CN113667674A - 一种靶向切除猪cd164基因编码区dna的成对编辑位点、使用方法及其应用 - Google Patents
一种靶向切除猪cd164基因编码区dna的成对编辑位点、使用方法及其应用 Download PDFInfo
- Publication number
- CN113667674A CN113667674A CN202111002995.1A CN202111002995A CN113667674A CN 113667674 A CN113667674 A CN 113667674A CN 202111002995 A CN202111002995 A CN 202111002995A CN 113667674 A CN113667674 A CN 113667674A
- Authority
- CN
- China
- Prior art keywords
- editing
- gene
- coding region
- sites
- porcine
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 101150028830 Cd164 gene Proteins 0.000 title claims abstract description 53
- 108091026890 Coding region Proteins 0.000 title claims abstract description 23
- 238000000034 method Methods 0.000 title claims abstract description 11
- 108091033409 CRISPR Proteins 0.000 claims abstract description 22
- 108020004414 DNA Proteins 0.000 claims abstract description 14
- 238000009395 breeding Methods 0.000 claims abstract description 8
- 230000001488 breeding effect Effects 0.000 claims abstract description 8
- 201000010099 disease Diseases 0.000 claims abstract description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 7
- 230000008685 targeting Effects 0.000 claims abstract description 7
- 210000000349 chromosome Anatomy 0.000 claims abstract description 6
- 230000002759 chromosomal effect Effects 0.000 claims abstract description 3
- 210000004027 cell Anatomy 0.000 claims description 49
- 210000002950 fibroblast Anatomy 0.000 claims description 13
- 238000012163 sequencing technique Methods 0.000 claims description 11
- 238000012258 culturing Methods 0.000 claims description 9
- 239000013598 vector Substances 0.000 claims description 9
- 108091027544 Subgenomic mRNA Proteins 0.000 claims description 8
- 238000011160 research Methods 0.000 claims description 6
- 102100035102 E3 ubiquitin-protein ligase MYCBP2 Human genes 0.000 claims description 4
- 238000012216 screening Methods 0.000 claims description 4
- 241000282887 Suidae Species 0.000 claims description 3
- 230000000694 effects Effects 0.000 claims description 3
- 238000005516 engineering process Methods 0.000 abstract description 10
- 238000010354 CRISPR gene editing Methods 0.000 abstract description 7
- 230000001404 mediated effect Effects 0.000 abstract description 4
- 239000000463 material Substances 0.000 abstract description 3
- 108700026244 Open Reading Frames Proteins 0.000 abstract description 2
- 238000010353 genetic engineering Methods 0.000 abstract description 2
- 102000053602 DNA Human genes 0.000 abstract 2
- 238000010586 diagram Methods 0.000 description 9
- 108090000623 proteins and genes Proteins 0.000 description 8
- 230000006870 function Effects 0.000 description 7
- 238000010362 genome editing Methods 0.000 description 7
- 238000004113 cell culture Methods 0.000 description 6
- 238000004520 electroporation Methods 0.000 description 6
- 239000012634 fragment Substances 0.000 description 6
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 6
- 238000010363 gene targeting Methods 0.000 description 5
- 238000001890 transfection Methods 0.000 description 5
- 108020005004 Guide RNA Proteins 0.000 description 4
- 238000003209 gene knockout Methods 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 238000010453 CRISPR/Cas method Methods 0.000 description 3
- 108010063954 Mucins Proteins 0.000 description 3
- 102000015728 Mucins Human genes 0.000 description 3
- 108091034117 Oligonucleotide Proteins 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 3
- 230000005782 double-strand break Effects 0.000 description 3
- 238000007877 drug screening Methods 0.000 description 3
- 239000013604 expression vector Substances 0.000 description 3
- 210000003734 kidney Anatomy 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 229950010131 puromycin Drugs 0.000 description 3
- 239000013049 sediment Substances 0.000 description 3
- 208000035240 Disease Resistance Diseases 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 2
- 102000019997 adhesion receptor Human genes 0.000 description 2
- 108010013985 adhesion receptor Proteins 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 239000012154 double-distilled water Substances 0.000 description 2
- 230000003828 downregulation Effects 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000002744 homologous recombination Methods 0.000 description 2
- 230000006801 homologous recombination Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 230000008439 repair process Effects 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 241000252212 Danio rerio Species 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- 108090000144 Human Proteins Proteins 0.000 description 1
- 102000003839 Human Proteins Human genes 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 102000007365 Sialoglycoproteins Human genes 0.000 description 1
- 108010032838 Sialoglycoproteins Proteins 0.000 description 1
- 238000010459 TALEN Methods 0.000 description 1
- 108010043645 Transcription Activator-Like Effector Nucleases Proteins 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- -1 aminobenzyl Chemical group 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000000181 anti-adherent effect Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000001120 cytoprotective effect Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 230000034431 double-strand break repair via homologous recombination Effects 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 238000003205 genotyping method Methods 0.000 description 1
- 230000011132 hemopoiesis Effects 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 229940051875 mucins Drugs 0.000 description 1
- 230000006780 non-homologous end joining Effects 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000008263 repair mechanism Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000001177 retroviral effect Effects 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 125000005629 sialic acid group Chemical group 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 230000000392 somatic effect Effects 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
- A01K67/0276—Knock-out vertebrates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0684—Cells of the urinary tract or kidneys
- C12N5/0686—Kidney cells
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/15—Humanized animals
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/108—Swine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Urology & Nephrology (AREA)
- Cell Biology (AREA)
- Environmental Sciences (AREA)
- Veterinary Medicine (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Toxicology (AREA)
- Immunology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Animal Behavior & Ethology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Medicinal Chemistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明涉及生物技术和基因工程的技术领域，具体涉及一种靶向切除猪CD164基因编码区DNA的成对编辑位点、使用方法及其应用，所述编辑位点包括T1和T2两个编辑位点，所述T1和T2两个编辑位点位于猪1号染色体CD164基因编码区的2外显子区域；其中T1编辑位点染色体坐标为75441814‑75441833，编码区坐标为246‑265；T2编辑位点染色体坐标为75442009‑75442028，编码区坐标为441‑460。本发明的两个能基于CRISPR技术有效编辑猪CD164基因的靶位点，可用于Cas9介导的猪CD164基因打靶，为研究CD164基因在猪抗病育种中提供实验材料。
Description
技术领域
本发明涉及生物技术和基因工程的技术领域，具体涉及一种靶向切除猪CD164基因编码区DNA的成对编辑位点、使用方法及其应用。
背景技术
基因转移技术根据外源基因整合的位点特异性可分为两大类，一类是非定点的，即导入的外源基因在靶细胞基因组中整合的位点一般是随机的，包括显微注射、电穿孔、磷酸钙沉淀、逆转录病毒载体感染等：另一类则是定点的，即外源基因导入靶细胞后整合到预先确定的位点或对细胞内靶位点进行定点修饰，这就是依赖于同源重组的基因打靶技术。将外源基因精准敲入猪体细胞的基因组中，一直都是极为困难的，因为早期仅基于同源重组的基因打靶技术效率极低，应用受到很大的限制。直到近年来人工核酸内切酶(engineered endonuclease,EEN)的出现，才彻底改变了这一现状。
CRISPR/Cas技术作为第三代基因组编辑技术在近年迅速发展起来，与基因打靶、ZFN、TALEN等基因修饰技术相比，CRISPR/Cas9具有操作简单、效率高等诸多优势，利用CRISPR/Cas9对重要经济性状基因进行修饰已经成为家畜新品种培育的重要手段。CRISPR/Cas9技术介导的基因突变是指Cas9蛋白通过sgRNA靶向识别并定点切割基因组DNA，导致DNA双链断裂(double strand break,DSB)，细胞通过非同源末端连接修复机制修复DSB时所引发的切割位点处DNA碱基的插入或缺失。目前该技术被成功应用于人类细胞、斑马鱼、小鼠、猴、猪、山羊等的基因组精确修饰；通过对基因的修复机制包括非同源末端连接修复、同源重组修复等，由于CRISPR/Cas突变效率高、制作简单及成本低的特点，被认为是一种具有广阔应用前景的基因组定点改造工具。
唾液酸粘蛋白是一种异质的分泌或膜相关的粘蛋白组，在体内似乎起着两个关键但相反的作用:首先其作为细胞保护剂或抗粘附剂，其次作用为粘附受体。CD164(人类白细胞分化抗原64)，又称内溶双体蛋白，唾液酸核心粘蛋白24，是一种由CD164基因编码的人体蛋白质，其功能为黏附分子，CD164作为I型整合跨膜唾液黏蛋白，作为粘附受体起作用。研究表明CD164在机体造血和调节正常细胞生长分化过程中发挥一定调控作用，并且参与机体免疫应答、细胞附着、异嗜性细胞附着、负调控细胞附着、信号转导、发育、负调控细胞增殖等生理活动。因此，获取CD164基因修饰动物，可为深入研究该基因功能以及相关疾病的治疗提供可靠的动物模型，并为研究该基因在动物抗病育种的作用提供实验材料。
发明内容
本发明的目的之一在于提供一种靶向切除猪CD164基因编码区DNA的成对编辑位点，提供两个能基于CRISPR技术有效编辑猪CD164基因的靶位点，该靶位点可用于Cas9介导的猪CD164基因打靶，并能够将CD164基因功能失活。
本发明的目的之二在于提供一种靶向切除猪CD164基因编码区DNA的成对编辑位点的使用方法，能够成功敲除CD164基因。
本发明的目的之三在于提供一种靶向切除猪CD164基因编码区DNA的成对编辑位点的的应用。
本发明的目的之四在于提供一种靶向切除猪CD164基因编码区DNA的成对编辑位点的应用。
本发明实现目的之一所采用的方案是：一种靶向切除猪CD164基因编码区DNA的成对编辑位点，所述编辑位点包括T1和T2两个编辑位点，所述T1和T2两个编辑位点位于猪1号染色体CD164基因编码区的2外显子区域；其中T1编辑位点染色体坐标为75441814-75441833，编码区坐标为246-265；T2编辑位点染色体坐标为75442009-75442028，编码区坐标为441-460。
优选地，所述T1编辑位点3’端含有AGG，作为Cas9核酸内切酶特异识别的PAM序列，T2编辑位点5’端含有CCT作为Cas9核酸内切酶特异识别的PAM序列。
本发明实现目的之二所采用的方案是：一种靶向切除猪CD164基因编码区DNA的成对编辑位点的使用方法包括以下步骤：
(1)设计编辑靶位点T1和T2的向导序列sgRNA；
(2)构建含有编辑位点T1和T2的sgRNA打靶载体；
(3)培养猪原代成纤维细胞；
(4)转染含有T1和T2编辑位点的打靶载体至猪原代成纤维细胞；
(5)筛选阳性单克隆；
(6)提取阳性单克隆细胞DNA进行PCR鉴定并通过测序验证准确序列。
本发明实现目的之三所采用的方案是：一种靶向切除猪CD164基因编码区DNA的成对编辑位点的应用，利用该编辑位点构建CD164基因敲除的猪成纤维细胞系。
本发明实现目的之四所采用的方案是：一种靶向切除猪CD164基因编码区DNA的成对编辑位点的应用利用该编辑位点构建CD164基因敲除的猪成纤维细胞系研究CD164基因在猪抗病育种的作用。
本发明具有以下优点和有益效果：
本发明提供的两个能特异识别猪CD164基因的编辑靶位点T1(246-265)、T2(441-460)，能基于CRISPR技术有效编辑猪CD164基因的靶位点，该靶位点可用于Cas9介导的猪CD164基因打靶，并能够将CD164基因功能失活。
利用本发明的编辑位点能成功构建CD164基因敲除猪成纤维细胞系，实现CD164基因的大片段敲除，可作为猪抗病育种研究的理想细胞模型，同时可用于创制CD164基因编辑猪，为研究CD164基因在猪抗病育种中的功能验证、揭示CD164基因在猪疾病抗性中的作用机制提供实验材料，并且能够用于研究CD164基因在猪抗病育种中的功能。
本发明采用CRISPR/Cas9基因编辑技术，构建插入有猪CD164基因编辑位点T1和T2的Cas9 gRNA表达载体，将构建好的含有靶位点的表达载体转染至猪成纤维细胞中，并通过筛选获得阳性单克隆，即CD164基因敲除的单克隆体，结合PCR扩增和测序的方法进行阳性细胞鉴定。
附图说明
图1是本发明中猪CD164基因双gRNA基因编辑示意图；
图2是本发明中电转24小时细胞示意图；
图3是本发明中细胞转染筛选后的细胞单克隆示意图；
图4是本发明中单克隆细胞基因型检测结果示意图；
图5本发明中阳性单克隆测序结果示意图。
具体实施方式
为更好的理解本发明，下面的实施例是对本发明的进一步说明，但本发明的内容不仅仅局限于下面的实施例。
【实施例1】猪CD164基因双sgRNA的设计
选定猪CD164基因(GeneBank登录号：NC_010443.5)第2外显子为打靶区域，利用麻省理工学院张峰实验室开发的网站(http://crispr.mit.edu)对打靶区域设计sgRNA，并从候选序列中选择2个比较合适的sgRNA，并分别命名为g1和g2。
表1 gRNAs靶位点核苷酸序列
【实施例2】猪CD164基因编辑载体构建
以pX459-puro载体表达载体，将g1和g2分别连接到PX459上，命名为PX459-g1、PX459-g2。根据g1、g2序列合成互补配对的寡聚核苷酸：
CD164-g1-F：caccCCGAGTACGACTACACCCTG，
CD164-g1-R：aaacCAGGGTGTAGTCGTACTCGG；
CD164-g2-F：caccGCGGGGGGAGCGAAATCTCG，
CD164-g2-R：aaacCGAGATTTCGCTCCCCCCGC。
载体构建步骤：(1)将两对寡核苷酸稀释至10μmol/L，分别取1μL，加16μL ddH2O，2μL10×NEB Buffer 3，混匀后95℃反应5min，自然冷却至室温的条件下对其进行退火；(2)37℃条件下，用限制性内切酶BbsI对含有Cas9序列的PX459载体酶切2h，切胶回收线性化片段；(3)将寡核苷酸双链插入到线性化的PX459，比例为：1μL线性化的PX459，3μL退火产物，5μLSolutionⅠ，加ddH2O至10μL，混匀后在37℃连接2h；(4)转化：将5μL连接产物加入到50μL感受态细胞DH5α中，轻弹混匀，冰置30min，42℃热激90s，取出后再冰置5min，先加入1mL无抗体的培养基摇配复苏45min，再涂布于含氨苄抗体的平板上，挑取单克隆继续培养，提取质粒后酶切验证，并通过公司测序检测是否连接正确，测序引物为通用引物U6。
【实施例3】猪肾成纤维细胞的培养及转染
39℃水浴复苏猪肾成纤维细胞于6孔细胞培养板中，用含15％胎牛血清、1％双抗的DMEM培养基培养，培养条件：5％CO2，39℃，饱和湿度。当细胞汇合度达到85％左右时即可进行转染，试验用电穿孔的方法进行转染，收集细胞沉淀至1.5mLEP管中，将要进行转染的质粒按照4mg/50μL的工作浓度加入细胞沉淀所在的EP管中，加入电解液补充混合液至50μL，细胞沉淀可视体积估算为1～2μL，电转混合液转移至预热后的电转杯中，按照电穿孔仪的操作进行电转。电转参数：120，脉冲1，时间为3ms。
【实施例4】单克隆细胞的筛选及培养
电转后的细胞培养24h更换新鲜培养基，继续培养24h后，按照6孔细胞培养板中1孔细胞量约5000-10000个接种细胞，继续培养细胞24-48h后，加入工作浓度1.75μg/mL的嘌呤霉素筛选细胞。期间2d更换新鲜培养基，随时注意观察。显微镜下观察细胞团数量及大小，药筛2-3d后细胞换液，以1/2嘌呤霉素药筛浓度继续培养1-2d，之后换液，以1/4嘌呤霉素药筛浓度继续培养至显微镜下的细胞团大小适中，挑选单克隆至96孔细胞培养板中，一孔1个单克隆细胞团。96孔细胞培养皿中单克隆细胞团培养至汇合度70％-80％后，收集每孔全部细胞，转移至48孔细胞培养皿中继续培养。依次类推将单克隆细胞转移至6孔细胞培养皿中继续培养待用。
【实施例5】单克隆细胞的基因型鉴定
提取未转染的细胞、单克隆扩繁的细胞的DNA进行PCR扩增。引物在第2外显子两侧设计，引物序列分别为
CD164-F：5-CAGGTTTCCTCTCGATGTTCCTG-3’，
CD164-R：5’-CTACGGGCTTCCTACGCTCTTC-3’。
PCR扩增体系：Premix Taq 10μL，CD164-F 0.5μL，CD164-R 0.5μL，DNA模板100ng，ddH2O补充至20μL；PCR扩增条件：94℃预变性5min，94℃变性30s，58℃退火45s，72℃延伸1min，35个循环，72℃总延伸7min。单克隆细胞的PCR产物送至生工生物工程股份有限公司武汉分公司进行测序，测序结果与猪CD164基因序列进行比对，鉴定每个单克隆细胞的基因型。
【实施例6】测序分析单克隆细胞敲除片段准确定
将实施例5中的PCR产物通过公司测序检测敲除片段的准确性，测序引物为通用引物U6。
图1是本发明中猪CD164基因双gRNA基因编辑示意图。
图2是本发明中电转24小时细胞示意图，从图中可以看出，电转24小时后的细胞活力较好。
图3是本发明中细胞转染筛选后的细胞单克隆示意图。
图4是本发明中单克隆细胞基因型检测结果示意图，从图中可以看出，单克隆5号、6号、7号、8号、13号、14号、15号和16号均为敲除阳性克隆细胞。
图5本发明中阳性单克隆测序结果示意图，从图中可以看出，利用该编辑位点，可以达到猪肾成纤维细胞CD164基因大片段敲除的目的，且敲除片段位于编辑位点区间。
以上所述是本发明的优选实施方式而已，当然不能以此来限定本发明之权利范围，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和变动，这些改进和变动也视为本发明的保护范围。
Claims (5)
1.一种靶向切除猪CD164基因编码区DNA的成对编辑位点，其特征在于：所述编辑位点包括T1和T2两个编辑位点，所述T1和T2两个编辑位点位于猪1号染色体CD164基因编码区的2外显子区域；其中T1编辑位点染色体坐标为75441814-75441833，编码区坐标为246-265；T2编辑位点染色体坐标为75442009-75442028，编码区坐标为441-460。
2.根据权利要求1所述的靶向切除猪CD164基因编码区DNA的成对编辑位点，其特征在于：所述T1编辑位点3’端含有AGG，作为Cas9核酸内切酶特异识别的PAM序列，T2编辑位点5’端含有CCT作为Cas9核酸内切酶特异识别的PAM序列。
3.根据权利要求1所述的靶向切除猪CD164基因编码区DNA的成对编辑位点的使用方法，其特征在于，包括以下步骤：
(1)设计编辑靶位点T1和T2的向导序列sgRNA；
(2)构建含有编辑位点T1和T2的sgRNA打靶载体；
(3)培养猪原代成纤维细胞；
(4)转染含有T1和T2编辑位点的打靶载体至猪原代成纤维细胞；
(5)筛选阳性单克隆；
(6)提取阳性单克隆细胞DNA进行PCR鉴定并通过测序验证准确序列。
4.根据权利要求1所述的靶向切除猪CD164基因编码区DNA的成对编辑位点的应用，其特征在于：利用该编辑位点构建CD164基因敲除的猪成纤维细胞系。
5.根据权利要求4所述的靶向切除猪CD164基因编码区DNA的成对编辑位点的应用，其特征在于：利用该编辑位点构建CD164基因敲除的猪成纤维细胞系研究CD164基因在猪抗病育种的作用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111002995.1A CN113667674B (zh) | 2021-08-30 | 2021-08-30 | 一种靶向切除猪cd164基因编码区dna的成对编辑位点、使用方法及其应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111002995.1A CN113667674B (zh) | 2021-08-30 | 2021-08-30 | 一种靶向切除猪cd164基因编码区dna的成对编辑位点、使用方法及其应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113667674A true CN113667674A (zh) | 2021-11-19 |
| CN113667674B CN113667674B (zh) | 2024-02-27 |
Family
ID=78547320
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111002995.1A Active CN113667674B (zh) | 2021-08-30 | 2021-08-30 | 一种靶向切除猪cd164基因编码区dna的成对编辑位点、使用方法及其应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113667674B (zh) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107699571A (zh) * | 2017-09-06 | 2018-02-16 | 湖北省农业科学院畜牧兽医研究所 | 一种猪生长抑素基因编辑位点及其应用 |
| CN110358766A (zh) * | 2019-06-10 | 2019-10-22 | 云南农业大学 | 一种基于全基因组CRISPR/Cas9文库筛选异种移植抗原基因的方法 |
| CN111363743A (zh) * | 2020-02-20 | 2020-07-03 | 中国农业科学院北京畜牧兽医研究所 | Scd基因敲除的猪胎儿成纤维细胞系及其构建方法 |
| US20200323182A1 (en) * | 2019-04-09 | 2020-10-15 | Shandong Landsee Genetics Co., Ltd. | Method for Preparing Porcine Fibroblasts with Both CD163 Gene and CD13 Gene Being Knocked-out |
| US20200325597A1 (en) * | 2017-07-03 | 2020-10-15 | Huazhong Argicultural University | Pig genome-wide specific sgrna library, preparation method therefor and application thereof |
| CN111926037A (zh) * | 2020-08-27 | 2020-11-13 | 湖北省农业科学院畜牧兽医研究所 | 一种利用双sgRNA技术敲除MSTN基因的质粒及敲除MSTN基因的方法 |
-
2021
- 2021-08-30 CN CN202111002995.1A patent/CN113667674B/zh active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20200325597A1 (en) * | 2017-07-03 | 2020-10-15 | Huazhong Argicultural University | Pig genome-wide specific sgrna library, preparation method therefor and application thereof |
| CN107699571A (zh) * | 2017-09-06 | 2018-02-16 | 湖北省农业科学院畜牧兽医研究所 | 一种猪生长抑素基因编辑位点及其应用 |
| US20200323182A1 (en) * | 2019-04-09 | 2020-10-15 | Shandong Landsee Genetics Co., Ltd. | Method for Preparing Porcine Fibroblasts with Both CD163 Gene and CD13 Gene Being Knocked-out |
| CN110358766A (zh) * | 2019-06-10 | 2019-10-22 | 云南农业大学 | 一种基于全基因组CRISPR/Cas9文库筛选异种移植抗原基因的方法 |
| CN111363743A (zh) * | 2020-02-20 | 2020-07-03 | 中国农业科学院北京畜牧兽医研究所 | Scd基因敲除的猪胎儿成纤维细胞系及其构建方法 |
| CN111926037A (zh) * | 2020-08-27 | 2020-11-13 | 湖北省农业科学院畜牧兽医研究所 | 一种利用双sgRNA技术敲除MSTN基因的质粒及敲除MSTN基因的方法 |
Non-Patent Citations (1)
| Title |
|---|
| ZHAO CHANGZHI等: "CRISPR screening of porcine sgRNA library identifies host factors associated with Japanese encephalitis virus replication", vol. 11, pages 1 - 15 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113667674B (zh) | 2024-02-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106191113B (zh) | 一种mc3r基因敲除猪的制备方法 | |
| CN108642055B (zh) | 能有效编辑猪miR-17-92基因簇的sgRNA | |
| CN108949824A (zh) | 基于HMEJ的方法介导Ipr1定点插入获取转基因牛胎儿成纤维细胞的方法 | |
| CN109475109A (zh) | 用于使用多个引导rna来破坏免疫耐受性的方法 | |
| CN108165581B (zh) | 采用单链核苷酸片段体外修复hba2基因突变的方法 | |
| CN113278619B (zh) | 双sgRNA、基因敲除载体、基因敲除STING基因的猪成纤维细胞系及其构建方法 | |
| CN111778252B (zh) | 一种靶向敲除SST基因的sgRNA及其CRISPR/Cas9系统和应用 | |
| CN106086031B (zh) | 猪肌抑素基因编辑位点及其应用 | |
| CN107267516B (zh) | 双sgRNA介导的基因精确修饰方法及应用 | |
| CN106754949B (zh) | 猪肌抑素基因编辑位点864-883及其应用 | |
| CN108048487A (zh) | 一种编辑猪属胎儿成纤维细胞中的bmpr-ib基因方法 | |
| CN108949832A (zh) | 一种用于敲除猪ghr基因的打靶载体及其应用 | |
| CN110938629B (zh) | 特异性识别猪Wip1基因的成套sgRNA及其应用和产品 | |
| CN112011539A (zh) | 一种基于CRISPR-Cas9技术靶向敲除猪GDPD2基因的细胞系及其构建方法 | |
| CN111733160A (zh) | 使用CRISPR-Cas9系统对间充质干细胞进行CKIP-1基因敲除的方法 | |
| CN113667674A (zh) | 一种靶向切除猪cd164基因编码区dna的成对编辑位点、使用方法及其应用 | |
| CN106591364B (zh) | 一种获取转基因牛胎儿成纤维细胞的方法 | |
| CN110862988A (zh) | 一种sgRNA及其构建的CREBRF点图变型巴马香猪和应用 | |
| CN111088253A (zh) | 针对hbb-28地中海贫血基因的crispr单碱基供体修复体系 | |
| CN111893119B (zh) | 利用CRISPR/Cas9系统与显微注射获得SCD1基因编辑山羊胚胎的方法 | |
| CN116162653A (zh) | 靶向切除猪SlamF1基因编码区DNA的成对编辑位点、使用方法及其应用 | |
| CN111944811A (zh) | 靶向敲除FRZB基因的双sgRNA和敲除FRZB基因的猪成纤维细胞系及其应用 | |
| CN114891786B (zh) | 犬Rosa26基因及其应用 | |
| CN115948477B (zh) | 一种提高CRISPR/Cas9的同源重组修复效率的诱导剂、方法以及应用 | |
| CN113913435B (zh) | 基于p53基因获得小型猪肿瘤疾病模型的方法 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |