CN107177595A - 用于猪CD163基因编辑的靶向sgRNA、修饰载体及其制备方法和应用 - Google Patents
用于猪CD163基因编辑的靶向sgRNA、修饰载体及其制备方法和应用 Download PDFInfo
- Publication number
- CN107177595A CN107177595A CN201710424822.6A CN201710424822A CN107177595A CN 107177595 A CN107177595 A CN 107177595A CN 201710424822 A CN201710424822 A CN 201710424822A CN 107177595 A CN107177595 A CN 107177595A
- Authority
- CN
- China
- Prior art keywords
- pig
- sgrna
- carrier
- preparation
- gene
- 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.)
- Pending
Links
- 101100382101 Sus scrofa CD163 gene Proteins 0.000 title claims abstract description 44
- 238000010362 genome editing Methods 0.000 title claims abstract description 28
- 108091027544 Subgenomic mRNA Proteins 0.000 title claims abstract description 26
- 238000002360 preparation method Methods 0.000 title claims abstract description 25
- 230000008685 targeting Effects 0.000 title claims abstract description 11
- 230000004048 modification Effects 0.000 title abstract description 8
- 238000012986 modification Methods 0.000 title abstract description 8
- 108091033409 CRISPR Proteins 0.000 claims abstract description 30
- 238000012239 gene modification Methods 0.000 claims abstract description 29
- 230000005017 genetic modification Effects 0.000 claims abstract description 29
- 235000013617 genetically modified food Nutrition 0.000 claims abstract description 29
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 20
- 210000004027 cell Anatomy 0.000 claims description 39
- 108020004414 DNA Proteins 0.000 claims description 33
- 239000000969 carrier Substances 0.000 claims description 31
- 230000000692 anti-sense effect Effects 0.000 claims description 20
- 230000029087 digestion Effects 0.000 claims description 16
- 108010008532 Deoxyribonuclease I Proteins 0.000 claims description 12
- 102000007260 Deoxyribonuclease I Human genes 0.000 claims description 12
- 230000000295 complement effect Effects 0.000 claims description 9
- 101150087379 CD163 gene Proteins 0.000 claims description 8
- 239000013604 expression vector Substances 0.000 claims description 8
- 239000003550 marker Substances 0.000 claims description 7
- 102000004169 proteins and genes Human genes 0.000 claims description 7
- 102000004190 Enzymes Human genes 0.000 claims description 6
- 108090000790 Enzymes Proteins 0.000 claims description 6
- 239000013599 cloning vector Substances 0.000 claims description 6
- 238000010276 construction Methods 0.000 claims description 6
- 238000000034 method Methods 0.000 claims description 6
- 238000012546 transfer Methods 0.000 claims description 4
- 238000012408 PCR amplification Methods 0.000 claims description 3
- 210000001161 mammalian embryo Anatomy 0.000 claims description 3
- 230000008569 process Effects 0.000 claims description 3
- 210000001082 somatic cell Anatomy 0.000 claims description 3
- 238000005034 decoration Methods 0.000 claims 1
- 241000700605 Viruses Species 0.000 abstract description 11
- 230000001850 reproductive effect Effects 0.000 abstract description 11
- 230000000241 respiratory effect Effects 0.000 abstract description 11
- 208000011580 syndromic disease Diseases 0.000 abstract description 11
- 238000010354 CRISPR gene editing Methods 0.000 abstract description 9
- 238000005516 engineering process Methods 0.000 abstract description 7
- 230000001413 cellular effect Effects 0.000 abstract description 3
- 230000002068 genetic effect Effects 0.000 abstract description 3
- 230000004083 survival effect Effects 0.000 abstract description 3
- 208000015181 infectious disease Diseases 0.000 abstract description 2
- 241000282898 Sus scrofa Species 0.000 description 46
- 241000894006 Bacteria Species 0.000 description 6
- 208000032625 disorder of ear Diseases 0.000 description 6
- 108091008146 restriction endonucleases Proteins 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- 229940088598 enzyme Drugs 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 101150044508 key gene Proteins 0.000 description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- 108010019160 Pancreatin Proteins 0.000 description 3
- 238000000137 annealing Methods 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 239000013592 cell lysate Substances 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 210000002950 fibroblast Anatomy 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 229940055695 pancreatin Drugs 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 2
- 102000018233 Fibroblast Growth Factor Human genes 0.000 description 2
- 108050007372 Fibroblast Growth Factor Proteins 0.000 description 2
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 2
- 102100024785 Fibroblast growth factor 2 Human genes 0.000 description 2
- 239000012097 Lipofectamine 2000 Substances 0.000 description 2
- 208000005342 Porcine Reproductive and Respiratory Syndrome Diseases 0.000 description 2
- 241001135989 Porcine reproductive and respiratory syndrome virus Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 239000012154 double-distilled water Substances 0.000 description 2
- 210000002257 embryonic structure Anatomy 0.000 description 2
- 210000003754 fetus Anatomy 0.000 description 2
- 239000002502 liposome Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 206010000234 Abortion spontaneous Diseases 0.000 description 1
- 241001313855 Bletilla Species 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- 208000000059 Dyspnea Diseases 0.000 description 1
- 206010013975 Dyspnoeas Diseases 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 208000006399 Premature Obstetric Labor Diseases 0.000 description 1
- 206010036600 Premature labour Diseases 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 101150063416 add gene Proteins 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000000090 biomarker Substances 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
- 239000006143 cell culture medium Substances 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 230000001351 cycling effect Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 229940126864 fibroblast growth factor Drugs 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000001215 fluorescent labelling Methods 0.000 description 1
- 101150110946 gatC gene Proteins 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 244000144980 herd Species 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000001638 lipofection Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 208000015994 miscarriage Diseases 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 208000026440 premature labor Diseases 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 108020001580 protein domains Proteins 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 208000000995 spontaneous abortion Diseases 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 238000000844 transformation Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000003146 transient transfection Methods 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
- 230000009385 viral infection Effects 0.000 description 1
Classifications
-
- 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
- C12N15/1131—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 against viruses
-
- 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
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
- C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian 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
- 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
- A01K2267/02—Animal zootechnically ameliorated
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Environmental Sciences (AREA)
- Virology (AREA)
- Cell Biology (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Mycology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明提供了一种用于猪CD163基因编辑的靶向sgRNA、修饰载体及其制备方法和应用,涉及基因工程技术领域,本发明提供的sgRNA和基因修饰载体,特异性强,能够非常高效地通过CRISPR/Cas9n***在细胞水平上对猪CD163基因进行编辑。本发明提供的抗蓝耳病猪的制备方法,破坏了蓝耳病病毒受体,除了对目的基因CD163进行编辑外不会引入其他任何外源基因,也不会对基因组上非CD163基因的区域进行非特异的编辑,遗传背景干净清晰,极大的减少了后期转基因安全评估的工作。利用本发明获得的抗蓝耳病的猪,在能够保证正常存活的同时,抵抗蓝耳病病毒的感染,极大的减少了养猪业因蓝耳病造成的经济损失。
Description
技术领域
本发明涉及基因工程技术领域,尤其是涉及一种用于猪CD163基因编辑的靶向sgRNA、修饰载体及其制备方法和应用。
背景技术
猪蓝耳病(猪繁殖与呼吸综合症,PRRS)是严重危害养猪业的第一大病毒性传染病,每年给全球养猪业带来数十亿美元的损失。猪蓝耳病是由猪繁殖与呼吸综合症病毒(PRRSV)引起的,主要侵害妊娠母猪、仔猪和种公猪,引起妊娠母猪早产、流产和死胎,仔猪呼吸困难甚至死亡。
国外的研究表明,猪CD163基因的第七外显子编码的蛋白质区域是蓝耳病病毒受体结合蓝耳病病毒的区域,完全删除了第七外显子的CD163基因,其表达的CD163蛋白恰巧缺失了结合PRRSV的结构域,而其他的生理功能却不受影响,由此可见,对CD163基因进行编辑是生产能够抵抗蓝耳病病毒感染的猪的重要候选手段。
CRISPR/Cas9***是近年来被广泛应用的一种基因编辑技术,可以精确的对基因组进行定点删除、***或置换等改造。CRISPR/Cas9***主要由两个重要的元件构成——导向RNA(sgRNA)和Cas9核酸内切酶(Cas9nuclease)。很多研究表明,与Cas9核酸内切酶相比,使用突变了一个活性域的Cas9切口酶(Cas9 nickase)和两条sgRNA对目的基因进行编辑可以极大地降低CRISPR/Cas9***的脱靶效应,这一***被称之为CRISPR/Cas9n***。
虽然,目前越来越多的基因作为家畜疾病的生物标志物及治疗靶点被应用,但在针对猪蓝耳病中的关键基因及其编辑修饰的方法尚未在我国得到应用。
有鉴于此,特提出本发明。
发明内容
本发明的第一个目的在于提供一种用于猪CD163基因编辑的靶向sgRNA;
本发明的第二个目的在于提供一种猪CD163基因修饰载体;
本发明的第三个目的在于提供一种猪CD163基因修饰载体的制备方法;
本发明的第四个目的在于提供一种猪CD163基因修饰载体在制备抗蓝耳病猪中的应用;
本发明的第五个目的在于提供一种抗蓝耳病猪的制备方法;
本发明的第六个目的在于提供一种抗蓝耳病的猪;以缓解现有技术中存在的针对猪蓝耳病中的关键基因及其编辑修饰的方法尚未得到应用的技术问题。
本发明提供的一种用于猪CD163基因编辑的靶向sgRNA,包括sgRNA-C161和sgRNA-C185;
所述sgRNA-C161的正义链和反义链分别为:
C161-Fwd:5’-AAAGTGAGCTCCCCCCAGGA-3’(SEQ ID NO.1);
C161-Rev:5’-TCCTGGGGGGAGCTCACTTT-3’(SEQ ID NO.2);
所述sgRNA-C185的正义链和反义链分别为:
C185-Fwd:5’-GAGAAGGAAGTGGACAGATC-3’(SEQ ID NO.3);
C185-Rev:5’-GATCTGTCCACTTCCTTCTC-3’(SEQ ID NO.4)。
本发明还提供了一种猪CD163基因修饰载体,包括上述的两条sgRNA、Cas9切口酶及荧光标记蛋白。
进一步地,所述猪CD163基因修饰为针对猪CD163基因的第七外显子进行的基因编辑。
本发明还提供了一种上述的猪CD163基因修饰载体的制备方法,包括以下步骤:
步骤(a):将所述sgRNA-C161的正义链和反义链进行互补配对,形成C161双链DNA分子;
步骤(b):将所述sgRNA-C185的正义链和反义链进行互补配对,形成C185双链DNA分子;
步骤(c):将所述C161双链DNA分子连接到表达载体上,构建表达sgRNA-C161的载体;
步骤(d):将所述C185双链DNA分子连接到表达载体上,构建表达sgRNA-C185的载体;
步骤(e):以所述表达sgRNA-C161的载体为模板,PCR扩增含有U6启动子的所述sgRNA-C161序列,并连接到克隆载体上,再经过酶切后获得U6-sgRNA-C161序列;
步骤(f):将所述U6-sgRNA-C161序列连接到所述表达sgRNA-C185的载体上,构建完成表达所述sgRNA-C161和sgRNA-C185的载体。
进一步地,在步骤(c)和步骤(d)中,所述表达载体为带有Cas9切口酶、荧光标记蛋白及U6启动子的经过BbsⅠ酶切的PX461载体。
进一步地,在步骤(e)中,所述克隆载体为pMD18-T载体。
本发明还提供了上述的猪CD163基因修饰载体在制备抗蓝耳病猪中的应用。
本发明还提供了一种抗蓝耳病猪的制备方法,应用上述的猪CD163基因修饰载体。
进一步地,将所述猪CD163基因修饰载体转入猪的体细胞中,获得CD163基因编辑阳性细胞克隆,以所述阳性细胞为供体细胞进行体细胞克隆和胚胎移植,获得所述抗蓝耳病猪。
另外,本发明还提供了一种抗蓝耳病的猪,应用上述的抗蓝耳病猪的制备方法制备得到。
本发明提供的用于猪CD163基因编辑的靶向sgRNA和包括上述两条sgRNA、Cas9切口酶及荧光标记蛋白的猪CD163基因修饰载体,特异性强,能够非常高效地通过CRISPR/Cas9n***在细胞水平上对猪CD163基因进行编辑。本发明提供的抗蓝耳病猪的制备方法,应用了本发明提供的猪CD163基因修饰载体,针对猪蓝耳病中的关键基因CD163进行基因编辑,从而破坏蓝耳病病毒受体,并且,除了对目的基因CD163进行编辑外不会引入其他任何外源基因,也不会对基因组上非CD163基因的区域进行非特异的编辑,遗传背景干净清晰,极大的减少了后期转基因安全评估的工作。本发明提供的抗蓝耳病的猪,应用了本发明提供的抗蓝耳病猪的制备方法制备得到,在能够保证正常存活的同时,抵抗蓝耳病病毒的感染,极大的减少了养猪业因蓝耳病造成的经济损失。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案,下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图是本发明的一些实施方式,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为本发明实施例1提供的猪CD163基因修饰载体的制备方法的流程图。
具体实施方式
下面将结合附图对本发明的技术方案进行清楚、完整地描述,显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
本发明提供了一种用于猪CD163基因编辑的靶向sgRNA,包括sgRNA-C161和sgRNA-C185;
所述sgRNA-C161的正义链和反义链分别为:
C161-Fwd:5’-AAAGTGAGCTCCCCCCAGGA-3’(SEQ ID NO.1);
C161-Rev:5’-TCCTGGGGGGAGCTCACTTT-3’(SEQ ID NO.2);
所述sgRNA-C185的正义链和反义链分别为:
C185-Fwd:5’-GAGAAGGAAGTGGACAGATC-3’(SEQ ID NO.3);
C185-Rev:5’-GATCTGTCCACTTCCTTCTC-3’(SEQ ID NO.4)。
在本发明中,用于猪CD163基因编辑的靶向sgRNA能够起到特异性识别猪CD163基因的作用。
本发明还提供了一种猪CD163基因修饰载体,包括上述的两条sgRNA、Cas9切口酶及荧光标记蛋白,特异性强,能够非常高效地通过CRISPR/Cas9n***在细胞水平上对猪CD163基因进行编辑。要生产抗病的猪是需要特异性的对靶基因进行编辑,绝对不能对基因组的其他区域进行编辑,相比于脱靶效应高,在非靶点序列的区域也能进行基因编辑的Cas9***,本发明选择CRISPR/Cas9n***而非传统的CRISPR/Cas9***,利用本发明提供的猪CD163基因修饰载体对CD163基因进行编辑,旨在在对CD163基因进行特异的基因编辑,效率高特异性强的同时,彻底杜绝由于脱靶效应对基因组上其他序列进行基因编辑的可能。
其中,荧光标记蛋白为GFP。
在本发明中,猪CD163基因修饰为针对猪CD163基因的第七外显子进行的基因编辑,从而破坏蓝耳病病毒受体,降低猪感染蓝耳病病毒的风险。
本发明还提供了上述猪CD163基因修饰载体的制备方法,包括以下步骤:
步骤(a):将sgRNA-C161的正义链和反义链进行互补配对,形成C161双链DNA分子;
步骤(b):将sgRNA-C185的正义链和反义链进行互补配对,形成C185双链DNA分子;
步骤(c):将C161双链DNA分子连接到表达载体上,构建表达sgRNA-C161的载体;
步骤(d):将C185双链DNA分子连接到表达载体上,构建表达sgRNA-C185的载体;
步骤(e):以表达sgRNA-C161的载体为模板,PCR扩增含有U6启动子的sgRNA-C161序列,并连接到克隆载体上,再经过酶切后获得U6-sgRNA-C161序列;
步骤(f):将U6-sgRNA-C161序列连接到表达sgRNA-C185的载体上,构建完成表达sgRNA-C161和sgRNA-C185的载体。
其中,在步骤(c)和步骤(d)中,表达载体为带有Cas9切口酶、荧光标记蛋白及U6启动子的经过BbsⅠ酶切的PX461载体;表达sgRNA-C161的载体为PX461-C161,表达sgRNA-C185的载体为PX461-C185。
在步骤(e)中,克隆载体为pMD18-T载体,酶切为KpnⅠ酶切。
将连接有sgRNA-C161序列的pMD18-T载体进行KpnⅠ酶切后,获得两端含有KpnⅠ酶切位点的U6-sgRNA-C161序列。
在步骤(f)中,表达sgRNA-C185的载体为经过KpnⅠ酶切的PX461-C185,表达sgRNA-C161和sgRNA-C185的载体为PX461-C185+C161。
将两端含有KpnⅠ酶切位点的U6-sgRNA-C161序列连接到KpnⅠ酶切后的PX461-C185上,构建完成同时表达sgRNA-C161和sgRNA-C185以及Cas9切口酶和GFP的载体PX461-C185+C161。
本发明还提供了上述的猪CD163基因修饰载体在制备抗蓝耳病猪中的应用。
本发明还提供了一种抗蓝耳病猪的制备方法,应用上述的猪CD163基因修饰载体。
在本发明中,将猪CD163基因修饰载体转入猪的体细胞,获得CD163基因编辑阳性细胞克隆,以阳性细胞为供体细胞进行体细胞克隆和胚胎移植,获得抗蓝耳病猪。
其中,将猪CD163基因修饰载体转入猪的体细胞的方法例如可以为,但不限于电穿孔法、显微注射法、磷酸钙共沉淀法或脂质体转染法。
在一个优选的实施方式中,猪的体细胞为猪成纤维细胞,在一个更优选的实施方式中,猪的体细胞为猪胎儿成纤维细胞。
将CD163基因编辑后的猪的体细胞作为供体细胞,***作为受体细胞,通过体细胞核移植技术获得克隆胚胎;将克隆胚胎移入供体猪子宫内妊娠获得CD163基因编辑的抗蓝耳病的猪。
本发明提供的抗蓝耳病猪的制备方法,应用了本发明提供的猪CD163基因修饰载体,针对猪蓝耳病中的关键基因CD163进行基因编辑,从而破坏蓝耳病病毒受体,并且,除了对目的基因CD163进行编辑外不会引入其他任何外源基因,也不会对基因组上非CD163基因的区域进行非特异的编辑,遗传背景干净清晰,极大的减少了后期转基因安全评估的工作。
另外,本发明还提供了一种抗蓝耳病的猪,应用上述的抗蓝耳病猪的制备方法制备得到。
本发明提供的抗蓝耳病的猪,应用了本发明提供的抗蓝耳病猪的制备方法制备得到,在能够保证正常存活的同时,抵抗蓝耳病病毒的感染,极大的减少了养猪业因蓝耳病造成的经济损失。
为了有助于更清楚的理解本发明的内容,现结合具体的实施例详细介绍如下。如未明确指出,以下实施例中采用的PX461载体购自Addgene,货号48140;pMD18-T载体购自TaKaRa公司;宿主菌大肠杆菌DH5α购自TaKaRa公司;引物合成由上海生工完成;序列测定由上海睿迪公司完成;质粒小提试剂盒购自TaKaRa公司;LA Taq酶购自TaKaRa公司;T4DNA连接酶购自TaKaRa公司;KpnⅠ内切酶购自TaKaRa公司;BbsⅠ内切酶购自NEB公司;细胞培养基DMEM、PBS、胎牛血清、胰酶和成纤维生长因子(bFGF)购自Life Technologies;脂质体Lipofectamine 2000购自Invitrogen公司;细胞培养板及培养皿购自Thermo Scientific。
实施例1sgRNA的设计和载体的构建
根据猪CD163基因的第七外显子、第八外显子及部分上下游内含子序列(如SEQ IDNO.5所示)和mRNA序列(NCBI NM_213976.1,如SEQ ID NO.6所示),在CD163基因的第七外显子上设计两条sgRNA,为sgRNA-C161和sgRNA-C185,分别在其两端加上粘性接头序列。在每条sgRNA序列F链的5’端加上接头序列CACC,其反向互补序列R链的5’端添加接头序列AAAC,如果sgRNA序列的5’端第一个碱基不是G,那么应先在sgRNA序列F链的5’端添加一个G,再加上接头序列CACC,相应地,其反向互补序列R链的3’端再增加一个C,以便能够与经BbsⅠ酶切的PX461载体的粘性末端互补。
用于构建猪CD163基因修饰载体的sgRNA-C161的正义链和反义链分别为:
C161-Fwd:5’-CACCGAAAGTGAGCTCCCCCCAGGA-3’(SEQ ID NO.7);
C161-Rev:5’-AAACTCCTGGGGGGAGCTCACTTTC-3’(SEQ ID NO.8);
sgRNA-C185的正义链和反义链分别为:
C185-Fwd:5’-CACCGAGAAGGAAGTGGACAGATC-3’(SEQ ID NO.9);
C185-Rev:5’-AAACGATCTGTCCACTTCCTTCTC-3’(SEQ ID NO.10)。
两条sgRNA的正义链和反义链分别在上海生工合成。
将sgRNA-C161和sgRNA-C185的正义链和反义链用水溶解为浓度为200μM的溶液,退火体系如下:
200μM正义链 | 5μL |
200μM反义链 | 5μL |
10×退火缓冲液 | 2μL |
DNase/RNase-free的水 | 8μL |
总体积 | 20μL |
注:10×退火缓冲液的组成包括100mM Tris-HCl(pH8.0),10mM EDTA(pH8.0)和1MNaCl。
在94℃变性5min后,取出样品室温放置10min使sgRNA的正义链和反义链进行互补配对,形成C161双链DNA分子和C185双链DNA分子,-20℃保存。
如图1所示,PX461载体用限制性内切酶BbsⅠ进行酶切回收后,与C161双链DNA分子和C185双链DNA分子连接,连接体系如下:
16℃放置2小时,然后利用常规方法将连接产物转化大肠杆菌DH5α感受态细胞并涂板。待菌落长成后,挑取单菌落扩大培养。将培养好的菌液抽提质粒,测序鉴定sgRNA-C161和sgRNA-C185是否连入载体PX461上。
构建完成同时表达sgRNA-C161、Cas9切口酶以及GFP的载体PX461-C161和同时表达sgRNA-C185、Cas9切口酶以及GFP的载体PX461-C185。
以PX461-C161为模板,sgRNA-Fwd和sgRNA-Rev分别为上下游引物,PCR扩增含有U6启动子的sgRNA-C161序列。
其中,PCR引物序列为:
sgRNA-Fwd:5’-GAGGGCCTATTTCCCATGATTCC-3’(SEQ ID NO.11);
sgRNA-Rev:5’-GGGGTACCTCTAGAGCCATTTG-3’(SEQ ID NO.12)。
PCR的反应体系为:
试剂 | 体积(μL) |
PX461-C161模板(1ng/μL) | 1 |
上游引物sgRNA-Fwd(10μM) | 1 |
下游引物sgRNA-Rev(10μM) | 1 |
dNTP Mix(2.5mM each) | 4 |
10×LA Taq Buffer | 5 |
TaKaRa LA Taq(5U/μL) | 0.25 |
ddH2O | 37.75 |
PCR循环条件如下:
PCR反应完成后,将含有U6启动子的sgRNA-C161序列连接到pMD18-T载体上,然后利用常规方法将连接产物转化大肠杆菌DH5α感受态细胞并涂板。待菌落长成后,挑取单菌落扩大培养。将培养好的菌液抽提质粒,再经过KpnⅠ酶切后获得两端含有KpnⅠ酶切位点的U6-sgRNA-C161序列。
将两端含有KpnⅠ酶切位点的U6-sgRNA-C161序列连接到KpnⅠ酶切后的PX461-C185上,构建完成同时表达sgRNA-C161和sgRNA-C185以及Cas9切口酶和GFP的载体PX461-C185+C161。
实施例2猪的体细胞的转染及CD163基因编辑细胞克隆点的筛选
细胞培养及瞬时转染:
接种猪胎儿成纤维细胞于6孔细胞培养中,在含有15%胎牛血清的DMEM培养基中培养至50-70%汇合度时,按照说明书要求,用Lipofectamine 2000脂质体将实施例1提供的载体PX461-C185+C161转入细胞中。
转染后细胞的流式分选及单克隆培养:
转染后的细胞在37℃培养箱中培养48小时,用0.1%的胰酶消化后,流式细胞仪分选表达GFP的阳性细胞,按照50-100个细胞/100mm培养皿的密度接种细胞,在含有15%胎牛血清及2.5ng/mL成纤维细胞生长因子(bFGF)的DMEM培养基中培养9至12天,至长出明显的单细胞克隆点。
单细胞克隆点CD163基因编辑情况的鉴定:
将生长状态良好的单细胞克隆点细胞用0.1%的胰酶消化后转接到48孔细胞培养板中,待细胞长满需要传代时,取1/10的细胞裂解后为模板做细胞PCR,剩余细胞接种到24孔细胞培养板中继续培养至长满然后冻存到液氮中保存。
用来鉴定细胞克隆点CD163基因编辑情况的引物为:
引物名称 | 引物序列(5’-3’) | SEQ ID NO. |
CD163-39F | TCCCTGCTCTGGTCGTGTTG | 13 |
CD163-527R | CTTCCATGTCCCAGTGAGAGTT | 14 |
dEX7-Fb | TTGGTGAGGGCCAATTGTGTAT | 15 |
dEX7-Rb | GGATAGAAAGGGCAACTCCACA | 16 |
dEX7-Fs | ACCTTGATGATTGCGCTCTT | 17 |
dEX7-Rs | TGTCCCAGTGAGAGTTGCAG | 18 |
由于用来进行鉴定的细胞数比较少,所以细胞裂解物先用CD163-39F和CD163-527R引物进行PCR,没有扩增出特异性条带的细胞裂解物再分别用dEX7-Fb/Rb和dEX7-Fs/Rs两对引物做巢式PCR。
其中,PCR的反应体系为:
试剂 | 体积(μL) |
细胞裂解物或第一次PCR产物(1:100稀释) | 5 |
上游引物(10μM) | 1 |
下游引物(10μM) | 1 |
dNTP Mix(2.5mM each) | 4 |
10×LA Taq Buffer | 5 |
TaKaRa LA Taq(5U/μL) | 0.25 |
ddH2O | 33.75 |
PCR的循环条件为:
随机挑取20个细胞克隆点的PCR产物,胶回收后连接到pMD18-T载体上,测序分析后发现有18个细胞克隆点发生了CD163基因的编辑,为阳性细胞。其中有一个克隆点的细胞完全删除了CD163基因的第七外显子,基因编辑效率高达90%。因此,本发明提供的同时表达两条sgRNA和Cas9切口酶的猪CD163基因修饰载体,可以非常高效地对靶基因CD163进行基因编辑。发生了基因编辑的细胞克隆点CD163基因型结果如下表所示。
最后应说明的是:以上各实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述各实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分或者全部技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
SEQUENCE LISTING
<110> 浙江大学
<120> 用于猪CD163基因编辑的靶向sgRNA、修饰载体及其制备方法和应用
<160> 18
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213> 人工序列
<400> 1
aaagtgagct ccccccagga 20
<210> 2
<211> 20
<212> DNA
<213> 人工序列
<400> 2
tcctgggggg agctcacttt 20
<210> 3
<211> 20
<212> DNA
<213> 人工序列
<400> 3
gagaaggaag tggacagatc 20
<210> 4
<211> 20
<212> DNA
<213> 人工序列
<400> 4
gatctgtcca cttccttctc 20
<210> 5
<211> 1800
<212> DNA
<213> Sus scrofa(猪)
<400> 5
ctacaaggtg cggccttaaa aggaaaaaaa aaaaaattaa atcaaggact caagagtctt 60
tcattatttg tgttgtggaa gctatatttg ttttaaagtc ttagttgtgt ttagaaagca 120
agatgttctt caactcaaat ttgggaggga acttgtttca tacattttta atggataagt 180
ggcaaaattt tcatgctgag gtgatctata gtgttgtaat gcagaatata gtcagatctt 240
gaacatttta ggaagttggt gagggccaat tgtgtatctg tgccatgctg ataagaatgt 300
caagggatca caagaattcg tgttatttga cagcagtcat ctttaaaagg catttgagaa 360
agtccaattt caaatgcatt tcctttcttt aaaagataaa ttgaagaaaa taagtcttta 420
tttcccaagt aaattgaatt gcctctcagt ctgttaaaag aaactcttac cttgatgatt 480
gcgctcttaa cctggcaaag attgtcttta aaatctgagc tccatgtctt ctgctttatt 540
tctggtgtgc ctttgactcc agattacagt aaatggagga ctgagtatag ggctaaaaag 600
tagagagaat ggatgcatat tatctgtggt ctccaatgtg atgaatgaag taggcaaata 660
ctcaaaggaa agagaaagca tgctccaaga attatgggtt ccagaaggca aagtcccaga 720
attgtctcca gggaaggaca gggaggtcta gaatcggcta agcccactgt aggcagaaaa 780
accaagaggc atgaatggct tccctttctc acttttcact ctctggctta ctcctatcat 840
gaaggaaaat attggaatca tattctccct caccgaaatg ctatttttca gcccacagga 900
aacccaggct ggttggaggg gacattccct gctctggtcg tgttgaagta caacatggag 960
acacgtgggg caccgtctgt gattctgact tctctctgga ggcggccagc gtgctgtgca 1020
gggaactaca gtgcggcact gtggtttccc tcctgggggg agctcacttt ggagaaggaa 1080
gtggacagat ctgggctgaa gaattccagt gtgaggggca cgagtcccac ctttcactct 1140
gcccagtagc accccgccct gacgggacat gtagccacag cagggacgtc ggcgtagtct 1200
gctcaagtga gacccaggga atgtgttcac tttgttccca tgccatgaag agggtagggt 1260
taggtagtca cagacatctt tttaaagccc tgtctccttc caggatacac acaaatccgc 1320
ttggtgaatg gcaagacccc atgtgaagga agagtggagc tcaacattct tgggtcctgg 1380
gggtccctct gcaactctca ctgggacatg gaagatgccc atgttttatg ccagcagctt 1440
aaatgtggag ttgccctttc tatcccggga ggagcacctt ttgggaaagg aagtgagcag 1500
gtctggaggc acatgtttca ctgcactggg actgagaagc acatgggaga ttgttccgtc 1560
actgctctgg gcgcatcact ctgttcttca gggcaagtgg cctctgtaat ctgctcaggt 1620
aagagaataa gggcagccag tgatgagcca ctcatgacgg tgccttaaga gtgggtgtac 1680
ctaggagttc ccattgtggc tcagtggtaa caaactcgac tggtatccat gagggtatgg 1740
gtttgatccc tggccttgct caatgggtta aggatccagc attgctgtga gctgtggtat 1800
<210> 6
<211> 3400
<212> RNA
<213> Sus scrofa(猪)
<400> 6
atggtgctac ttgaagactc tggatctgca gactttagaa gatgttctgc ccatttaagt 60
tccttcactt ttgctgtagt cgctgttctc agtgcctgct tggtcactag ttctcttgga 120
ggaaaagaca aggagctgag gctaacgggt ggtgaaaaca agtgctctgg aagagtggag 180
gtgaaagtgc aggaggagtg gggaactgtg tgtaataatg gctgggacat ggatgtggtc 240
tctgttgttt gtaggcagct gggatgtcca actgctatca aagccactgg atgggctaat 300
tttagtgcag gttctggacg catttggatg gatcatgttt cttgtcgagg gaatgagtca 360
gctctctggg actgcaaaca tgatggatgg ggaaagcata actgtactca ccaacaggat 420
gctggagtaa cctgctcaga tggatctgat ttagagatga ggctggtgaa tggaggaaac 480
cggtgcttag gaagaataga agtcaaattt caagagcggt ggggaacagt gtgtgatgat 540
aacttcaaca taaatcatgc ttctgtggtt tgtaaacaac ttgaatgtgg aagtgctgtc 600
agtttctctg gttcagctaa ttttggagaa ggttctggac caatctggtt tgatgatctt 660
gtatgcaatg gaaatgagtc agctctctgg aactgcaaac atgaaggatg gggaaagcac 720
aattgcgatc atgctgagga tgctggagtg atttgcttaa atggagcaga cctgaaactg 780
agagtggtag atggactcac tgaatgttca ggaagattgg aagtgaaatt ccaaggagaa 840
tggggaacaa tctgtgatga tggctgggat agtgatgatg ccgctgtggc atgtaagcaa 900
ctgggatgtc caactgctgt cactgccatt ggtcgagtta acgccagtga gggaactgga 960
cacatttggc ttgacagtgt ttcttgccat ggacacgagt ctgctctctg gcagtgtaga 1020
caccatgaat ggggaaagca ttattgcaat cataatgaag atgctggtgt gacatgttct 1080
gatggatcag atctggaact gagacttaaa ggtggaggca gccactgtgc tgggacagtg 1140
gaggtggaaa ttcagaaact ggtaggaaaa gtgtgtgata gaagctgggg actgaaagaa 1200
gctgatgtgg tttgcaggca gctgggatgt ggatctgcac tcaaaacatc atatcaagtt 1260
tattccaaaa ccaaggcaac aaacacatgg ctgtttgtaa gcagctgtaa tggaaatgaa 1320
acttctcttt gggactgcaa gaattggcag tggggtggac ttagttgtga tcactatgac 1380
gaagccaaaa ttacctgctc agcccacagg aaacccaggc tggttggagg ggacattccc 1440
tgctctggtc gtgttgaagt acaacatgga gacacgtggg gcaccgtctg tgattctgac 1500
ttctctctgg aggcggccag cgtgctgtgc agggaactac agtgcggcac tgtggtttcc 1560
ctcctggggg gagctcactt tggagaagga agtggacaga tctgggctga agaattccag 1620
tgtgaggggc acgagtccca cctttcactc tgcccagtag caccccgccc tgacgggaca 1680
tgtagccaca gcagggacgt cggcgtagtc tgctcaagat acacacaaat ccgcttggtg 1740
aatggcaaga ccccatgtga aggaagagtg gagctcaaca ttcttgggtc ctgggggtcc 1800
ctctgcaact ctcactggga catggaagat gcccatgttt tatgccagca gcttaaatgt 1860
ggagttgccc tttctatccc gggaggagca ccttttggga aaggaagtga gcaggtctgg 1920
aggcacatgt ttcactgcac tgggactgag aagcacatgg gagattgttc cgtcactgct 1980
ctgggcgcat cactctgttc ttcagggcaa gtggcctctg taatctgctc agggaaccag 2040
agtcagacac tatccccgtg caattcatca tcctcggacc catcaagctc tattatttca 2100
gaagaaagtg gtgttgcctg catagggagt ggtcaacttc gcctggtcga tggaggtggt 2160
cgttgtgctg ggagagtaga ggtctatcct ggggcatcct ggggcaccat ctgtgatgac 2220
agctgggacc tgaatgatgc ccatgtggtg tgcaaacagc tgagctgtgg atgggccatt 2280
aatgccactg gttctgctca ttttggggaa ggaacagggc ccatttggct ggatgagata 2340
aactgtaatg gaaaagaatc tcatatttgg caatgccact cacatggttg ggggcggcac 2400
aattgcaggc ataaggagga tgcaggagtc atctgctcag agttcatgtc tctgagactg 2460
atcagtgaaa acagcagaga gacctgtgca gggcgcctgg aagtttttta caacggagct 2520
tggggcagcg ttggcaggaa tagcatgtct ccagccacag tgggggtggt atgcaggcag 2580
ctgggctgtg cagacagagg ggacatcagc cctgcatctt cagacaagac agtgtccagg 2640
cacatgtggg tggacaatgt tcagtgtcct aaaggacctg acacactatg gcagtgcccc 2700
tcatctccat ggaagaagag actggccagc ccctcagagg agacatggat cacatgtgcc 2760
aacaaaataa gacttcaaga aggaaacact aattgttctg gacgtgtgga gatctggtac 2820
ggaggttcct ggggcactgt gtgtgacgac tcctgggacc ttgaagatgc tcaggtggtg 2880
tgccgacagc tgggctgtgg ctcagctttg gaggcaggaa aagagcccgc atttggccag 2940
gggactgggc ccatatggct caatgaagtg aagtgcaagg ggaatgaacc ctccttgtgg 3000
gattgtcctg ccagatcctg gggccacagt gactgtggac acaaggagga tgctgctgtg 3060
acgtgctcag aaattgcaaa gagccgagaa tccctacatg ccacaggtcg ctcatctttt 3120
gttgcacttg caatctttgg ggtcattctg ttggcctgtc tcatcgcatt cctcatttgg 3180
actcagaagc gaagacagag gcagcggctc tcagttttct caggaggaga gaattctgtc 3240
catcaaattc aataccggga gatgaattct tgcctgaaag cagatgaaac ggatatgcta 3300
aatccctcag gagaccactc tgaagtacaa tgaaaaggaa aatgggaatt ataacctggt 3360
gagttcagcc tttaagatac cttgatgaag acctggacta 3400
<210> 7
<211> 25
<212> DNA
<213> 人工序列
<400> 7
caccgaaagt gagctccccc cagga 25
<210> 8
<211> 25
<212> DNA
<213> 人工序列
<400> 8
aaactcctgg ggggagctca ctttc 25
<210> 9
<211> 24
<212> DNA
<213> 人工序列
<400> 9
caccgagaag gaagtggaca gatc 24
<210> 10
<211> 24
<212> DNA
<213> 人工序列
<400> 10
aaacgatctg tccacttcct tctc 24
<210> 11
<211> 23
<212> DNA
<213> 人工序列
<400> 11
gagggcctat ttcccatgat tcc 23
<210> 12
<211> 22
<212> DNA
<213> 人工序列
<400> 12
ggggtacctc tagagccatt tg 22
<210> 13
<211> 20
<212> DNA
<213> 人工序列
<400> 13
tccctgctct ggtcgtgttg 20
<210> 14
<211> 22
<212> DNA
<213> 人工序列
<400> 14
cttccatgtc ccagtgagag tt 22
<210> 15
<211> 22
<212> DNA
<213> 人工序列
<400> 15
ttggtgaggg ccaattgtgt at 22
<210> 16
<211> 22
<212> DNA
<213> 人工序列
<400> 16
ggatagaaag ggcaactcca ca 22
<210> 17
<211> 20
<212> DNA
<213> 人工序列
<400> 17
accttgatga ttgcgctctt 20
<210> 18
<211> 20
<212> DNA
<213> 人工序列
<400> 18
tgtcccagtg agagttgcag 20
Claims (10)
1.一种用于猪CD163基因编辑的靶向sgRNA,其特征在于,所述sgRNA包括sgRNA-C161和sgRNA-C185;
所述sgRNA-C161的正义链和反义链分别为:
C161-Fwd:5’-AAAGTGAGCTCCCCCCAGGA-3’(SEQ ID NO.1);
C161-Rev:5’-TCCTGGGGGGAGCTCACTTT-3’(SEQ ID NO.2);
所述sgRNA-C185的正义链和反义链分别为:
C185-Fwd:5’-GAGAAGGAAGTGGACAGATC-3’(SEQ ID NO.3);
C185-Rev:5’-GATCTGTCCACTTCCTTCTC-3’(SEQ ID NO.4)。
2.一种猪CD163基因修饰载体,其特征在于,包括权利要求1所述的两条sgRNA、Cas9切口酶及荧光标记蛋白。
3.根据权利要求2所述的猪CD163基因修饰载体,其特征在于,所述猪CD163基因修饰为针对猪CD163基因的第七外显子进行的基因编辑。
4.如权利要求2或3所述的猪CD163基因修饰载体的制备方法,其特征在于,包括以下步骤:
步骤(a):将所述sgRNA-C161的正义链和反义链进行互补配对,形成C161双链DNA分子;
步骤(b):将所述sgRNA-C185的正义链和反义链进行互补配对,形成C185双链DNA分子;
步骤(c):将所述C161双链DNA分子连接到表达载体上,构建表达sgRNA-C161的载体;
步骤(d):将所述C185双链DNA分子连接到表达载体上,构建表达sgRNA-C185的载体;
步骤(e):以所述表达sgRNA-C161的载体为模板,PCR扩增含有U6启动子的所述sgRNA-C161序列,并连接到克隆载体上,再经过酶切后获得U6-sgRNA-C161序列;
步骤(f):将所述U6-sgRNA-C161序列连接到所述表达sgRNA-C185的载体上,构建完成表达所述sgRNA-C161和sgRNA-C185的载体。
5.根据权利要求4所述的制备方法,其特征在于,在步骤(c)和步骤(d)中,所述表达载体为带有Cas9切口酶、荧光标记蛋白及U6启动子的经过BbsⅠ酶切的PX461载体。
6.根据权利要求4所述的制备方法,其特征在于,在步骤(e)中,所述克隆载体为pMD18-T载体。
7.如权利要求2或3所述的猪CD163基因修饰载体在制备抗蓝耳病猪中的应用。
8.一种抗蓝耳病猪的制备方法,其特征在于,应用权利要求2或3所述的猪CD163基因修饰载体。
9.根据权利要求8所述的抗蓝耳病猪的制备方法,其特征在于,将所述猪CD163基因修饰载体转入猪的体细胞中,获得CD163基因编辑阳性细胞克隆,以所述阳性细胞为供体细胞进行体细胞克隆和胚胎移植,获得所述抗蓝耳病猪。
10.一种抗蓝耳病的猪,其特征在于,应用权利要求8或9所述的抗蓝耳病猪的制备方法制备得到。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201710424822.6A CN107177595A (zh) | 2017-06-07 | 2017-06-07 | 用于猪CD163基因编辑的靶向sgRNA、修饰载体及其制备方法和应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201710424822.6A CN107177595A (zh) | 2017-06-07 | 2017-06-07 | 用于猪CD163基因编辑的靶向sgRNA、修饰载体及其制备方法和应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN107177595A true CN107177595A (zh) | 2017-09-19 |
Family
ID=59835790
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201710424822.6A Pending CN107177595A (zh) | 2017-06-07 | 2017-06-07 | 用于猪CD163基因编辑的靶向sgRNA、修饰载体及其制备方法和应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN107177595A (zh) |
Cited By (29)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107937345A (zh) * | 2017-11-16 | 2018-04-20 | 山东蓝思种业股份有限公司 | 一种制备同时敲除cd163基因和cd13基因的猪成纤维细胞的方法 |
US9999671B2 (en) | 2013-09-06 | 2018-06-19 | President And Fellows Of Harvard College | Delivery of negatively charged proteins using cationic lipids |
US10113163B2 (en) | 2016-08-03 | 2018-10-30 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
US10167457B2 (en) | 2015-10-23 | 2019-01-01 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
CN109666646A (zh) * | 2018-11-15 | 2019-04-23 | 广东省农业科学院农业生物基因研究中心 | 一种cd163基因编辑的猪胚胎成纤维细胞系的制备及应用 |
US10323236B2 (en) | 2011-07-22 | 2019-06-18 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
US10465176B2 (en) | 2013-12-12 | 2019-11-05 | President And Fellows Of Harvard College | Cas variants for gene editing |
US10508298B2 (en) | 2013-08-09 | 2019-12-17 | President And Fellows Of Harvard College | Methods for identifying a target site of a CAS9 nuclease |
US10597679B2 (en) | 2013-09-06 | 2020-03-24 | President And Fellows Of Harvard College | Switchable Cas9 nucleases and uses thereof |
US10704062B2 (en) | 2014-07-30 | 2020-07-07 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
US10745677B2 (en) | 2016-12-23 | 2020-08-18 | President And Fellows Of Harvard College | Editing of CCR5 receptor gene to protect against HIV infection |
US10858639B2 (en) | 2013-09-06 | 2020-12-08 | President And Fellows Of Harvard College | CAS9 variants and uses thereof |
CN112779292A (zh) * | 2021-02-22 | 2021-05-11 | 杭州合欣源生物科技有限公司 | 构建瘦肉率高、生长快且抗蓝耳病和系列腹泻病的优质猪核移植供体细胞的方法及其应用 |
US11046948B2 (en) | 2013-08-22 | 2021-06-29 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
CN113151291A (zh) * | 2020-05-05 | 2021-07-23 | 吉纳斯公司 | 通过对cd163靶向灭活来改善猪类健康的方法 |
US11268082B2 (en) | 2017-03-23 | 2022-03-08 | President And Fellows Of Harvard College | Nucleobase editors comprising nucleic acid programmable DNA binding proteins |
US11306324B2 (en) | 2016-10-14 | 2022-04-19 | President And Fellows Of Harvard College | AAV delivery of nucleobase editors |
US11319532B2 (en) | 2017-08-30 | 2022-05-03 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
CN114774468A (zh) * | 2022-04-20 | 2022-07-22 | 温氏食品集团股份有限公司 | 一种新的等位基因分子标记及抗蓝耳病猪群体组建方法 |
US11447770B1 (en) | 2019-03-19 | 2022-09-20 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
US11542509B2 (en) | 2016-08-24 | 2023-01-03 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
US11542496B2 (en) | 2017-03-10 | 2023-01-03 | President And Fellows Of Harvard College | Cytosine to guanine base editor |
US11560566B2 (en) | 2017-05-12 | 2023-01-24 | President And Fellows Of Harvard College | Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation |
US11661590B2 (en) | 2016-08-09 | 2023-05-30 | President And Fellows Of Harvard College | Programmable CAS9-recombinase fusion proteins and uses thereof |
US11732274B2 (en) | 2017-07-28 | 2023-08-22 | President And Fellows Of Harvard College | Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE) |
US11795443B2 (en) | 2017-10-16 | 2023-10-24 | The Broad Institute, Inc. | Uses of adenosine base editors |
US11898179B2 (en) | 2017-03-09 | 2024-02-13 | President And Fellows Of Harvard College | Suppression of pain by gene editing |
US11912985B2 (en) | 2020-05-08 | 2024-02-27 | The Broad Institute, Inc. | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
US12031126B2 (en) | 2023-12-08 | 2024-07-09 | The Broad Institute, Inc. | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104263754A (zh) * | 2014-08-29 | 2015-01-07 | 中国科学院广州生物医药与健康研究院 | 白化病模型猪的重构卵及其构建方法和模型猪的构建方法 |
CN104593422A (zh) * | 2015-01-08 | 2015-05-06 | 中国农业大学 | 一种抗蓝耳病克隆猪的制备方法 |
-
2017
- 2017-06-07 CN CN201710424822.6A patent/CN107177595A/zh active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104263754A (zh) * | 2014-08-29 | 2015-01-07 | 中国科学院广州生物医药与健康研究院 | 白化病模型猪的重构卵及其构建方法和模型猪的构建方法 |
CN104593422A (zh) * | 2015-01-08 | 2015-05-06 | 中国农业大学 | 一种抗蓝耳病克隆猪的制备方法 |
Non-Patent Citations (4)
Title |
---|
BURKARD CHRISTINE等: "《Precision engineering for PRRSV resistance in pigs:Macrophages from genome edited pigs lacking CD163 SRCR5 domain are fully resistant to both PRRSV genotypes while maintaining biological function》", 《PLOS PATHOGENS》 * |
KRISTIN M. WHITWORTH等: "《Use of the CRISPR/Cas9 System to Produce Genetically Engineered Pigs from In Vitro-Derived Oocytes and Embryos》", 《BIOLOGY OF REPRODUCTION》 * |
WHITWORTH KRISTIN M.等: "《Gene-edited pigs are protected from porcine reproductive》", 《NATURE BIOTECHNOLOGY》 * |
赵盼盼等: "《提高CRISPR/Cas9***靶向编辑效率方法的研究进展》", 《江苏农业科学》 * |
Cited By (50)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US12006520B2 (en) | 2011-07-22 | 2024-06-11 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
US10323236B2 (en) | 2011-07-22 | 2019-06-18 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
US10508298B2 (en) | 2013-08-09 | 2019-12-17 | President And Fellows Of Harvard College | Methods for identifying a target site of a CAS9 nuclease |
US11920181B2 (en) | 2013-08-09 | 2024-03-05 | President And Fellows Of Harvard College | Nuclease profiling system |
US10954548B2 (en) | 2013-08-09 | 2021-03-23 | President And Fellows Of Harvard College | Nuclease profiling system |
US11046948B2 (en) | 2013-08-22 | 2021-06-29 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
US11299755B2 (en) | 2013-09-06 | 2022-04-12 | President And Fellows Of Harvard College | Switchable CAS9 nucleases and uses thereof |
US9999671B2 (en) | 2013-09-06 | 2018-06-19 | President And Fellows Of Harvard College | Delivery of negatively charged proteins using cationic lipids |
US10912833B2 (en) | 2013-09-06 | 2021-02-09 | President And Fellows Of Harvard College | Delivery of negatively charged proteins using cationic lipids |
US10597679B2 (en) | 2013-09-06 | 2020-03-24 | President And Fellows Of Harvard College | Switchable Cas9 nucleases and uses thereof |
US10682410B2 (en) | 2013-09-06 | 2020-06-16 | President And Fellows Of Harvard College | Delivery system for functional nucleases |
US10858639B2 (en) | 2013-09-06 | 2020-12-08 | President And Fellows Of Harvard College | CAS9 variants and uses thereof |
US11124782B2 (en) | 2013-12-12 | 2021-09-21 | President And Fellows Of Harvard College | Cas variants for gene editing |
US11053481B2 (en) | 2013-12-12 | 2021-07-06 | President And Fellows Of Harvard College | Fusions of Cas9 domains and nucleic acid-editing domains |
US10465176B2 (en) | 2013-12-12 | 2019-11-05 | President And Fellows Of Harvard College | Cas variants for gene editing |
US10704062B2 (en) | 2014-07-30 | 2020-07-07 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
US11578343B2 (en) | 2014-07-30 | 2023-02-14 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
US10167457B2 (en) | 2015-10-23 | 2019-01-01 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
US11214780B2 (en) | 2015-10-23 | 2022-01-04 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
US11999947B2 (en) | 2016-08-03 | 2024-06-04 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
US11702651B2 (en) | 2016-08-03 | 2023-07-18 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
US10947530B2 (en) | 2016-08-03 | 2021-03-16 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
US10113163B2 (en) | 2016-08-03 | 2018-10-30 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
US11661590B2 (en) | 2016-08-09 | 2023-05-30 | President And Fellows Of Harvard College | Programmable CAS9-recombinase fusion proteins and uses thereof |
US11542509B2 (en) | 2016-08-24 | 2023-01-03 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
US11306324B2 (en) | 2016-10-14 | 2022-04-19 | President And Fellows Of Harvard College | AAV delivery of nucleobase editors |
US10745677B2 (en) | 2016-12-23 | 2020-08-18 | President And Fellows Of Harvard College | Editing of CCR5 receptor gene to protect against HIV infection |
US11820969B2 (en) | 2016-12-23 | 2023-11-21 | President And Fellows Of Harvard College | Editing of CCR2 receptor gene to protect against HIV infection |
US11898179B2 (en) | 2017-03-09 | 2024-02-13 | President And Fellows Of Harvard College | Suppression of pain by gene editing |
US11542496B2 (en) | 2017-03-10 | 2023-01-03 | President And Fellows Of Harvard College | Cytosine to guanine base editor |
US11268082B2 (en) | 2017-03-23 | 2022-03-08 | President And Fellows Of Harvard College | Nucleobase editors comprising nucleic acid programmable DNA binding proteins |
US11560566B2 (en) | 2017-05-12 | 2023-01-24 | President And Fellows Of Harvard College | Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation |
US11732274B2 (en) | 2017-07-28 | 2023-08-22 | President And Fellows Of Harvard College | Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE) |
US11319532B2 (en) | 2017-08-30 | 2022-05-03 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
US11932884B2 (en) | 2017-08-30 | 2024-03-19 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
US11795443B2 (en) | 2017-10-16 | 2023-10-24 | The Broad Institute, Inc. | Uses of adenosine base editors |
CN107937345A (zh) * | 2017-11-16 | 2018-04-20 | 山东蓝思种业股份有限公司 | 一种制备同时敲除cd163基因和cd13基因的猪成纤维细胞的方法 |
CN107937345B (zh) * | 2017-11-16 | 2019-01-29 | 山东蓝思种业股份有限公司 | 一种制备同时敲除cd163基因和cd13基因的猪成纤维细胞的方法 |
CN109666646A (zh) * | 2018-11-15 | 2019-04-23 | 广东省农业科学院农业生物基因研究中心 | 一种cd163基因编辑的猪胚胎成纤维细胞系的制备及应用 |
US11643652B2 (en) | 2019-03-19 | 2023-05-09 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
US11795452B2 (en) | 2019-03-19 | 2023-10-24 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
US11447770B1 (en) | 2019-03-19 | 2022-09-20 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
CN113151291A (zh) * | 2020-05-05 | 2021-07-23 | 吉纳斯公司 | 通过对cd163靶向灭活来改善猪类健康的方法 |
CN117487855A (zh) * | 2020-05-05 | 2024-02-02 | 吉纳斯公司 | 通过对cd163靶向灭活来改善猪类健康的方法 |
US11912985B2 (en) | 2020-05-08 | 2024-02-27 | The Broad Institute, Inc. | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
CN112779292B (zh) * | 2021-02-22 | 2023-03-10 | 南京启真基因工程有限公司 | 构建瘦肉率高、生长快且抗蓝耳病和系列腹泻病的优质猪核移植供体细胞的方法及其应用 |
CN112779292A (zh) * | 2021-02-22 | 2021-05-11 | 杭州合欣源生物科技有限公司 | 构建瘦肉率高、生长快且抗蓝耳病和系列腹泻病的优质猪核移植供体细胞的方法及其应用 |
CN114774468B (zh) * | 2022-04-20 | 2022-12-20 | 温氏食品集团股份有限公司 | 一种等位基因分子标记及抗蓝耳病猪群体组建方法 |
CN114774468A (zh) * | 2022-04-20 | 2022-07-22 | 温氏食品集团股份有限公司 | 一种新的等位基因分子标记及抗蓝耳病猪群体组建方法 |
US12031126B2 (en) | 2023-12-08 | 2024-07-09 | The Broad Institute, Inc. | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN107177595A (zh) | 用于猪CD163基因编辑的靶向sgRNA、修饰载体及其制备方法和应用 | |
CN107034218A (zh) | 用于猪APN基因编辑的靶向sgRNA、修饰载体及其制备方法和应用 | |
CN107099533A (zh) | 一种特异靶向猪IGFBP3基因的sgRNA导向序列及应用 | |
CN104232669A (zh) | 一种采用基因敲除法构建基于鱼类CRISPR/Cas9***的载体及其构建方法 | |
CN107338266A (zh) | 一种鉴定桑树MmPDS基因的VIGS沉默体系及其构建方法和应用 | |
EP3714038A1 (en) | Cellular compositions and methods of treatment i | |
Chen et al. | Three novel insect-associated species of Simplicillium (Cordycipitaceae, Hypocreales) from Southwest China | |
CN110241178A (zh) | 一种单细胞转录组测序高通量快速文库制备方法及检测试剂盒 | |
CN109706148A (zh) | 一种用于敲除BCL11A基因或者BCL11A基因增强子的gRNA、gRNA组合物以及电转方法 | |
CN105861551B (zh) | 联合表达microRNAs抑制乳腺癌细胞增殖的载体及其构建方法和应用 | |
CN103882043B (zh) | 羊驼TGF-β1-3’UTR双荧光素酶报告基因载体及其构建和应用 | |
WO2021243881A1 (zh) | 用于猪MBP基因敲除的sgRNA组合物及用途 | |
CN106244590B (zh) | 经修饰的siRNA分子、RNAi分子混合物及其应用 | |
Doria-Borrell et al. | Generation of knockout mouse trophoblast stem cells by CRISPR/Cas9 | |
CN108949809A (zh) | 一种连翘叶片trv载体介导病毒诱导基因沉默的方法 | |
CN104672317B (zh) | 调节Oct4基因表达的转录因子ZNF312b及其应用 | |
CN106566872B (zh) | 基于测序基因分型技术的猪snp标记位点的分析方法 | |
CN101358199A (zh) | 一种表达人cdc25 b3质粒及其构建方法 | |
CN102304580A (zh) | 一种筛选猪抗病育种的方法及其应用 | |
CN108893486A (zh) | 一种可用于丝状真菌基因敲除的载体及应用 | |
CN103194441A (zh) | 获取miRNA候选靶基因的方法及其专用反转录引物 | |
Sun et al. | Coexpression of recombinant adenovirus carrying GDNF and EDNRB genes in neural stem cells in vitro | |
CN107794264A (zh) | 用于构建多倍体植物突变体库的接头序列及鉴定方法 | |
CN111269301B (zh) | 香蕉转录因子MaARF12、MaARF24及其在抑制MaSBE2.3表达上的应用 | |
CN111171121B (zh) | 一种转录因子及其在激活香蕉MaSBE2.3表达上的应用 |
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 | ||
WD01 | Invention patent application deemed withdrawn after publication | ||
WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20170919 |