranded DNA molecule, respectively.
  • Streptococcus pyogenes Cas9 protein the amino acid sequence of which can be found in SwissProt database accession number Q99ZW2; Neisseria meningitides Cas9 protein, whose amino acid sequence can be found in 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.
  • gRNA generally refers to guide RNA (guide RNA), a type of RNA molecule.
  • guide RNA guide RNA
  • crRNA and tracrRNA usually exist as two separate RNA molecules, constituting gRNA.
  • CRISPR RNA CRISPR RNA
  • tracrRNA generally refers to a scaffold-type RNA that can bind to Cas nucleases.
  • gRNA can also be called single-stranded guide RNA (sgRNA), sgRNA has become the most common form of gRNA used in CRISPR technology by those skilled in the art, hence the term "sgRNA” and "gRNA” may have the same meaning herein.
  • sgRNAs can be synthesized artificially or prepared from DNA templates in vitro or in vivo. The sgRNA can bind to the Cas nuclease or target the target DNA, which can guide the Cas nuclease to cleave the DNA site complementary to the gRNA. The degree of complementarity between the gRNA and its corresponding target sequence is at least about 50%.
  • backbone sequence generally refers to the part of the gRNA, other than the part that recognizes or hybridizes to the target sequence, and may include the sequence between the gRNA pairing sequence and the transcription terminator in the sgRNA.
  • the backbone sequence generally does not change due to changes in the target sequence, nor does it affect the recognition of the target sequence by the gRNA.
  • the backbone sequence can be any sequence available in the art.
  • the structure of the backbone sequence can be found in A and B in Figure 1, A, B, C in Figure 3, and A, B, C, D, The part described in E except the spacer sequence.
  • target nucleic acid In this application, the terms “target nucleic acid”, “target nucleic acid” and “target region” are used interchangeably, and usually refer to a nucleic acid sequence that can be recognized by a gRNA.
  • the target nucleic acid can refer to a double-stranded nucleic acid or a single-stranded nucleic acid.
  • isolated nucleic acid molecule generally refers to a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases, or analogs thereof, read from the 5' to 3' end.
  • An isolated nucleic acid molecule can be isolated from the usual or natural environment, or it can be produced synthetically. Such an isolated nucleic acid molecule is removed or isolated from its normal or natural environment, or the molecule is produced in such a way that it is not present in its normal or natural environment, which is different from its normal or natural environment isolated polypeptides, peptides, lipids, carbohydrates, other polynucleotides or other materials.
  • the isolated nucleic acid molecules of the present application can encode RNA, eg, can encode a gRNA that specifically targets the RPGR gene.
  • CMAH generally refers to the gene encoding cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMP-Neu5Ac hydroxylase).
  • CMP-Neu5Ac hydroxylase A functional cytidine monophosphate-N-acetylneuraminic acid hydroxylase catalyzes the conversion of sialic acid N-acetylneuraminic acid (Neu5Ac) to N-glycolylneuraminic acid (Neu5Gc).
  • Neu5Gc residues are epitopes or antigens recognized by the human immune system.
  • Cytidine monophosphate-N-acetylneuraminic acid hydroxylases described herein can include full-length proteins, functional fragments, homologues, and/or functional variants (eg, splice variants).
  • the nucleotide sequence of the CMAH gene described in this application includes its functional variants, derivatives, analogs, homologues and fragments thereof.
  • the amino acid sequence of the porcine CMAH protein can be found in the NCBI database under accession number NP_001106486.1, and the porcine CMAH nucleotide sequence can be found in the NCBI database under the accession number NM_001113015.1.
  • GGTA1 may also be referred to as ⁇ Gal, GGTA, GGT1, GT, ⁇ GT, GGTA1, and generally refers to the gene encoding ⁇ 1,3 galactosyltransferase ( ⁇ Gal, GT).
  • Functional ⁇ 1,3 galactosyltransferases can catalyze the formation of galactose ⁇ 1,3-galactose ( ⁇ Gal, Gal, Gal, gall, 3gal, or gal1-3gal) residues on glycoproteins.
  • Galactose ⁇ 1,3-galactose ( ⁇ Gal) residues are epitopes or antigens recognized by the human immune system.
  • the al,3 galactosyltransferases described herein can include full-length proteins, functional fragments, homologues, and/or functional variants (eg, splice variants).
  • the nucleotide sequence of the GGTA1 gene described in this application includes its functional variants, derivatives, analogs, homologues and fragments thereof.
  • the amino acid sequence of the porcine GGTA1 protein can be found in the NCBI database under accession number NP_001309984.1, and the porcine GGTA1 nucleotide sequence can be found in the NCBI database under the accession number NM_001323055.1.
  • ⁇ 4GalNT2 generally refers to the gene encoding ⁇ -1,4N-acetylgalactosaminyltransferase 2 ( ⁇ 4GalNT2, ⁇ 4GalNT2, ⁇ 1,4GalNT2, ⁇ 1,4GalNT2), a functional ⁇ 4GalNT2 that produces Sda-like Glycans.
  • the beta-1,4N-acetylgalactosaminyltransferase 2 described herein can include full-length proteins, functional fragments, homologues, and/or functional variants (eg, splice variants).
  • the nucleotide sequence of the ⁇ 4GalNT2 gene described in this application includes its functional variants, derivatives, analogs, homologues and fragments thereof.
  • the amino acid sequence of the porcine ⁇ 4GalNT2 protein can be found in the NCBI database under accession number NP_001231259.1, and the porcine ⁇ 4GalNT2 nucleotide sequence can be found in the NCBI database under the accession number NM_001244330.1.
  • the present application provides a gRNA that specifically targets the ⁇ 4GalNT2 gene, wherein the gRNA can specifically bind to the nucleotide sequence shown in any one of SEQ ID NO. 1-2.
  • the gRNA that specifically targets the ⁇ 4GalNT2 gene can specifically bind to the nucleotide sequence shown in any one of SEQ ID NO. 1-2 with at least 70% (eg, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) nucleotide sequences of sequence identity.
  • the gRNA specifically targeting the ⁇ 4GalNT2 gene can specifically bind to a nucleotide sequence complementary to the nucleotide sequence set forth in any one of SEQ ID NOs: 1-2.
  • the gRNA can specifically bind to at least 70% (eg, at least 75%, at least 80%, at least 85%) of the nucleotide sequence set forth in any one of SEQ ID NOs. , at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) of sequence identity
  • the gRNAs specifically targeting the ⁇ 4GalNT2 gene described in this application can bind to sequences in the target nucleic acid.
  • a gRNA can interact with a target nucleic acid in a sequence-specific manner by hybridization (ie, base pairing).
  • the nucleotide sequence of the gRNA specifically targeting the ⁇ 4GalNT2 gene may vary according to the sequence of the target nucleic acid.
  • the gRNA may comprise at least 70% (eg, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) sequence identity nucleotide sequences.
  • the gRNA that specifically targets the ⁇ 4GalNT2 gene may comprise the nucleotide sequence shown in any one of SEQ ID NO. 6-7.
  • the gRNA may comprise at least 70% (eg, at least 75%, at least 80%, at least 85%, at least 90%) of the nucleotide sequence shown in any one of SEQ ID NO. 6-7 , at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) nucleotide sequences of sequence identity .
  • the gRNA specifically targeting the ⁇ 4GalNT2 gene may comprise a backbone sequence
  • the backbone sequence used in the present application may be derived from any commercially available plasmid as long as it can achieve the expression of Cas nuclease and the transcription of the gRNA.
  • the backbone sequence can be that from pX330.
  • the backbone sequence can be the nucleotide sequence set forth in SEQ ID NO:17.
  • the gRNA comprises the nucleotide sequence-backbone sequence-3' shown in 5'-(X)n-SEQ ID NO: 16, wherein X is selected from A, U, C and G any base, and n is any integer from 0-15.
  • n is 0.
  • the gRNA described herein may comprise the nucleotide sequence shown in 5'-SEQ ID NO:6-the nucleotide sequence shown in SEQ ID NO:17-3', or 5'-SEQ ID NO:7 Nucleotide sequence shown - Nucleotide sequence shown in SEQ ID NO: 17 - 3'.
  • the gRNA that specifically targets the ⁇ 4GalNT2 gene may be a single-stranded guide RNA (sgRNA).
  • sgRNA single-stranded guide RNA
  • the present application provides a gRNA combination, which may include the gRNA specifically targeting the ⁇ 4GalNT2 gene, the gRNA specifically targeting the GGTA1 gene and the gRNA specifically targeting the CMAH gene described in the present application.
  • the gRNA that specifically targets the GGTA1 gene may comprise the nucleotide sequence shown in SEQ ID NO.9.
  • the gRNA that specifically targets the GGTA1 gene may comprise at least 70% (eg, at least 75%, at least 80%, at least 85% of the nucleotide sequence shown in any one of SEQ ID NO. 9) %, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) sequence identity nucleotide sequence.
  • the gRNA that specifically targets the CMAH gene may comprise the nucleotide sequence shown in SEQ ID NO.10.
  • the gRNA specifically targeting the CMAH gene may comprise at least 70% (eg, at least 75%, at least 80%, at least 85% of the nucleotide sequence shown in any one of SEQ ID NO. 10) %, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) sequence identity nucleotide sequence.
  • the application provides one or more isolated nucleic acid molecules encoding the gRNAs described herein that specifically target the ⁇ 4GalNT2 gene.
  • the present application provides a vector, which can comprise the nucleic acid molecule encoding the gRNA specifically targeting the ⁇ 4GalNT2 gene described in the present application.
  • the two nucleic acid molecules may be located in the same vector, or may be located in different vectors.
  • the two nucleic acid molecules encoding the gRNA that specifically targets the ⁇ 4GalNT2 gene can be located in the same vector.
  • nucleic acid molecules encoding the gRNA specifically targeting the ⁇ 4GalNT2 gene nucleic acid molecules encoding the gRNA specifically targeting the GGTA1 gene, and encoding the specific targeting CMAH
  • Each of the nucleic acid molecules of the gene's gRNA can be located in 1, 2, 3 or 4 vectors.
  • nucleic acid molecules encoding the gRNA specifically targeting the ⁇ 4GalNT2 gene nucleic acid molecules encoding the gRNA specifically targeting the GGTA1 gene, and encoding the specific targeting CMAH
  • Each of the nucleic acid molecules of the gene's gRNA can be located in a different vector.
  • nucleic acid molecules encoding the gRNA specifically targeting the ⁇ 4GalNT2 gene nucleic acid molecules encoding the gRNA specifically targeting the GGTA1 gene, and encoding the specific targeting CMAH Two of the nucleic acid molecules of the gene's gRNA can be located in the same vector.
  • nucleic acid molecules encoding the gRNA specifically targeting the ⁇ 4GalNT2 gene nucleic acid molecules encoding the gRNA specifically targeting the GGTA1 gene, and encoding the specific targeting CMAH
  • nucleic acid molecules encoding the gRNA specifically targeting the GGTA1 gene nucleic acid molecules encoding the gRNA specifically targeting the GGTA1 gene
  • CMAH specific targeting CMAH
  • nucleic acid molecules encoding the specific targeting ⁇ 4GalNT2 gene gRNA nucleic acid molecules encoding the specific targeting GGTA1 gene gRNA, and encoding the specific
  • the nucleic acid molecules of the gRNA targeting the CMAH gene can be located in the same vector.
  • the vector can be any vector that can be used for CRISPR/Cas, for example, the vector is PX330 plasmid.
  • the present application provides cells that may comprise the gRNA specifically targeting the ⁇ 4GalNT2 gene, the nucleic acid molecule, the vector, and/or the in vitro transcription product of the vector.
  • the present application provides the gRNA that specifically targets the ⁇ 4GalNT2 gene, the nucleic acid molecule, the vector, the in vitro transcription product of the vector and/or the cell in which the ⁇ 4GalNT2 gene is knocked out use in or in the construction of animal models.
  • the application provides a ⁇ 4GalNT2 gene-deficient cell line, which uses the sgRNA specifically targeting the ⁇ 4GalNT2 gene, the nucleic acid molecule, the vector, the in vitro transcription product of the vector and / or obtained from the cell preparation.
  • the present application provides a method for constructing an animal model, the method may comprise administering to the cells of the animal at least two gRNAs specifically targeting the ⁇ 4GalNT2 gene described in the present application, thereby knocking out all of the ⁇ 4GalNT2 gene or part, wherein the gRNA specifically binds to the nucleotide sequence set forth in any one of SEQ ID NOs. 1-2.
  • the method may comprise the step of using one or more deoxyribonucleic acid (DNA) endonucleases to generate one or more single-strand breaks (SSBs) within or near the ⁇ 4GalNT2 gene ) or double-strand breaks (DSBs) resulting in the deletion of all or part of one or more exons of the ⁇ 4GalNT2 gene.
  • DNA deoxyribonucleic acid
  • SSBs single-strand breaks
  • DSBs double-strand breaks
  • the method may comprise administering the gRNA specifically targeting the GGTA1 gene described in the present application to the cells of the animal, thereby knocking out all or part of the GGTA1 gene.
  • the method may comprise administering to the cells of the animal the gRNA specifically targeting the CMAH gene described herein, thereby knocking out all or part of the CMAH gene.
  • the method may further comprise the step of: using one or more deoxyribonucleic acid (DNA) endonucleases to produce one or more in or near the GGTA1 gene and/or the CMAH gene More single-strand breaks (SSBs) or double-strand breaks (DSBs) resulting in all or partial deletion of one or more exons of the GGTA1 gene and/or the CMAH gene.
  • DNA deoxyribonucleic acid
  • SSBs single-strand breaks
  • DSBs double-strand breaks
  • the DNA endonucleases can include Endonuclease I, Endonuclease II, Endonuclease IV, Restriction Endonuclease, UvrABC Endonuclease, and/or Engineered Nuclease.
  • engineered nucleases include, but are not limited to, homing endonucleases (also known as meganucleases or meganucleases, Meganucleases), zinc finger nucleases (ZFNs), transcription activators transcription activator-like effector-based nuclease (TALEN), Clustered regularly interspaced short palindromic repeat (CRISPR).
  • the DNA endonuclease may include Cas nuclease.
  • the Cas nucleases can include Cas9 nucleases, homologues thereof, recombinants of naturally occurring molecules thereof, codon-optimized versions thereof, and/or modified versions thereof.
  • the DNA endonuclease can be modified or unmodified.
  • the gRNA, crRNA, tracrRNA or sgRNA can be modified or unmodified.
  • modifications known in the art that can be used. For example, deletion, insertion, translocation, inactivation and/or activation of nucleotides. Such modifications may include introducing one or more mutations (including single or multiple base pair changes), increasing the number of hairpins, cross-linking, breaking specific stretches of nucleotides, and other modifications. Modifications can include inclusion of at least one non-naturally occurring nucleotide, or a modified nucleotide, or an analog thereof.
  • the nucleotides may be modified at ribose, phosphate and/or base moieties.
  • the method may comprise: a) providing a cell comprising one or more in vitro transcription products comprising the vector described herein or the gRNA vector; b) subjecting the cell to culturing in a medium; c) transplanting the cultured cells into the fallopian tubes of a recipient female non-human mammal, allowing the cells to develop in the uterus of the female non-human mammal; and d) identifying step c ) Germline Transmission in Genetically Modified Non-Human Mammals of Offspring of Pregnant Females.
  • the animals may include pigs.
  • the pigs may be any species of pigs inhibited in the art, including but not limited to those as defined above.
  • the pigs may be selected from Bama pigs, Wuzhishan pigs and/or Landrace pigs.
  • the present application provides an animal model prepared according to the method for constructing an animal model described in the present application, wherein the animal does not express the ⁇ 4GalNT2 gene and/or ⁇ -1,4-N-acetylgalactosaminyltransferase 2 .
  • the animal may not express the GGTA1 gene and/or ⁇ Gal.
  • the animal may not express the CMAH gene and/or Neu5Gc.
  • the non-expression of ⁇ 4GalNT2 gene, GGTA1 and/or CMAH generally refers to the insertion, interruption or deletion of the nucleotide sequence of the gene, or the transcription and mRNA translation of the gene involved in the precursor or mature
  • the reduction or absence of function of the protein alternatively, encodes a polypeptide with fewer amino acid residues than the endogenous amino acid sequence or does not encode a polypeptide.
  • non-expression of ⁇ -1,4-N-acetylgalactosaminyltransferase 2, ⁇ Gal and/or Neu5Gc involves a reduction or elimination of activity or levels.
  • the expression level of a gene or protein can be detected by a variety of methods, including methods at the RNA level (including mRNA quantification by reverse transcriptase polymerase chain reaction (RT-PCR) or by Southern blotting, in situ hybridization) and at the protein level. horizontal methods (including histochemistry, immunoblot analysis, and in vitro binding studies). Furthermore, the expression level of the gene of interest can be quantified by ELISA techniques well known to those skilled in the art. Quantitative measurements can be done using a number of standard assays. For example, transcript levels can be measured using RT-PCR and hybridization methods including RNase protection, Southern blot analysis, RNA dot analysis.
  • Western blot analysis can also be used to assess the presence of the ⁇ 4GalNT2 gene and/or ⁇ -1,4-N-acetylgalactosaminyltransferase 2, GGTA1 gene and/or ⁇ Gal, and/or Or, CMAH gene and/or Neu5Gc.
  • the present application provides a method for preparing an animal model
  • the method may include: a) providing the animal model described in the present application; b) mating the animal model obtained in step a) with other animals or in vitro fertilization Or further perform gene editing on the animal model obtained in step a) or transplant human tissues and cells into the animal model obtained in step a), and perform screening to obtain an animal model.
  • the present application provides an animal model prepared according to the method for preparing an animal model.
  • the animal can include a pig.
  • the present application provides a cell or cell line or primary cell culture, wherein the cell or cell line or primary cell culture can be derived from the animal model described herein or its progeny.
  • Cell cultures can be isolated from non-human mammals or prepared from cell cultures established using standard cell transfection techniques using the same constructs.
  • the present application provides a tissue or organ or a culture thereof, wherein the tissue or organ or a culture thereof can be derived from the animal model or progeny thereof.
  • the present application provides a CRISPR/Cas9 system for specifically targeting the ⁇ 4GalNT2 gene, which may include using a DNA sequence containing the gRNA specifically targeting the ⁇ 4GalNT2 gene described in the present application.
  • the present application provides a nucleic acid molecule kit capable of specifically targeting the ⁇ 4GalNT2 gene, wherein the kit may include the gRNA specifically targeting the ⁇ 4GalNT2 gene.
  • the present application provides a set of nucleic acid molecules that can specifically target the ⁇ 4GalNT2 gene, wherein the set of nucleic acid molecules can include the sgRNA that specifically targets the ⁇ 4GalNT2 gene and the mRNA of Cas9 nuclease.
  • the present application provides said cells or cell lines or primary cell cultures, said tissues or organs or cultures thereof in organ and/or tissue transplantation product development, or as pharmacological, immunological and Model systems for medical research, or applications in the validation, evaluation or study of immune rejection.
  • the present application provides the use of the animal model in the development of organ and/or tissue transplantation products, or as a model system for pharmacological, immunological and medical research.
  • the organ and/or tissue transplantation product includes cells, tissues or organs from different species transplanted, implanted or infused into a recipient subject. Transplants in which the recipient is a human are particularly contemplated.
  • the transplant product can be isolated from transgenic animals with reduced expression of aGal, ⁇ -1,4N-acetylgalactosaminyltransferase 2 and Neu5Gc.
  • the organ and/or tissue transplant product can be isolated from prenatal, neonatal, immature or fully mature transgenic animals.
  • Transplant material can be used as a temporary or permanent organ replacement for human subjects in need of organ transplantation.
  • the present application provides the application of the animal model in verifying, evaluating or studying immune rejection.
  • Immune rejection occurs when the transplanted tissue, organ, cell or material is not accepted by the recipient's body. In immune rejection, the recipient's immune system attacks the transplanted material. There are various types of immune rejection and they can occur individually or together. Immune rejection includes, but is not limited to, hyperacute rejection (HAR), acute humoral xenograft rejection (AHXR), thrombocytopenia, acute humoral rejection, hyperacute vascular rejection, antibody mediated rejection, and graft versus host disease.
  • HAR hyperacute rejection
  • HXR acute humoral xenograft rejection
  • thrombocytopenia acute humoral rejection
  • hyperacute vascular rejection hyperacute vascular rejection
  • antibody mediated rejection and graft versus host disease.
  • Methods for analyzing symptoms associated with immune rejection may include, but are not limited to, laboratory assessments including CBC with platelet count, coagulation studies, liver function tests, flow cytometry, immunohistochemistry, standard diagnostic criteria, immunological methods, western blotting assay, immunoblotting, microscopy, confocal microscopy, transmission electron microscopy, IgG binding assay, IgM binding assay, expression assay, creatinine assay and endosome isolation.
  • sgRNAs single guide RNA
  • pX330 Additional plasmid 423230
  • the exon 3 exon3 of GGTA1 gene, exon 6 exon6 of CMAH gene and exon 8 exon8 of ⁇ 4GalNT2 gene were selected as CRISPR/Cas9 targets.
  • the 5' end is G
  • the 3' end is PAM sequence (NGG)
  • the sgRNA sequence designed to target GGTA1 is GAAAATAATGAATGTCAA (SEQ ID NO: 9)
  • the sgRNA sequence targeting CMAH is GAGTAAGGTACGTGATCTGT (SEQ ID NO: 9).
  • the sgRNA1 sequence targeting ⁇ 4GalNT2 is GGTAGTACTCACGAACACTC (SEQ ID NO:6) and the sgRNA2 sequence targeting ⁇ 4GalNT2 is CTACCCTTTCTTGCCCAGAG (SEQ ID NO:7).
  • the sgRNA sequence was cloned into the pX330 backbone vector (for targeting the ⁇ 4GalNT2 gene, when the two sgRNAs were in the same vector, the cloned fragment was U6 promoter- ⁇ 4GalNT2/sgRNA1-gRNA backbone-U6 promoter- ⁇ 4GalNT2/sgRNA2-gRNA skeleton), the specific steps are as follows:
  • the digested pX330 plasmid was separated by running on agarose gel (agarose gel concentration was 1%, that is, 1 g of agarose gel was added to 100 mL of electrophoresis buffer), and the digested product was purified and recovered with a gel recovery kit (QIAGEN). ;
  • step 5 (2) Add 15 ⁇ L of the misconnection-removing plasmid solution obtained in step 5 to the centrifuge tube containing competent cells, mix well, and let stand in an ice bath for 30 minutes;
  • the CRSAPR/Cas9 targeting vectors were obtained, and the vectors were named as GGTA1-CRISPR/Cas9 ( Figure 1, the full nucleotide sequence is shown in SEQ ID NO: 4), CMAH--CRISPR/Cas9 ( Figure 2, the full nucleotide sequence). shown in SEQ ID NO: 5) and ⁇ 4GalNT2-CRISPR/Cas9 ( Figure 3, the full nucleotide sequence is shown in SEQ ID NO: 3), the nucleotide sequence is shown.
  • the GGTA1-CRISPR/Cas9 targeting vector, CMAH-CRISPR/Cas9 targeting vector and ⁇ 4GalNT2-CRISPR/Cas9 targeting vector were simultaneously transfected into male Landrace pig fetal fibroblasts, and single-cell clones with triple gene knockout were obtained by G418 screening. Specific steps are as follows.
  • the formula of cell complete medium is: 16% fetal bovine serum (Gibco) + 84% DMEM medium (Gibco), 16% and 84% are volume percentages.
  • GGTA1-CRISPR/Cas9 targeting vector CMAH-CRISPR/Cas9 targeting vector, ⁇ 4GalNT2-CRISPR/Cas9 targeting vector and tdTomato plasmid (Clontech, PT4069-5) to co-transfect primary porcine fibroblasts
  • the cell suspension prepared in 2.1 was washed twice with DPBS Dulbecco's Phosphate Buffered Saline (Gibco), digested at 37°C for 2 min, terminated with DMEM complete medium containing 10% fetal bovine serum by volume, and centrifuged at 1500 rpm for 5 min , discard the supernatant, and resuspend the cells with the plasmid-containing nuclear transfer reaction solution in step 2, avoiding the generation of air bubbles during the resuspension process;
  • PCR primer sequences are:
  • the forward primer is: 5'-CCTTAGTATCCTTCCCAACCCAGAC-3' (SEQ ID NO:11)
  • the reverse primer is: 5'-GCTTTCTTTACGGTGTCAGTGAATCC-3' (SEQ ID NO:12)
  • the length of the PCR target product is 428bp
  • the forward primer is: 5'-CTTGGAGGTGATTTGAGTTGGG-3' (SEQ ID NO:13)
  • the reverse primer is: 5'-CATTTTCTTCGAGTTGAGGGC-3' (SEQ ID NO:14)
  • PCR target product length is 485bp
  • the forward primer is: 5'-CCCAAGGATCCTGCTGCC-3' (SEQ ID NO:15)
  • the reverse primer is: 5'-CGCCGTGTAAAGAAACCTCC-3'; (SEQ ID NO:8)
  • PCR target product length is 406bp
  • the amplification of the CMAH target gene is the same as the above steps; the amplification of the ⁇ 4GalNT2 target gene is the same as the above steps.
  • TAKARA pMDTM 18-T Vector Cloning Kit to link the recovered PCR product to the T vector.
  • the T vector reaction system is as follows:
  • the TAKARA pMD TM 18-T Vector Cloning Kit instruction manual requires 0.1 to 0.3 pM for the amount of Insert DNA (this time, the PCR product is recovered by gel), and this time 0.2 pM is selected.
  • reaction conditions for T carrier linking are 16°C for 30min;
  • TIANGEN Use competent cells (TIANGEN) to transform the T vector linked product obtained in this step 5. After the transformation, the competent cells are spread on Amp-resistant LB agar solid medium, and cultured in a 37°C constant temperature incubator overnight;
  • ⁇ 4GalNT2 CRISPR/Cas9 targeting vector was constructed using ⁇ 4GalNT2 sgRNA (SEQ ID NO:6)
  • CCTA1 CRISPR/Cas9 targeting vector was constructed using GGTA1 sgRNA (SEQ ID NO:9)
  • a double knockout ⁇ 4GalNT2 and GGTA1 targeting vector was constructed.
  • Bama pig and Wuzhishan pig cells were cloned and sequenced to obtain knockout efficiency and genotype of double knockout cell clones.
  • the knockdown efficiency of ⁇ 4GalNT2 using a single sgRNA of ⁇ 4GalNT2 was 21.88% (Bama pig) and 17.65% (Wuzhishan pig).
  • the double knockout efficiency of ⁇ 4GalNT2 and GGTA1 was 21.88% (Bama pig) and 13.24% (Wuzhishan pig).
  • sgRNAs of ⁇ 4GalNT2 (SEQ ID NO: 6 and 7) were used to construct the ⁇ 4GalNT2 CRISPR/Cas9 targeting vector
  • sgRNA of GGTA1 (SEQ ID NO: 9) was used to construct the CCTA1 CRISPR/Cas9 targeting vector
  • sgRNA of CMAH was used (SEQ ID NO: 10)
  • a CMAH CRISPR/Cas9 targeting vector was constructed, and a landrace pig cell clone with triple knockout of ⁇ 4GalNT2, GGTA1 and CMAH was constructed and sequenced to obtain the knockout efficiency and the genotype of the triple knockout cell clone.
  • the efficiency of using two sgRNAs for ⁇ 4GalNT2 to knock out ⁇ 4GalNT2 was 30.51% (landrace male) and 39.21% (landrace female), compared with the use of 1 sgRNA in Example 3, the knockdown of ⁇ 4GalNT2 using two sgRNAs to raise efficiency.
  • the triple knockout efficiency of ⁇ 4GalNT2, GGTA1 and CMAH was 25.42% (Landrace male), which was higher than the double gene knockout efficiency (21.88%) in Example 2, indicating that the sgRNA of the present application not only achieved triple gene knockout, but also The knockout efficiency is higher than that of the double gene.

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Abstract

提供了一种特异性靶向β4GalNT2基因的gRNA，该gRNA特异性结合SEQ ID NO.1-2中任一项所示的核苷酸序列。还提供了使用该gRNA构建的动物模型，以及它们在生物医药领域中的应用。

Description

敲除猪异种抗原的基因的gRNA及其应用 技术领域
本申请涉及生物医药领域，具体的涉及特异性靶向β4GalNT2基因的gRNA及其应用。
背景技术
目前，细胞、组织和/或器官移植是许多疾病(包括肾、心、肺、肝和其他器官疾病或皮肤损伤)的常规治疗选择。为了克服缺乏足够的移植物、无法满足临床需求的问题，出现了将来源于一种物种(例如，猪)的移植物移植到另一物种(例如，人类)的异种移植。但是，使用标准的未修饰的异种动物的移植物可能伴随严重的免疫排斥反应。
为了解决现有的异种移植治疗中出现的免疫排斥反应，可以通过基因编辑敲除编码某些引起免疫***反应的蛋白的基因。
发明内容
本申请提供了一种特异性靶向β4GalNT2基因的gRNA，所述gRNA包含SEQ ID NO.6-7中任一项所示的核苷酸序列。本申请所述的gRNA显著提高了基因的敲除效率。使用本申请所述的特异性靶向β4GalNT2基因的gRNA，可以使β4GalNT2基因的敲除效率达到至少20％以上(例如至少21％以上、22％以上、23％以上、24％以上、25％以上、26％以上、27％以上、28％以上、29％以上或更高)。与使用一种gRNA敲除相比，使用两种所述sgRNA可以提高敲除效率，例如，可提高至少5％以上(例如，至少10％以上、15％以上、20％以上、25％以上、30％以上、35％以上40％以上、45％以上、50％以上或更高)。本申请所述的特异性靶向β4GalNT2基因的gRNA还可以和特异性靶向GGTA1和/或CMAH基因的gRNA共同用于基因敲除，可以使三个基因同时敲除的敲除效率达到至少10％以上(例如，至少11％以上、12％以上、13％以上、14％以上、15％以上、16％以上、17％以上、18％以上、19％以上、20％以上、25％以上或更高)。本申请所述的sgRNA可以具有较高的敲除效率和/或稳定的敲除效率。
本申请还提供了编码所述gRNA的核酸分子、包括所述核酸分子或sgRNA的细胞、载体、载体转录物、试剂盒和/或***，和根据所述gRNA制备的动物模型、细胞、组织和/或器官，以及他们的应用。
一方面，本申请提供了一种特异性靶向β4GalNT2基因的gRNA，其中所述gRNA特异 性结合SEQ ID NO.1-2中任一项所示的核苷酸序列。
在某些实施方式中，所述gRNA包含SEQ ID NO.16所示的核苷酸序列。
在某些实施方式中，所述gRNA包含SEQ ID NO.6-7中任一项所示的核苷酸序列。
在某些实施方式中，所述sgRNA包含5’-(X)n-SEQ ID NO.16所示的核苷酸序列-骨架序列-3’，其中X为选自A、U、C和G中任一个的碱基，且n为0-15中的任一整数。
在某些实施方式中，所述gRNA为单链向导RNA(sgRNA)。
另一方面，本申请提供了一种gRNA组合，其包括本申请所述的特异性靶向β4GalNT2基因的gRNA，特异性靶向GGTA1基因的gRNA和特异性靶向CMAH基因的gRNA。
在某些实施方式中，所述特异性靶向GGTA1基因的gRNA包含SEQ ID NO.9所示的核苷酸序列。
在某些实施方式中，所述特异性靶向CMAH基因的gRNA包含SEQ ID NO.10所示的核苷酸序列。
另一方面，本申请提供了一种或多种分离的核酸分子，其编码本申请所述的特异性靶向β4GalNT2基因的gRNA。
另一方面，本申请提供了一种载体，其包含本申请所述的核酸分子。
在某些实施方式中，本申请的编码所述特异性靶向β4GalNT2基因的gRNA的一种和/或两种核酸分子，编码所述特异性靶向GGTA1基因的gRNA的核酸分子，以及编码所述特异性靶向CMAH基因的gRNA的核酸分子位于同一载体中。
另一方面，本申请提供了细胞，其包含所述特异性靶向β4GalNT2基因的gRNA，所述的核酸分子，所述的载体，和/或所述的载体的体外转录产物。
另一方面，本申请提供了所述特异性靶向β4GalNT2基因的gRNA，所述的核酸分子，所述的载体，所述的载体的体外转录产物和/或所述的细胞在敲除β4GalNT2基因中的用途，或者在构建动物模型中的用途。
另一方面，本申请提供了一种β4GalNT2基因缺失细胞株，其是使用所述特异性靶向β4GalNT2基因的sgRNA，所述的核酸分子，所述的载体，所述的载体的体外转录产物和/或所述的细胞制备获得的。
另一方面，本申请提供了一种构建动物模型的方法，所述方法包括向动物的细胞施用至少两种特异性靶向β4GalNT2基因的gRNA，从而敲除β4GalNT2基因的全部或部分，其中所述gRNA特异性结合SEQ ID NO.1-2中任一项所示的核苷酸序列。
在某些实施方式中，所述特异性靶向β4GalNT2基因的gRNA包含SEQ ID NO.16所示的 核苷酸序列。
在某些实施方式中，所述特异性靶向β4GalNT2基因的gRNA包含SEQ ID NO.6-7中任一项所示的核苷酸序列。
在某些实施方式中，所述特异性靶向β4GalNT2基因的gRNA包含5’-(X)n-SEQ ID NO.16所示的核苷酸序列-骨架序列-3’，其中X为选自A、U、C和G中任一个的碱基，且n为0-15中的任一整数。
在某些实施方式中，所述gRNA为单链向导RNA(sgRNA)。
在某些实施方式中，所述方法包括以下步骤：使用一种或多种脱氧核糖核酸(DNA)核酸内切酶以在所述β4GalNT2基因内或其附近产生一个或更多个单链断裂(SSB)或双链断裂(DSB)，从而使得所述β4GalNT2基因的一个或更多个外显子全部或部分缺失。
在某些实施方式中，所述方法还包括向动物的细胞施用特异性靶向GGTA1基因的gRNA，从而敲除GGTA1基因的全部或部分。
在某些实施方式中，所述特异性靶向GGTA1基因的gRNA包含SEQ ID NO.9所示的核苷酸序列。
在某些实施方式中，所述方法还包括向动物的细胞施用特异性靶向CMAH基因的gRNA，从而敲除CMAH基因的全部或部分。
在某些实施方式中，所述特异性靶向CMAH基因的gRNA包含SEQ ID NO.10所示的核苷酸序列。
在某些实施方式中，所述方法包括以下步骤：使用一种或多种脱氧核糖核酸(DNA)核酸内切酶以在所述GGTA1基因和/或所述CMAH基因内或其附近产生一个或更多个单链断裂(SSB)或双链断裂(DSB)，从而使得所述GGTA1基因和/或所述CMAH基因的一个或更多个外显子全部或部分缺失。
在某些实施方式中，所述DNA核酸内切酶包括Cas核酸酶。
在某些实施方式中，所述Cas核酸酶包括Cas9核酸酶、其同源物、其天然存在分子的重组体、其密码子优化版本，和/或其经修饰版本。
在某些实施方式中，所述方法包括：a)提供一种细胞，所述细胞包含一种或多种包含本申请所述的载体或所述gRNA载体的体外转录产物；b)将所述细胞在培养液中进行培养；c)将培养后的细胞移植至受体雌性非人哺乳动物的输卵管内，允许所述细胞在所述雌性非人哺乳动物的子宫中发育；和d)鉴定步骤c)的怀孕雌性的后代基因改造的非人哺乳动物中的种系传递。
在某些实施方式中，所述动物包括猪。
另一方面，本申请提供了根据本申请所述的构建动物模型的方法制备获得动物模型，其中所述动物不表达β4GalNT2基因和/或β-1,4-N-乙酰氨基半乳糖转移酶2。
在某些实施方式中，所述动物不表达GGTA1基因和/或αGal。
在某些实施方式中，所述动物不表达CMAH基因和/或Neu5Gc。
另一方面，本申请提供了一种制备动物模型的方法，所述方法包括：a)提供本申请所述的动物模型；b)将步骤a)获得的动物模型与其它动物交配或体外授精或对基于步骤a)获得的动物模型进一步进行基因编辑或将人组织、细胞移植至步骤a)获得的动物模型中，并进行筛选，得到的动物模型。
另一方面，本申请提供了根据所述制备动物模型的方法制备获得动物模型。
在某些实施方式中，所述动物包括猪。
另一方面，本申请提供了一种细胞或细胞系或原代细胞培养物，其中所述的细胞或细胞系或原代细胞培养物来源于本申请所述的动物模型或其后代。
另一方面，本申请提供了一种组织或器官或其培养物，其中所述组织或器官或其培养物来源于所述的动物模型或其后代。
另一方面，本申请提供了一种特异靶向敲除β4GalNT2基因的CRISPR/Cas9***，其包括使用含有本申请所述的特异性靶向β4GalNT2基因的gRNA的DNA序列。
另一方面，本申请提供了一种能够特异性靶向β4GalNT2基因的核酸分子试剂盒，其中，所述试剂盒包括所述的特异性靶向β4GalNT2基因的gRNA。
另一方面，本申请提供了一种能够特异的靶向β4GalNT2基因的成套核酸分子，其中，所述成套核酸分子包括所述的特异性靶向β4GalNT2基因的sgRNA和Cas9mRNA。
另一方面，本申请提供了所述的细胞或细胞系或原代细胞培养物，所述的组织或器官或其培养物在器官和/或组织移植产品开发，或者作为药理学、免疫学和医学研究的模型***，或在验证、评价或研究免疫排斥反应中的应用。
另一方面，本申请提供了所述的动物模型在器官和/或组织移植产品开发，或者作为药理学、免疫学和医学研究的模型***中的应用。
另一方面，本申请提供了所述的动物模型在验证、评价或研究免疫排斥反应方面的应用。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉 及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明书如下：
图1显示的是GGTA1-CRISPR/Cas9打靶载体的示意图。
图2显示的是CMAH-CRISPR/Cas9打靶载体的示意图。
图3显示的是双sgRNA β4GalNT2-CRISPR/Cas打靶载体的示意图。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“单链断裂(SSB)”通常是指DNA分子的一条链被切割发生断裂的现象。当DNA双螺旋的两条链中只有一条具有缺陷时，另一条链可以用作模板来指导受损链的校正。DNA核酸内切酶可以引起单链断裂。
在本申请中，术语“双链断裂(DSB)”通常是指双股DNA分子的两条单链在同一位置被切割时发生的现象。双股断裂可诱发DNA修复，可能造成遗传重组，细胞也有一些***作用于其他时候造成的双股断裂。双链断裂可在正常细胞复制周期中定期发生，也可以在某些情况下增强，例如紫外线、DNA断裂的诱导剂(例如，各种化学诱导剂)。许多诱导剂可以导致DSB在基因组中无差别地发生，并且DSB可以在正常细胞中有规律地诱导和修复。在修复过程中，可以完全保真地重建原始序列，但是，在某些情况下，会在DSB站点引入小的***或缺失(称为“indels”)。在某些情形中，还可以在特定的位置特异性诱导双链断裂，其可以用于在选定的染色***置引起定向或优先的基因修饰。在许多情况下，可以利用DNA修复(以及复制)过程中同源序列易于重组的趋势，这是基因编辑***(如 CRISPR)应用的基础。该同源性指导的修复用于将通过使用“供体”多核苷酸提供的目的序列***所需的染色***置。
在本申请中，术语“体外转录产物”通常是指通过DNA体外转录合成，或者在处理后变成信使RNA(mRNA)的产物。体外转录产物可包括前体信使RNA(前mRNA)和经处理的mRNA自身。在DNA链转录为转录产物后，新近合成的初级转录物可以以若干方式改性，以转化为它们的成熟功能形式，以产生不同的蛋白质和RNA(如mRNA、tRNA、rRNA、lncRNA、miRNA等)。术语“体外转录产物”可包括外显子、内含子、5’UTR和3’UTR。
在本申请中，所述“载体”通常是指能够在合适的宿主中自我复制的核酸分子，用以将***的核酸分子转移到宿主细胞中和/或宿主细胞之间。所述载体可包括主要用于将DNA或RNA***细胞中的载体、主要用于复制DNA或RNA的载体，以及主要用于DNA或RNA的转录和/或翻译的表达的载体。所述载体还包括具有多种上述功能的载体。所述载体可以是当引入合适的宿主细胞时能够转录并翻译成多肽的多核苷酸。通常，通过培养包含所述载体的合适的宿主细胞，所述载体可以产生期望的表达产物。载体可涵盖除转基因***序列和主链以外的额外特征：启动子、遗传标记、抗生素抗性、报告基因、靶向序列、蛋白质纯化标签。称为表达载体(表达构建体)的载体具体地讲用于在靶细胞中表达转基因，且通常具有控制序列。本申请所述的载体可以是表达载体，可包括病毒载体(慢病毒载体和/或逆转录病毒载体)、噬菌体载体、噬菌粒、粘粒、cosmid、人工染色体如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)和/或质粒。
在本申请中，术语“猪”通常是指本领域中己知的任何猪，包括但不限于：野生猪、家养猪、迷你猪、野猪(Sus scrofa pig)、家猪(Sus scrofa domesticus pig)以及近亲交配猪。不受限制，所述猪可选自包含以下的组：兰德瑞斯猪(Landrace，也称为长白猪)、约克郡猪(Yorkshire)、汉普郡猪(Hampshire)、杜洛克猪(Duroc)、中国眉山猪(Chinese Meishan)、切斯特白猪(Chester White)、伯克希尔戈廷根猪(Berkshire Goettingen)、兰德瑞斯/约克郡/切斯特白猪、尤卡坦猪(Yueatan)、巴马香猪(Barna)、五指山猪(Wuzhishan)、西双版纳猪(Xi Shuang Banna)和皮特兰猪(Pietrain)。
在本申请中，术语“脱氧核糖核酸(DNA)核酸内切酶”通常是指可水解DNA分子链内部的磷酸二酯键，从而生成寡核苷酸的酶。DNA核酸内切酶可包括不具碱基特异性的酶和能够识别并切断特定的碱基或碱基序列的酶。
在本申请中，术语“Cas核酸酶”通常是指CRISPR相关核酸酶，一种DNA核酸内切酶，其可在特定的DNA序列处形成双链断裂。Cas核酸酶通常可以与CRISPR序列互补，能够使用CRISPR序列作为指导(guide)，从而识别和切割特定的DNA链。Cas核酸酶的实例可包括但不限于下组：Casl、CaslB、Cas2、Cas3、Cas4、Cas5、Cas6、Cas7、Cas8、Cas9(也称为Csnl和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，和/或他们的同系物、或其修饰形式。
在本申请中，术语“Cas9核酸酶”也可称为，Csn1或Csx12，通常是指与II型CRISPR(有规律地间隔的短回文重复序列)自适应免疫***相关的RNA引导的DNA核酸内切酶。Cas9核酸酶还可包括野生型蛋白质，直系同源物，及其功能性和非功能性突变体。所述Cas9核酸酶可源于任何合适的细菌。Cas9核酸酶通常包括RuvC核酸酶结构域和HNH核酸酶结构域，分别切割双链DNA分子的两条不同的链。已经在不同的细菌物种如嗜热链球菌(S.thermophiles)、无害利斯特氏菌(Listeria innocua)(Gasiunas，Barrangou et al.2012；Jinek，Chylinski et al.2012)和化脓性链球菌(S.Pyogenes)(Deltcheva，Chylinski et al.2011)中描述了Cas9核酸酶。例如，化脓链球菌(Streptococcus pyogenes)Cas9蛋白，其氨基酸序列参见SwissProt数据库登录号Q99ZW2；脑膜炎奈瑟氏菌(Neisseria meningitides)Cas9蛋白，其氨基酸序列见UniProt数据库编号A1IQ68；嗜热链球菌(Streptococcus thermophilus)Cas9蛋白，其氨基酸序列见UniProt数据库编号Q03LF7；金黃色葡萄球菌(Staphylococcus aureus)Cas9蛋白，其氨基酸序列见UniProt数据库编号J7RUA5。
在本申请中，术语“gRNA”通常是指指导RNA(guide RNA)，一种RNA分子。在自然界中，crRNA和tracrRNA通常作为两个独立的RNA分子存在，组成gRNA。术语“crRNA”也称为CRISPR RNA，通常是指与所靶向的目标DNA互补的一段核苷酸序列，术语“tracrRNA”通常是指可与Cas核酸酶结合的支架型RNA。crRNA和tracRNA也可以融合成为单链，此时gRNA也可称为单链指导RNA(sgRNA)，sgRNA已成为本领域技术人员在CRISPR技术中使用的gRNA的最常见的形式，因此术语“sgRNA”和“gRNA”在本文中可具有相同的含义。sgRNA可以人工合成，也可以在体外或体内由DNA模板制备。sgRNA可以结合Cas核酸酶，也可以靶向目标DNA，其可引导Cas核酸酶切割与gRNA互补的DNA位点。gRNA与其相应的靶序列之间的互补程度至少为约50％。
在本申请中，术语“骨架序列”通常是指gRNA中，除识别或杂交靶序列的部分的其他部分，可包括sgRNA中gRNA配对序列与转录终止子之间的序列。骨架序列一般不会因为靶序列的变化而变化，也不影响gRNA对靶序列的识别。因此，骨架序列可以是现有技术中任何可行的序列。骨架序列的结构可参见如文献Nowak et al.Nucleic Acids Research 2016.44:9555-9564中的Figure 1中A和B，Figure 3中A、B、C，以及Figure 4中A、B、C、D、E中所记载的除spacer序列之外的部分。
在本申请中，术语“靶核酸”、“靶核酸”和“靶区域”可以互换的使用，通常是指可以被gRNA识别的核酸序列，所述靶核酸可以指双链核酸，也可以指单链核酸。
在本申请中，术语“分离的核酸分子”通常是指从5’至3’末端阅读的脱氧核糖核苷酸或核糖核苷酸碱基的单链或双链聚合物或其类似物。分离的核酸分子可以是从通常的或天然的环境中分离出的，也可以是人工合成的方式生产而成的。这种分离的核酸分子从其通常的或天然的环境中移出的或分离的，或者生产所述分子的方式使其不存在于其通常的或天然的环境中，其与通常的或天然的环境中的多肽、肽、脂质、糖类、其他的多核苷酸或其它材料分离。本申请中的分离的核酸分子可编码RNA，例如，可编码特异性靶向RPGR基因的gRNA。
在本申请中，术语“CMAH”通常是指编码胞苷单磷酸-N-乙酰神经氨酸羟化酶(CMP-Neu5Ac羟化酶)的基因。功能性的胞苷单磷酸-N-乙酰神经氨酸羟化酶可以催化唾液酸N-乙酰神经氨酸(Neu5Ac)转化为N-羟乙酰神经氨酸(Neu5Gc)。Neu5Gc残基是被人免疫***识别的抗原表位或抗原。本申请所述胞苷单磷酸-N-乙酰神经氨酸羟化酶可包括全长蛋白、功能性片段、同源物和/或功能性变体(如，剪接变体)。本申请所述的CMAH基因的核苷酸序列包括其功能性变体、衍生物、类似物、同源物及其片段。猪CMAH蛋白的氨基酸序列可参见NCBI数据库的登录号NP_001106486.1下，猪CMAH核苷酸序列可参见NCBI数据库的登录号NM_001113015.1下。
在本申请中，术语“GGTA1”也可称为αGal、GGTA、GGTl、GT、αGT、GGTA 1，通常是指编码α1,3半乳糖基转移酶(αGal，GT)的基因。功能性α1,3半乳糖基转移酶可以催化在糖蛋白上形成半乳糖α1,3-半乳糖(αGal、Gal、Gal、gall、3gal或gal1-3gal)残基。半乳糖α1,3-半乳糖(αGal)残基是人免疫***识别的抗原表位或抗原。本申请所述α1,3半乳糖基转移酶可包括全长蛋白、功能性片段、同源物和/或功能性变体(如，剪接变体)。本申请所述的GGTA1基因的核苷酸序列包括其功能性变体、衍生物、类似物、同源物及其片段。猪GGTA1蛋白的氨基酸序列可参见NCBI数据库的登录号NP_001309984.1下，猪GGTA1核苷酸序列可参见NCBI数据库的登录号NM_001323055.1下。
在本申请中，术语“β4GalNT2”通常是指编码β-1,4N-乙酰氨基半乳糖转移酶2(β4GalNT2、β4GalNT2、βl,4GalNT2、β1,4GalNT2)的基因，功能性的β4GalNT2可产生Sda样聚糖。本申请所述的β-1,4N-乙酰氨基半乳糖转移酶2可包括全长蛋白、功能性片段、同源物和/或功能性变体(如，剪接变体)。本申请所述的β4GalNT2基因的核苷酸序列包括其功能性变体、衍生物、类似物、同源物及其片段。猪β4GalNT2蛋白的氨基酸序列可参见NCBI数据库的登录号NP_001231259.1下，猪β4GalNT2核苷酸序列可参见NCBI数据库的登录号NM_001244330.1下。
发明详述
一方面，本申请提供了一种特异性靶向β4GalNT2基因的gRNA，其中所述gRNA可以特异性结合SEQ ID NO.1-2中任一项所示的核苷酸序列。在某些情形中，所述特异性靶向β4GalNT2基因的gRNA可特异性结合与SEQ ID NO.1-2中任一项所示的核苷酸序列具有至少70％(例如，至少75％，至少80％，至少85％，至少90％，至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％，至少99％，或至少100％)序列同一性的核苷酸序列。
在某些情形中，所述特异性靶向β4GalNT2基因的gRNA可特异性结合与SEQ ID NO:1-2中任一项所示的核苷酸序列互补的核苷酸序列。在某些情形中，所述gRNA可特异性结合与SEQ ID NO.1-2中任一项所示的核苷酸序列具有至少70％(例如，至少75％，至少80％，至少85％，至少90％，至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％，至少99％，或至少100％)序列同一性的核苷酸序列互补的核苷酸序列。
本申请所述的特异性靶向β4GalNT2基因的gRNA可以与目标靶核酸中的序列结合。gRNA可以通过杂交(即碱基配对)以序列特异性的方式与靶核酸相互作用。所述特异性靶向β4GalNT2基因的gRNA的核苷酸序列可以根据目标靶核酸的序列而变化。
在本申请中，所述特异性靶向β4GalNT2基因的gRNA可包含SEQ ID NO.16所示的核苷酸序列：n 1n 2n 3n 4n 5n 6n 7n 8tcn 11n 12gn 14n 15can 18n 19n 20，其中n 1＝c或g，n 2＝g或u，n 3＝a或u，n 4＝a或c，n 5＝c或g，n 6＝c或u，n 7＝a或u，n 8＝c或u，n 11＝a或u，n 12＝c或u，n 14＝a或c，n 15＝a或c，n 18＝c或g，n 19＝a或u，且n 20＝c或g。本申请中，所述gRNA可包括与SEQ ID NO.16中任一项所示的核苷酸序列具有至少70％(例如，至少75％，至少80％，至少85％，至少90％，至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％，至少99％，或至少100％)序列同一性的核苷酸序列。
在本申请中，所述特异性靶向β4GalNT2基因的gRNA可包含SEQ ID NO.6-7中任一项所示的核苷酸序列。本申请中，所述gRNA可包括与SEQ ID NO.6-7中任一项所示的核苷酸序列具有至少70％(例如，至少75％，至少80％，至少85％，至少90％，至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％，至少99％，或至少100％)序列同一性的核苷酸序列。
在本申请中，所述特异性靶向β4GalNT2基因的gRNA可包含骨架序列，本申请使用的骨架序列可以来自任何商购可得的质粒，只要其可实现Cas核酸酶的表达和gRNA的转录即可。例如，所述骨架序列可以是来自pX330的骨架序列。例如，所述骨架序列可以是SEQ ID NO:17所示的核苷酸序列。在本申请中，所述gRNA包含5’-(X)n-SEQ ID NO:16中所示的核苷酸序列-骨架序列-3’，其中X为选自A、U、C和G中任一个的碱基，且n为0-15中的任一整数。例如，n为0。例如，本申请所述gRNA可包含5’-SEQ ID NO:6所示的核苷酸序列-SEQ ID NO:17所示的核苷酸序列-3’，或5’-SEQ ID NO:7所示的核苷酸序列-SEQ ID NO:17所示的核苷酸序列-3’。
在本申请中，所述特异性靶向β4GalNT2基因的gRNA可以为单链向导RNA(sgRNA)。
另一方面，本申请提供了一种gRNA组合，其可包括本申请所述的特异性靶向β4GalNT2基因的gRNA，特异性靶向GGTA1基因的gRNA和特异性靶向CMAH基因的gRNA。
在本申请中，所述特异性靶向GGTA1基因的gRNA可包含SEQ ID NO.9所示的核苷酸序列。本申请中，所述特异性靶向GGTA1基因的gRNA可包括与SEQ ID NO.9中任一项所示的核苷酸序列具有至少70％(例如，至少75％，至少80％，至少85％，至少90％，至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％，至少99％，或至少100％)序列同一性的核苷酸序列。
在本申请中，所述特异性靶向CMAH基因的gRNA可包含SEQ ID NO.10所示的核苷酸序列。本申请中，所述特异性靶向CMAH基因的gRNA可包括与SEQ ID NO.10中任一项所示的核苷酸序列具有至少70％(例如，至少75％，至少80％，至少85％，至少90％，至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％，至少99％，或至少100％)序列同一性的核苷酸序列。
另一方面，本申请提供了一种或多种分离的核酸分子，其可编码本申请所述的特异性靶向β4GalNT2基因的gRNA。
另一方面，本申请提供了一种载体，其可包含本申请所述编码特异性靶向β4GalNT2基因的gRNA的核酸分子。
当使用本申请的编码所述特异性靶向β4GalNT2基因的gRNA的两种核酸分子时，这两种核酸分子可以位于同一载体中，或者，也可以位于不同的载体中。例如，编码所述特异性靶向β4GalNT2基因的gRNA的两种核酸分子可以位于同一载体中。
在申请中，编码所述特异性靶向β4GalNT2基因的gRNA的一种和/或两种核酸分子，编码所述特异性靶向GGTA1基因的gRNA的核酸分子，以及编码所述特异性靶向CMAH基因的gRNA的核酸分子中的每一个核酸分子可以位于1个、2个、3个或4个载体中。
在申请中，编码所述特异性靶向β4GalNT2基因的gRNA的一种和/或两种核酸分子，编码所述特异性靶向GGTA1基因的gRNA的核酸分子，以及编码所述特异性靶向CMAH基因的gRNA的核酸分子中的每一个核酸分子可以位于不同的载体中。
在申请中，编码所述特异性靶向β4GalNT2基因的gRNA的一种和/或两种核酸分子，编码所述特异性靶向GGTA1基因的gRNA的核酸分子，以及编码所述特异性靶向CMAH基因的gRNA的核酸分子中的其中两个可以位于同一载体中。
在申请中，编码所述特异性靶向β4GalNT2基因的gRNA的一种和/或两种核酸分子，编码所述特异性靶向GGTA1基因的gRNA的核酸分子，以及编码所述特异性靶向CMAH基因的gRNA的核酸分子中的其中三个可以位于同一载体中。
在申请中，本申请的编码所述特异性靶向β4GalNT2基因的gRNA的一种和/或两种核酸分子，编码所述特异性靶向GGTA1基因的gRNA的核酸分子，以及编码所述特异性靶向CMAH基因的gRNA的核酸分子可以位于同一载体中。
在本申请中，所述载体可以是任意可用于CRISPR/Cas的载体，例如，所述载体为PX330质粒。
另一方面，本申请提供了细胞，其可包含所述特异性靶向β4GalNT2基因的gRNA，所述的核酸分子，所述的载体，和/或所述的载体的体外转录产物。
另一方面，本申请提供了所述特异性靶向β4GalNT2基因的gRNA，所述的核酸分子，所述的载体，所述的载体的体外转录产物和/或所述的细胞在敲除β4GalNT2基因中的用途，或者在构建动物模型中的用途。
另一方面，本申请提供了一种β4GalNT2基因缺失细胞株，其是使用所述特异性靶向β4GalNT2基因的sgRNA，所述的核酸分子，所述的载体，所述的载体的体外转录产物和/或所述的细胞制备获得的。
另一方面，本申请提供了一种构建动物模型的方法，所述方法可包括向动物的细胞施用至少两种本申请所述的特异性靶向β4GalNT2基因的gRNA，从而敲除β4GalNT2基因的全部 或部分，其中所述gRNA特异性结合SEQ ID NO.1-2中任一项所示的核苷酸序列。
在本申请中，所述方法可包括以下步骤：使用一种或多种脱氧核糖核酸(DNA)核酸内切酶以在所述β4GalNT2基因内或其附近产生一个或更多个单链断裂(SSB)或双链断裂(DSB)，从而使得所述β4GalNT2基因的一个或更多个外显子全部或部分缺失。
在本申请中，所述方法可包括向动物的细胞施用本申请所述特异性靶向GGTA1基因的gRNA，从而敲除GGTA1基因的全部或部分。
在本申请中，所述方法可包括向动物的细胞施用本申请所述特异性靶向CMAH基因的gRNA，从而敲除CMAH基因的全部或部分。
在本申请中，所述方法还可包括以下步骤：使用一种或多种脱氧核糖核酸(DNA)核酸内切酶以在所述GGTA1基因和/或所述CMAH基因内或其附近产生一个或更多个单链断裂(SSB)或双链断裂(DSB)，从而使得所述GGTA1基因和/或所述CMAH基因的一个或更多个外显子全部或部分缺失。
所述DNA核酸内切酶可以包括脱氧核糖核酸内切酶I，脱氧核糖核酸内切酶II，脱氧核糖核酸内切酶IV，限制性内切酶，UvrABC核酸内切酶，和/或工程化核酸酶。工程化核酸酶的例子有，包括但不限于，归巢核酸内切酶(也称为兆核酸酶或大范围核酸酶，Meganuclease)，锌指核酸酶(zinc finger nuclease，ZFN)，转录激活子样效应因子核酸酶(transcription activator-like effector-based nuclease，TALEN)，规律性间隔的短回文序列重复簇(Clustered regularly interspaced short palindromic repeat，CRISPR)。
在本申请中，所述DNA核酸内切酶可以包括Cas核酸酶。在某些情形中，所述Cas核酸酶可以包括Cas9核酸酶、其同源物、其天然存在分子的重组体、其密码子优化版本，和/或其经修饰版本。
所述DNA核酸内切酶可以被修饰或未被修饰。同样，gRNA、crRNA、tracrRNA或sgRNA可以被修饰或未被修饰。本领域中存在许多已知并可被使用的修饰。例如，核苷酸的缺失、***、转位、失活和/或激活。所述修饰可包括引入一个或多个突变(包括单个或多个碱基对改变)、增加发夹的数目、交联、断开具体的核苷酸段以及其他修饰。修饰可以包括包含至少一个非天然存在的核苷酸、或一个经修饰的核苷酸、或其类似物。所述核苷酸可以在核糖、磷酸和/或碱基部分处被修饰。
在本申请中，所述方法可包括：a)提供一种细胞，所述细胞包含一种或多种包含本申请所述的载体或所述gRNA载体的体外转录产物；b)将所述细胞在培养液中进行培养；c)将培养后的细胞移植至受体雌性非人哺乳动物的输卵管内，允许所述细胞在所述雌性非人哺乳动物 的子宫中发育；和d)鉴定步骤c)的怀孕雌性的后代基因改造的非人哺乳动物中的种系传递。
在本申请中，所述动物可包括猪。所述猪可以为本领域抑制的任何种类的猪，包括但不限于如上文定义的那些。例如，所述猪可以选自巴马猪、五指山猪和/或长白猪。
另一方面，本申请提供了根据本申请所述的构建动物模型的方法制备获得动物模型，其中所述动物不表达β4GalNT2基因和/或β-1,4-N-乙酰氨基半乳糖转移酶2。在某些情形中，所述动物可以不表达GGTA1基因和/或αGal。在某些情形中，所述动物可以不表达CMAH基因和/或Neu5Gc。
在本申请中，所述不表达β4GalNT2基因、GGTA1和/或CMAH通常是指所述基因的核苷酸序列的***、中断或缺失，或涉及到该基因的转录和mRNA翻译为前体或成熟蛋白质的功能的降低或缺失，或者，编码具有比内源性氨基酸序列少的氨基酸残基的多肽或者不编码多肽。在本申请中，β-1,4-N-乙酰氨基半乳糖转移酶2、αGal和/或Neu5Gc的不表达涉及活性或水平的降低或消除。
可以通过多种方法检测基因或蛋白质的表达水平，包括在RNA水平的方法(包括通过逆转录酶聚合酶链式反应(RT-PCR)或通过Southern印迹、原位杂交的mRNA定量)和在蛋白质水平的方法(包括组织化学、免疫印迹分析和体外结合研究)。此外，可以通过本领域技术人员众所周知的ELISA技术来定量目的基因的表达水平。可以使用许多标准分析来完成定量测量。例如，可使用RT-PCR和包括RNA酶保护、Southern印迹分析、RNA斑点(RNAdot)分析在内的杂交方法测量转录水平。也可以使用免疫组织化学染色和流式细胞检测、Western印迹分析来评估是否存在β4GalNT2基因和/或β-1,4-N-乙酰氨基半乳糖转移酶2、GGTA1基因和/或αGal，和/或，CMAH基因和/或Neu5Gc。
另一方面，本申请提供了一种制备动物模型的方法，所述方法可以包括：a)提供本申请所述的动物模型；b)将步骤a)获得的动物模型与其它动物交配或体外授精或对基于步骤a)获得的动物模型进一步进行基因编辑或将人组织、细胞移植至步骤a)获得的动物模型中，并进行筛选，得到的动物模型。
另一方面，本申请提供了根据所述制备动物模型的方法制备获得动物模型。例如，所述动物可以包括猪。
另一方面，本申请提供了一种细胞或细胞系或原代细胞培养物，其中所述的细胞或细胞系或原代细胞培养物可以来源于本申请所述的动物模型或其后代。可以从非人类哺乳动物中分离细胞培养物，或从使用同样的构建体、经标准的细胞转染技术建立的细胞培养物中制备细胞培养物。
另一方面，本申请提供了一种组织或器官或其培养物，其中所述组织或器官或其培养物可以来源于所述的动物模型或其后代。
另一方面，本申请提供了一种特异靶向敲除β4GalNT2基因的CRISPR/Cas9***，其可以包括使用含有本申请所述的特异性靶向β4GalNT2基因的gRNA的DNA序列。
另一方面，本申请提供了一种能够特异性靶向β4GalNT2基因的核酸分子试剂盒，其中，所述试剂盒可以包括所述的特异性靶向β4GalNT2基因的gRNA。
另一方面，本申请提供了一种能够特异的靶向β4GalNT2基因的成套核酸分子，其中，所述成套核酸分子可以包括所述的特异性靶向β4GalNT2基因的sgRNA和Cas9核酸酶的mRNA。
另一方面，本申请提供了所述的细胞或细胞系或原代细胞培养物，所述的组织或器官或其培养物在器官和/或组织移植产品开发，或者作为药理学、免疫学和医学研究的模型***，或在验证、评价或研究免疫排斥反应中的应用。
另一方面，本申请提供了所述的动物模型在器官和/或组织移植产品开发，或者作为药理学、免疫学和医学研究的模型***中的应用。
所述器官和/或组织移植产品包括移植、植入或输注到接受者对象的来自不同物种的细胞、组织或器官。特别地考虑其中接受者是人的移植。所述移植产品可以从具有降低的aGal、β-1,4N-乙酰氨基半乳糖转移酶2和Neu5Gc表达的转基因动物中分离出来。
所述器官和/或组织移植产品可以从产前、新生、未成熟或完全成熟的转基因动物中分离出来。移植材料可用作需要器官移植的人对象的临时或永久性器官替代物。
另一方面，本申请提供了所述的动物模型在验证、评价或研究免疫排斥反应方面的应用。当移植的组织、器官、细胞或材料不被接受者的身体接受时，则发生免疫排斥。在免疫排斥中，接受者的免疫***攻击移植的材料。存在多种类型的免疫排斥并且其可以单独或一起发生。免疫排斥反应包括但不限于超急性排斥(HAR)、急性体液异种移植排斥反应(AHXR)、血小板减少症、急性体液排斥、超急性血管排斥、抗体介导的排斥反应和移植物抗宿主病。
评价、评估、分析、测量、量化或确定本领域中已知的排斥相关症状的任何方法可与本申请的组合物、试剂盒和方法一起使用。分析免疫排斥相关症状的方法可包括但不限于：包括具有血小板计数的CBC的实验室评估、凝血研究、肝功能测试、流式细胞术、免疫组织化学、标准诊断标准、免疫学方法、蛋白质印迹法、免疫印迹法、显微术、共聚焦显微术、透射电子显微术、IgG结合测定、IgM结合测定、表达测定、肌酐测定和吞体分离。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的融合蛋白、制备方法和用途等，而不用于限制本申请发明的范围。
实施例
实施例1 构建CRISPR/Cas9载体
首先根据GGTA1/CMAH/β4GalNT2基因的DNA序列，合成靶向GGTA1，CMAH和β4GalNT2基因的sgRNA(single guide RNA)，以pX330(Addgene plasmid 423230)为骨架质粒，分别构建GGTA1-CRISPR/Cas9载体、CMAH-CRISPR/Cas9载体和β4GalNT2-CRISPR/Cas9载体。
首先根据Genbank中公布的猪GGTA1、CMAH和β4GalNT2的基因序列，选取GGTA1基因的3号外显子exon3、CMAH基因的6号外显子exon6和β4GalNT2基因的8号外显子exon8作为CRISPR/Cas9靶点，根据cas9靶点设计原则：5’端为G，3’端为PAM序列(NGG)，设计靶向GGTA1的sgRNA序列为GAAAATAATGAATGTCAA(SEQ ID NO:9)，靶向CMAH的sgRNA序列为GAGTAAGGTACGTGATCTGT(SEQ ID NO:10)，以及靶向β4GalNT2的sgRNA1序列为GGTAGTACTCACGAACACTC(SEQ ID NO:6)和靶向β4GalNT2的sgRNA2序列为CTACCCTTTCTTGCCCAGAG(SEQ ID NO:7)。合成5’端磷酸化寡核苷酸链sgRNA序列。
将sgRNA序列克隆到pX330骨架载体上(对于靶向β4GalNT2基因，当两个sgRNA在同一载体中时，克隆的片段为U6启动子-β4GalNT2/sgRNA1-gRNA骨架-U6启动子-β4GalNT2/sgRNA2-gRNA骨架)，具体步骤如下：
1、用限制性内切酶BbsI消化1μg pX330质粒；
2、酶切的pX330质粒跑琼脂糖凝胶(琼脂糖凝胶浓度1％，即1g琼脂糖凝胶加入到100mL电泳缓冲液中)分离，用胶回收试剂盒(QIAGEN)纯化回收酶切产物；
3、将合成的5’端磷酸化寡核苷酸链sgRNA序列按照以下程序退火：
37℃30min
95℃5min然后以5℃/min的速率降至25℃。
4、按照以下体系启动连接反应：室温反应10min
Figure PCTCN2021105973-appb-000001
Figure PCTCN2021105973-appb-000002
5、用质粒安全核酸外切酶处理连接体系，去除错误链接质粒：
Figure PCTCN2021105973-appb-000003
37℃反应30min
6、转化
(1)取50μL感受态细胞(TIANGEN)置于冰浴中；
(2)向装有感受态细胞的离心管中加入15μL步骤5得到的去除错误连接质粒溶液，混匀后在冰浴中静置30min；
(3)将冰浴30min的感受态细胞置于42℃水浴中60～90s，然后迅速转移至冰浴中，使细胞冷却2～3min；
(4)向离心管中加入900μL无菌的LB培养基(不含抗生素)，混匀后置于37℃摇床150rpm振荡培养45min；
(5)将离心管放到离心机中12000rpm离心5min，然后弃去900μL上清，用剩余的100μL上清重悬感受态细胞沉淀，然后将重悬的感受态细胞加到含相应抗生素的LB固体琼脂培养基上，用无菌的涂布棒将感受态细胞涂布均匀；将涂布有感受态细胞的LB固体琼脂培养基倒置于37℃培养箱中培养12～16h。
7、小提质粒，测序，鉴定打靶质粒构建成功。
得到CRSAPR/Cas9打靶载体，载体分别命名为GGTA1-CRISPR/Cas9(图1，全核苷酸序列如SEQ ID NO:4所示)、CMAH--CRISPR/Cas9(图2，全核苷酸序列如SEQ ID NO:5所示)和β4GalNT2-CRISPR/Cas9(图3，全核苷酸序列如SEQ ID NO:3所示)，核苷酸序列如所示。
实施例2 构建GGTA1/CMAH/β4GalNT2三基因敲除克隆
将GGTA1-CRISPR/Cas9打靶载体，CMAH-CRISPR/Cas9打靶载体和β4GalNT2-CRISPR/Cas9打靶载体同时转染雄性长白猪的胎儿成纤维细胞，通过G418筛选获得三基因敲除的单细胞克隆。具体步骤如下。
2.1猪原代成纤维细胞复苏
1、从液氮中取出冻存的原代猪成纤维细胞，在37℃水浴中解冻；
2、将解冻的细胞转入无菌的15mL离心管中，然后加入3mL细胞培养基，1500rpm离心5min；
其中，细胞完全培养基的配方为：16％胎牛血清(Gibco)+84％DMEM培养基(Gibco)，16％和84％为体积百分比。
3、弃去上清，加入2mL完全培养基重悬细胞沉淀，然后将重悬的细胞铺入6cm细胞培养皿中，补加2mL完全培养基，置于37℃，5％CO 2(体积百分比)的恒温培养箱中进行培养；
4、将细胞培养至长满皿底90％左右时使用0.05％(5g/100mL)的胰蛋白酶将细胞消化下来，然后加入完全培养基终止消化，将细胞悬液转入15mL离心管中，1500rpm离心5min，弃去上清，使用2mL完全培养基重悬细胞，对细胞计数，将细胞总量调整至1.5×10 6以备下一步核转染实验。
2.2使用构建好的GGTA1-CRISPR/Cas9打靶载体，CMAH-CRISPR/Cas9打靶载体和β4GalNT2-CRISPR/Cas9打靶载体和tdTomato质粒(Clontech,PT4069-5)共转染猪原代成纤维细胞
使用哺乳动物成纤维细胞核转染试剂盒(Lonza)与Lonza Nucleofactor TM2b核转仪进行核转染实验
1、配制核转染反应液，体系如下：
核转染基本溶液      82μL
补充成分            8μL
2、将构建好的三个质粒与Tdtomato质粒分别按照质量比5:1的比例加入本步骤1获得的100μL核转反应液中混匀，过程中注意切勿产生气泡；
3、将2.1制备得到的细胞悬液使用DPBS杜氏磷酸缓冲液(Gibco)洗两遍，37℃消化2min，用含体积百分比为10％胎牛血清的DMEM完全培养基终止消化后，1500rpm离心5min，弃去上清，使用本步骤2中含有质粒的核转反应液重悬细胞，重悬过程中要避免气泡的产生；
4、将该核转体系小心加入到试剂盒带有的电转杯中，注意防止气泡。先用含有100μLPBS的电转杯放置于Lonza核转仪的杯槽内，选择U023核转程序调试程序后，将含有细胞的电转杯电击转染后立即在超净台内将电转杯中液体轻柔吸出，转入到1mL含体积百分比为16％胎牛血清的DMEM完全培养基中，轻轻混匀；
5、准备含8mL完全培养基的培养皿(10cm)若干，吸取核转后的细胞悬液加入含有完全培养基的培养皿中，混匀，在显微镜下观察细胞数量，计数，使得培养皿在显微镜下一个视野内约有50～60个细胞，其余皿均按照此细胞悬液最终用量加入，混匀后放置于37℃，5％CO 2的恒温培养箱中进行培养。
2.3三基因敲除细胞系的筛选
1、将2.2所得细胞培养24h后将细胞培养基更换为含有1mg/mL G418的完全培养基，放置于37℃，5％CO 2的恒温培养箱中进行培养，每2～3天更换一次细胞培养基，期间根据细胞生长状况逐渐降低G418的药物浓度，G418终浓度为0.3mg/mL，培养10～14天左右培养皿中会陆续长出G418抗性的单克隆细胞系；
2、使用克隆环挑取细胞系，将挑取的单克隆细胞系接种于铺有0.3mg/mLG418完全培养基的24孔板中，放置于37℃，5％CO 2的恒温培养箱中进行培养，每2～3天换一次细胞培养基；
3、待24孔板的孔中细胞长满孔底，使用胰蛋白酶消化并收集细胞，其中4/5细胞接种到含有0.3mg/mL G418完全培养基的12孔板或6孔板中(根据细胞量)，剩余的1/5的细胞留在24孔板中继续培养；
4、待12孔板或6孔板细胞铺满孔底后使用0.05％(5g/100mL)的胰蛋白酶消化并收集细胞，使用细胞冻存液(90％胎牛血清+10％DMSO，体积比)将细胞冻存；
2.4三基因敲除细胞系的基因鉴定
1、待24孔板中细胞长满孔底后使用0.05％(5g/100mL)的胰蛋白酶消化并收集细胞，然后在细胞中加入25ml NP-40裂解液裂解细胞提取细胞基因组DNA，裂解程序为：55℃ 60min——95℃ 5min——4℃，反应结束后基因组DNA于-20℃保存；
2、针对GGTA1/CMAH/β4GalNT2基因靶点信息设计相应的PCR引物，PCR引物序列分别为：
GGTA1
正向引物为：5’-CCTTAGTATCCTTCCCAACCCAGAC-3’(SEQ ID NO:11)
反向引物为：5’-GCTTTCTTTACGGTGTCAGTGAATCC-3’(SEQ ID NO:12)
PCR目的产物长度为428bp；
CMAH
正向引物为：5’-CTTGGAGGTGATTTGAGTTGGG-3’(SEQ ID NO:13)
反向引物为：5’-CATTTTCTTCGGAGTTGAGGGC-3’(SEQ ID NO:14)
PCR目的产物长度为485bp；
β4GalNT2
正向引物为：5’-CCCAAGGATCCTGCTGCC-3’(SEQ ID NO:15)
反向引物为：5’-CGCCGTGTAAAGAAACCTCC-3’；(SEQ ID NO:8)
PCR目的产物长度为406bp；
3、使用PCR反应扩增GGTA1/CMAH/β4GalNT2靶点基因，PCR反应体系如下：
Figure PCTCN2021105973-appb-000004
反应条件如下
Figure PCTCN2021105973-appb-000005
CMAH靶点基因的扩增同上述步骤；β4GalNT2靶点基因的扩增同上述步骤。
4、将PCR反应产物进行琼脂糖凝胶电泳(1％，即1g琼脂糖凝胶加入到100mL电泳缓冲液中)，电泳结束后在紫外线下切下目的条带，然后使用胶回收试剂盒(QIAGEN)回收目的条带，并使用NanoDrop 200测定回收的PCR产物的浓度；
5、将回收的PCR产物使用TAKARA pMD TM18-T Vector Cloning Kit链接T载体，T载体反应体系如下：
pMD18-T vector          1μL
胶回收PCR产物           81.7ng*
ddH 2O                   补齐体系至10uL
*注：TAKARA pMD TM18-T Vector Cloning Kit说明书上对Insert DNA(本次为胶回收PCR产物)用量的要求0.1～0.3pM，本次选取0.2pM，用量计算方法为：Insert DNA的使用 量(ng)＝nmol数×660×Insert DNA的bp数。
T载体链接的反应条件为16℃反应30min；
6、将本步骤5所得的T载体链接产物使用感受态细胞(TIANGEN)进行转化，转化后将感受态细胞涂布于Amp抗性的LB琼脂固体培养基上，37℃恒温培养箱培养过夜；
从培养过夜的培养基上挑取单克隆菌落送测序公司进行测序，然后将测序结果与靶点GGTA1/CMAH/β4GalNT2信息进行比对，从而判断该细胞系是否为GGTA1/CMAH/β4GalNT2基因敲除细胞系。
实施例3使用单个sgRNA敲除β4GalNT2
按照实施例2的方法使用β4GalNT2的sgRNA(SEQ ID NO:6)构建β4GalNT2CRISPR/Cas9打靶载体，使用GGTA1的sgRNA(SEQ ID NO:9)构建CCTA1CRISPR/Cas9打靶载体，构建双敲除β4GalNT2和GGTA1的巴马猪和五指山猪细胞克隆，测序，得到敲除效率和双敲除细胞克隆的基因型。
结果如表1-表4显示。在巴马猪的35个冻存克隆中，成功敲除β4GalNT2的克隆有7个，成功敲除β4GalNT2和GGTA1的克隆有7个，其中，双基因敲除的7个克隆的基因型请见表2。在五指山猪的68个冻存克隆中，成功敲除β4GalNT2的克隆有12个，成功敲除β4GalNT2和GGTA1的克隆有9个，其中，双基因敲除的9个克隆的基因型请见表4。使用单个β4GalNT2的sgRNA敲除β4GalNT2的效率为21.88％(巴马猪)和17.65％(五指山猪)。β4GalNT2和GGTA1双敲除的效率为21.88％(巴马猪)和13.24％(五指山猪)。
表1 巴马猪双敲除效率
Figure PCTCN2021105973-appb-000006
表2 巴马猪双敲除细胞克隆基因型
编号 GGTA1敲除 β4GalNT2敲除
3 -2bp -10bp
18 +1bp(T) +1-17bp
24 +1bp(C) -1bp(A)
27 +1-4bp +1bp(A)
41 +1bp(T) -10bp
42 +1bp(T) -10bp
68 +1bp(T) +1bp(A)
表3 五指山猪双敲除效率
Figure PCTCN2021105973-appb-000007
表4 五指山猪双敲除细胞克隆基因型
编号 GGTA1敲除 β4GalNT2敲除
4 +1bp(C)，-14bp +1bp(A)，-2bp
13 +1bp(C) -1bp(A)，-4bp
18 -3bp(ATG) +1bp(A)
20 -17bp +1bp，-1bp(A)，-5bp
37 -33bp，-26bp，+1bp(C) +1bp(C)，-10bp
40 +1bp(C) +1bp(C)，-1bp(A)
43 +1bp(C) -10bp，-4bp，-3bp
45 +1bp(C) +6-17bp
56 +1bp(C) -2bp(AC)
实施例4使用两个sgRNA敲除β4GalNT2
按照实施例2的方法使用β4GalNT2的2个sgRNA(SEQ ID NO:6和7)构建β4GalNT2CRISPR/Cas9打靶载体，使用GGTA1的sgRNA(SEQ ID NO:9)构建CCTA1CRISPR/Cas9打靶载体，使用CMAH的sgRNA(SEQ ID NO:10)构建CMAH CRISPR/Cas9打靶载体，构建三敲除β4GalNT2、GGTA1和CMAH的长白猪细胞克隆，测序，得到敲除效率和三敲除细胞克隆的基因型。
结果如表5-表7显示。在雄性长白猪59个冻存克隆中，成功敲除β4GalNT2的克隆有18个，成功敲除β4GalNT2、GGTA1和CMAH的克隆有15个，其中，三基因敲除的15个克隆的基因型请见表6。在雌性长白猪51个冻存克隆中，成功敲除β4GalNT2的克隆有20个。使用两个β4GalNT2的sgRNA敲除β4GalNT2的效率为30.51％(长白猪雄性)和39.21％(长白猪雌性)，与实施例3中使用1个sgRNA相比，使用两个sgRNA敲除β4GalNT2的敲除效 率提高。β4GalNT2、GGTA1和CMAH三敲除的效率为25.42％(长白猪雄性)，高于实施例2中的双基因敲除效率(21.88％)，说明本申请的sgRNA不仅实现了三基因敲除，且敲除效率比双基因的敲除效率更高。
表5 长白猪雄性敲除效率
Figure PCTCN2021105973-appb-000008
表6 长白猪雄性三敲除细胞克隆基因型
Figure PCTCN2021105973-appb-000009
Figure PCTCN2021105973-appb-000010
表7 长白猪雌性敲除效率
冻存克隆数(个) β4GalNT2敲除克隆数(个) β4GalNT2敲除效率
51 20 39.21％

Claims (43)

  1. 一种特异性靶向β4GalNT2基因的gRNA，其中所述gRNA特异性结合SEQ ID NO.1-2中任一项所示的核苷酸序列。
  2. 根据权利要求1所述的gRNA，其中所述gRNA包含SEQ ID NO.16所示的核苷酸序列。
  3. 根据权利要求1-2中任一项所述gRNA，其中所述gRNA包含SEQ ID NO.6-7中任一项所示的核苷酸序列。
  4. 根据权利要求1-3中任一项所述gRNA，其中所述sgRNA包含5’-(X)n-SEQ ID NO.16所示的核苷酸序列-骨架序列-3’，其中X为选自A、U、C和G中任一个的碱基，且n为0-15中的任一整数。
  5. 根据权利要求1-4中任一项所述gRNA，其中所述gRNA为单链向导RNA(sgRNA)。
  6. gRNA组合，其包括权利要求1-5中任一项所述的特异性靶向β4GalNT2基因的gRNA，特异性靶向GGTA1基因的gRNA和特异性靶向CMAH基因的gRNA。
  7. 根据权利要求6所述的gRNA组合，其中所述特异性靶向GGTA1基因的gRNA包含SEQ ID NO.9所示的核苷酸序列。
  8. 根据权利要求6-7中任一项所述的gRNA组合，其中所述特异性靶向CMAH基因的gRNA包含SEQ ID NO.10所示的核苷酸序列。
  9. 一种或多种分离的核酸分子，其编码权利要求1-8中任一项所述gRNA。
  10. 载体，其包含权利要求9所述的核酸分子。
  11. 根据权利要求10所述的载体，其中编码所述特异性靶向β4GalNT2基因的gRNA的一种和/或两种核酸分子，编码所述特异性靶向GGTA1基因的gRNA的核酸分子，以及编码所述特异性靶向CMAH基因的gRNA的核酸分子，其中，编码所述特异性靶向β4GalNT2基因的gRNA的一种和/或两种核酸分子位于同一载体中。
  12. 细胞，其包含权利要求1-8中任一项所述gRNA，权利要求9所述的核酸分子，权利要求10-11中任一项所述的载体，和/或权利要求10-11中任一项所述的载体的体外转录产物。
  13. 权利要求1-8中任一项所述gRNA，权利要求9所述的核酸分子，权利要求10-11中任一项所述的载体，权利要求10-11中任一项所述的载体的体外转录产物和/或权利要求12所述的细胞在敲除β4GalNT2基因中的用途，或者在构建动物模型中的用途。
  14. 一种β4GalNT2基因缺失细胞株，其是使用权利要求1-8中任一项所述sgRNA，权利要求9所述的核酸分子，权利要求10-11中任一项所述的载体，权利要求10-11中任一项所述的载体的体外转录产物和/或权利要求12所述的细胞制备获得的。
  15. 一种构建动物模型的方法，所述方法包括向动物的细胞施用至少两种特异性靶向β4GalNT2基因的gRNA，从而敲除β4GalNT2基因的全部或部分，其中所述gRNA特异性结合SEQ ID NO.1-2中任一项所示的核苷酸序列。
  16. 根据权利要求15所述的gRNA，其中所述gRNA包含SEQ ID NO.16所示的核苷酸序列。
  17. 根据权利要求15-16中任一项所述的方法，其中所述特异性靶向β4GalNT2基因的gRNA包含SEQ ID NO.6-7中任一项所示的核苷酸序列。
  18. 根据权利要求17所述的方法，其中所述特异性靶向β4GalNT2基因的gRNA包含5’-(X)n-SEQ ID NO.16所示的核苷酸序列-骨架序列-3’，其中X为选自A、U、C和G中任一个的碱基，且n为0-15中的任一整数。
  19. 根据权利要求15-18中任一项所述的方法，其中所述gRNA为单链向导RNA(sgRNA)。
  20. 根据权利要求15-19中任一项所述的方法，其中所述方法包括以下步骤：使用一种或多种脱氧核糖核酸(DNA)核酸内切酶以在所述β4GalNT2基因内或其附近产生一个或更多个单链断裂(SSB)或双链断裂(DSB)，从而使得所述β4GalNT2基因的一个或更多个外显子全部或部分缺失。
  21. 根据权利要求15-20中任一项所述的方法，其中所述方法还包括向动物的细胞施用特异性靶向GGTA1基因的gRNA，从而敲除GGTA1基因的全部或部分。
  22. 根据权利要求21所述的方法，其中所述特异性靶向GGTA1基因的gRNA包含SEQ ID NO.9所示的核苷酸序列。
  23. 根据权利要求15-22中任一项所述的方法，其中所述方法还包括向动物的细胞施用特异性靶向CMAH基因的gRNA，从而敲除CMAH基因的全部或部分。
  24. 根据权利要求23所述的方法，其中所述特异性靶向CMAH基因的gRNA包含SEQ ID NO.10所示的核苷酸序列。
  25. 根据权利要求15-24中任一项所述的方法，其中所述方法包括以下步骤：使用一种或多种脱氧核糖核酸(DNA)核酸内切酶以在所述GGTA1基因和/或所述CMAH基因内或其附近产生一个或更多个单链断裂(SSB)或双链断裂(DSB)，从而使得所述GGTA1基因和/或所述CMAH基因的一个或更多个外显子全部或部分缺失。
  26. 根据权利要求25所述的方法，其中所述DNA核酸内切酶包括Cas核酸酶。
  27. 根据权利要求26所述的方法，其中所述Cas核酸酶包括Cas9核酸酶、其同源物、其天然存在分子的重组体、其密码子优化版本，和/或其经修饰版本。
  28. 根据权利要求15-27中任一项所述的方法，所述方法包括：
    a)提供一种细胞，所述细胞包含一种或多种包含权利要求10-11中任一所述的载体或所述gRNA载体的体外转录产物；
    b)将所述细胞在培养液中进行培养；
    c)将培养后的细胞移植至受体雌性非人哺乳动物的输卵管内，允许所述细胞在所述雌性非人哺乳动物的子宫中发育；和
    d)鉴定步骤c)的怀孕雌性的后代基因改造的非人哺乳动物中的种系传递。
  29. 根据权利要求15-28中任一项所述的方法，其中所述动物包括猪。
  30. 根据权利要求18-29中任一项所述的方法制备获得动物模型，其中所述动物不表达β4GalNT2基因和/或β-1,4-N-乙酰氨基半乳糖转移酶2。
  31. 根据权利要求30所述的动物模型，其中所述动物不表达GGTA1基因和/或αGal。
  32. 根据权利要求30-31中任一项所述的动物模型，其中所述动物不表达CMAH基因和/或Neu5Gc。
  33. 一种制备动物模型的方法，所述方法包括：
    a)提供权利要求30-32中任一项所述的动物模型；
    b)将步骤a)获得的动物模型与其它动物交配或体外授精或对基于步骤a)获得的动物模型进一步进行基因编辑或将人组织、细胞移植至步骤a)获得的动物模型中，并进行筛选，得到的动物模型。
  34. 根据权利要求33所述的方法制备获得动物模型。
  35. 根据权利要求34所述的动物模型，其中所述动物包括猪。
  36. 一种细胞或细胞系或原代细胞培养物，其中所述的细胞或细胞系或原代细胞培养物来源于权利要求30-32和34-35中任一项所述的动物模型或其后代。
  37. 一种组织或器官或其培养物，其中所述组织或器官或其培养物来源于权利要求30-32和34-35中任一项所述的动物模型或其后代。
  38. 一种特异靶向敲除β4GalNT2基因的CRISPR/Cas9***，其特征在于，使用含有权利要求1-8中任一项所述的特异性靶向β4GalNT2基因的gRNA的DNA序列。
  39. 一种能够特异性靶向β4GalNT2基因的核酸分子试剂盒，其中，所述试剂盒包括权利要求1-8中任一所述的特异性靶向β4GalNT2基因的gRNA。
  40. 一种能够特异的靶向β4GalNT2基因的成套核酸分子，其中，所述成套核酸分子包括权利要求1-8中任一项所述的sgRNA和Cas9 mRNA。
  41. 权利要求36所述的细胞或细胞系或原代细胞培养物，权利要求37所述的组织或器官或其培养物在器官和/或组织移植产品开发，或者作为药理学、免疫学和医学研究的模型***，或在验证、评价或研究免疫排斥反应中的应用。
  42. 权利要求30-32和34-35中任一项所述的动物模型在器官和/或组织移植产品开发，或者作为药理学、免疫学和医学研究的模型***中的应用。
  43. 权利要求30-32和34-35中任一项所述的动物模型在验证、评价或研究免疫排斥反应方面的应用。
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