CN106916820A - 能有效编辑猪ROSA26基因的sgRNA及其应用 - Google Patents
能有效编辑猪ROSA26基因的sgRNA及其应用 Download PDFInfo
- Publication number
- CN106916820A CN106916820A CN201710341126.9A CN201710341126A CN106916820A CN 106916820 A CN106916820 A CN 106916820A CN 201710341126 A CN201710341126 A CN 201710341126A CN 106916820 A CN106916820 A CN 106916820A
- Authority
- CN
- China
- Prior art keywords
- gene
- sgrna
- porcine
- effectively
- rosa
- 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
- 108091027544 Subgenomic mRNA Proteins 0.000 title claims abstract description 51
- 235000011449 Rosa Nutrition 0.000 title claims abstract description 30
- 101150112497 26 gene Proteins 0.000 title claims abstract description 25
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 19
- 108700019146 Transgenes Proteins 0.000 claims abstract description 14
- 238000002360 preparation method Methods 0.000 claims abstract description 7
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 15
- 230000008685 targeting Effects 0.000 claims description 9
- 230000000295 complement effect Effects 0.000 claims description 6
- 239000013604 expression vector Substances 0.000 claims description 5
- 238000010363 gene targeting Methods 0.000 claims description 5
- 239000013600 plasmid vector Substances 0.000 claims description 5
- 108091034117 Oligonucleotide Proteins 0.000 claims description 4
- 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 claims description 4
- 241000282887 Suidae Species 0.000 claims description 3
- 238000000137 annealing Methods 0.000 claims description 3
- 238000003209 gene knockout Methods 0.000 claims description 3
- 102100035102 E3 ubiquitin-protein ligase MYCBP2 Human genes 0.000 claims description 2
- 239000000539 dimer Substances 0.000 claims description 2
- 210000004027 cell Anatomy 0.000 abstract description 20
- 108010048367 enhanced green fluorescent protein Proteins 0.000 abstract description 16
- 108091033409 CRISPR Proteins 0.000 abstract description 11
- 230000010354 integration Effects 0.000 abstract description 10
- 238000010354 CRISPR gene editing Methods 0.000 abstract description 6
- 238000005516 engineering process Methods 0.000 abstract description 6
- 210000002950 fibroblast Anatomy 0.000 abstract description 6
- 210000003754 fetus Anatomy 0.000 abstract description 3
- 239000003550 marker Substances 0.000 abstract description 3
- 235000021393 food security Nutrition 0.000 abstract description 2
- 230000001404 mediated effect Effects 0.000 abstract 1
- 239000013612 plasmid Substances 0.000 description 7
- 108020004414 DNA Proteins 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 238000012163 sequencing technique Methods 0.000 description 5
- 230000009471 action Effects 0.000 description 4
- 238000005520 cutting process Methods 0.000 description 4
- 238000010362 genome editing Methods 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- 230000001605 fetal effect Effects 0.000 description 3
- 238000012239 gene modification Methods 0.000 description 3
- 239000013598 vector Substances 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 230000005017 genetic modification Effects 0.000 description 2
- 235000013617 genetically modified food Nutrition 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 238000001000 micrograph Methods 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- OZFAFGSSMRRTDW-UHFFFAOYSA-N (2,4-dichlorophenyl) benzenesulfonate Chemical compound ClC1=CC(Cl)=CC=C1OS(=O)(=O)C1=CC=CC=C1 OZFAFGSSMRRTDW-UHFFFAOYSA-N 0.000 description 1
- 108091079001 CRISPR RNA Proteins 0.000 description 1
- 241000252212 Danio rerio Species 0.000 description 1
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 description 1
- 108091029865 Exogenous DNA Proteins 0.000 description 1
- 241000244206 Nematoda Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 108091027967 Small hairpin RNA Proteins 0.000 description 1
- 101150029220 Tprg1l gene Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000004721 adaptive immunity Effects 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- 239000012930 cell culture fluid Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 238000012869 ethanol precipitation Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 238000003198 gene knock in Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000000384 rearing effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000004055 small Interfering RNA Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 238000005406 washing Methods 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
-
- 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
-
- 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
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)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Plant Pathology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Environmental Sciences (AREA)
- Mycology (AREA)
- Cell Biology (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一条能有效编辑猪ROSA26基因的sgRNA及其应用，以猪基因组中的一个能特异识别猪ROSA26基因的sgRNA为前提，利用CRISPR/Cas9 介导基因敲入技术，成功地构建了EGFP定点整合的猪胎儿成纤维细胞的细胞系，结果显示该细胞系能稳定且高效的表达EGFP基因，且该细胞系的制备过程中没有引入任何的外源的启动子基因及正负筛选标记基因。这大大的增加的转基因猪的安全性，对去除转基因猪农产品的生物、食品安全隐患具有重要意义。
Description
技术领域
本发明公开了一条能有效编辑猪ROSA26基因的sgRNA及其应用，同时还公开了与该sgRNA相关的更加安全的外源基因的定点策略及可行性分析，属于生物技术领域。
技术背景
CRISPR/Cas9 是细菌和古细菌在长期演化过程中形成的一种适应性免疫防御，可用来对抗入侵的病毒及外源DNA。CRISPR/Cas9 系统通过将入侵噬菌体和质粒 DNA 的片段整合到 CRISPR 中，并利用相应的 CRISPR RNAs（crRNAs）来指导同源序列的降解，从而提供免疫性。后来，研究人员发现，它更是一种精确且万能基因编辑工具，可以用来删除、添加、激活或抑制其他生物体的目标基因，包括人、老鼠、猪、牛、羊、斑马鱼、细菌、果蝇、酵母、线虫和农作物等的基因。利用这种机制可以实现基因组的精确编辑，如条件性基因敲除、基因敲进、基因替换、点突变等等。CRISPR/Cas9 技术以自己操作的便捷性，高效的基因编辑能力获得青睐，该技术面世以后，弥补了传统基因编辑技术的诸多不足，在基因编辑领域有着广阔的应用前景。
转基因猪在农业新品种培育和生物医药研究中具有非常重要的应用价值。长期以来，由于缺乏猪胚胎干细胞，转基因猪的制备主要依赖于体细胞基因修饰和体细胞核移植技术。而目前，对体细胞的基因修饰主要依赖将外源基因随机插入猪基因组的方法，导致外源基因在猪基因组中的整合位点和拷贝数不可控，进而使外源基因猪体内表达不稳定，引发转基因猪个体之间表型不均一等问题。又因为传统转基因猪的制备过程中对外源启动子基因及正负筛选标记基因的依赖性，进一步增加了转基因猪的安全评价等问题。这些因素限制了转基因猪的培育与应用前景。
发明内容
本发明的目的是提供一条能有效编辑猪ROSA26基因的sgRNA，该sgRNA能用于特异识别和靶向编辑猪ROSA26基因及ROSA26基因定点整合转基因猪的制备。
本发明所提供了一种能有效编辑猪ROSA26基因的sgRNA，其特征在于：该sgRNA的RNA序列如：SEQ ID 1所示，互补链的RNA序列如：SEQ ID 2所示。
本发明能有效编辑猪ROSA26基因的sgRNA的DNA序列，其特征在于：该sgRNA的DNA序列如SEQ ID 3所示，互补链的DNA序列如：SEQ ID 4所示。
本发明所述的能有效编辑猪ROSA26基因的sgRNA的制备方法，包括以下步骤：
1）在基因库中调出猪ROSA26基因的序列，根据PAM序列（NGG）选择用于基因敲除的sgRNA靶向的区域；
2）根据靶位点的序列，设计并合成对应的引物序列；
3）引物退火形成寡聚核苷二聚体（oligoduplex）；
4）将寡核苷酸二聚体连接到相应的质粒载体中，即可获得sgRNA的表达载体；
本发明所述的能有效编辑猪ROSA26基因的sgRNA在猪ROSA26位点编辑的应用。
本发明所述的所述的能有效编辑猪ROSA26基因的sgRNA在猪ROSA26位点编辑的应用，其特征在于：能有效编辑猪ROSA26基因的sgRNA在特异识别和靶向编辑猪ROSA26基因中的应用以及在制备ROSA26基因定点整合转基因猪中的应用。
本发明的积极效果在于：
以猪基因组中的一个能特异识别猪ROSA26基因的sgRNA为前提，利用CRISPR/Cas9 介导基因敲入技术，成功地构建了EGFP定点整合的猪胎儿成纤维细胞的细胞系，结果显示该细胞系能稳定且高效的表达EGFP基因，且该细胞系的制备过程中没有引入任何的外源的启动子基因及正负筛选标记基因。这大大的增加的转基因猪的安全性，对去除转基因猪农产品的生物、食品安全隐患具有重要意义。
附图说明：
图1为本发明用于评估3条不同sgRNA切割效率的测序峰图；
图2为本发明EGFP定点打靶质粒载体示意图；
图3为本发明EGFP定点整合细胞克隆的荧光显微镜图；
图4为本发明EGFP定点整合的细胞克隆的PCR鉴定电泳图。
具体实施方式
通过以下实施例进一步举例描述本发明，并不以任何方式限制本发明，在不背离本发明的技术解决方案的前提下，对本发明所作的本领域普通技术人员容易实现的任何改动或改变都将落入本发明的权利要求范围之内。
实施例1
1-1、sgRNA序列的设计及PX330表达载体的构建
设计并合成了3条针对猪ROSA26位点的sgRNA序列。上述设计完成的shRNA序列合成；6条单链的sgRNA的DNA序列分别经过退火后形成3条靶向猪ROSA26第一内含子不同位点的sgRNA的寡核苷酸链；然后将该寡聚核苷酸连连入PX330质粒载体。
这3条sgRNA的序列及其作用位点的序列分别为：
SgRNA-86序列：5-ATCGCAGTGGTAGTCAAGAT-3；
SgRNA-86作用位点的序列：5-ATCTTGACTACCACTGCGAT-3；
SgRNA-89序列：5-ATCTTGAGCATAGGCCCAAC-3；
SgRNA-89作用位点的序列：5-GTTGGGCCTATGCTCAAGAT-3；
SgRNA-91序列：5-TACGGTCAGATAACTCTCAC-3；
SgRNA-91作用位点的序列：5-GTGAGAGTTATCTGACCGTA-3；
其中，本发明所涉及的
sgRNA的RNA序列为：5-UACGGUCAGAUAACUCUCAC-3 （SEQ ID NO.1）；
sgRNA的RNA序列的互补序列为：5-UACGGUCAGAUAACUCUCAC-3 （SEQ ID NO.2）；
其中，本发明所涉及的
sgRNA的DNA序列为：5-TACGGTCAGATAACTCTCAC-3 （SEQ ID NO.3）；
sgRNA的DNA序列的互补序列为：5-TACGGTCAGATAACTCTCAC-3 （SEQ ID NO.4）。
、高效sgRNA的评估和筛选
通过对3种sgRNA的表达载体进一步的测序后，进行质粒的大提和质粒的乙醇沉淀，将纯化后一定浓度的三种sgRNA的PX330的表达载体，通过电穿孔转染的方式引入到猪的胎儿成纤维细胞中，转染后的72小时，提取各组细胞的基因组，然后用特异性的检测突变效率的引物进行pcr反应，将所获得的PCR产物一方面送去测序通过对测序峰图的分析初步评估个sgRNA的切割效率，同时将剩下的PCR产物用于连T载体或同过T7E1分析来精确的评估各sgRNA对靶基因的切割效率。图1为本发明用于评估3条不同sgRNA切割效率的测序峰图。
实施例2
EGFP定点整合打靶载体的构建
根据筛选出的高效sgRNA设计并构建与该sgRNA配套的EGFP基因打靶载体（PUC57-EGFP-KI-Donor），该打靶载体的主要原件依次为：上游同源臂、SA位点、EGFP基因、PolyA位点、下游同源臂及原核表达的骨架载体。该EGFP定点整合打靶质粒与筛选出的sgRNA共同作用可以对猪基因组的ROSA26位点进行特异的基因改造，然后再结合荧光显微镜与PCR的方法可以很方便的分析外源基因在该sgRNA识别位点整合并表达的可行性。图2为本发明EGFP定点打靶质粒载体示意图。
实施例3
（1）PX330质粒与PUC57-EGFP-KI-Donor质粒的共转染
复苏原代猪的胎儿成纤维细胞，传至F3代时，消化F3代的猪的胎儿成纤维细胞，用DPBS洗2~3遍后，弃上清，加入电转染缓冲液，然后将PX330质粒与PUC57-EGFP-KI-Donor质粒按比例加入到细胞和缓冲液中，用移液器轻轻混匀后，轻轻的将混合液移入电极杯中，将电极杯放到电穿孔仪器上进行电击操作。电击完成后，将电极杯在4℃静置10分钟后，将电极杯中的混合液转入细胞培养皿中。最后将该细胞培养皿置于39℃二氧化碳培养箱中培养。培养12小时后，换液。
实施例4
EGFP细胞克隆的挑取与鉴定及荧光显微镜图片
电转染72h后，通过极限稀释的方法将猪的胎儿成纤维细胞铺到100mm细胞培养皿中，2~3天更换一次细胞培养液。9~10天后待细胞克隆长成后，在荧光显微镜下将发绿光的细胞克隆统一做上标记，然后将这些标记过的克隆挑取入24孔细胞培养板中接着培养。2~3天后，待24孔板中的细胞长至一定得汇合度，对细胞进行传代同时分出部分克隆的细胞，将这些细胞用NP40裂解液裂解后再通过PCR和测序的方法进一步验证定点EGFP的整合事件。图3为本发明EGFP定点整合细胞克隆的荧光显微镜图；图4为本发明EGFP定点整合的细胞克隆的PCR鉴定电泳图。
<110> 吉林大学
<120> 一条能有效编辑猪ROSA26基因的sgRNA及其应用
<160> 6
<210> 1
<211> 20
<212> RNA
<213> 人工序列
<400> 1
AUCGCAGUGGUAGUCAAGAU 20
<210> 2
<211> 20
<212> RNA
<213> 人工序列
<400> 2
AUCUUGAGCAUAGGCCCAAC 20
<210> 3
<211> 20
<212> RNA
<213> 人工序列
<400> 3
UACGGUCAGAUAACUCUCAC 20
<210> 4
<211> 20
<212> DNA
<213> 人工序列
<400> 4
ATCGCAGTGGTAGTCAAGAT 20
<210> 5
<211> 20
<212> DNA
<213> 人工序列
<400> 5
ATCTTGAGCATAGGCCCAAC 20
<210> 6
<211> 20
<212> DNA
<213> 人工序列
<400> 6
TACGGTCAGATAACTCTCAC 20
Claims (6)
1. 能有效编辑猪ROSA26基因的sgRNA的RNA序列，其特征在于：该sgRNA的RNA序列如：SEQ ID 1所示，互补链的RNA序列如：SEQ ID 2所示。
2. 能有效编辑猪ROSA26基因的sgRNA的DNA序列，其特征在于：该sgRNA的DNA序列如SEQ ID 3所示，互补链的DNA序列如：SEQ ID 4所示。
3.如权利要求2所述的能有效编辑猪ROSA26基因的sgRNA的制备方法，包括以下步骤：
1）在基因库中调出猪ROSA26基因的序列，根据PAM序列（NGG）选择用于基因敲除的sgRNA靶向的区域；
2）根据靶位点的序列，设计并合成对应的引物序列；
3）引物退火形成寡聚核苷二聚体（oligoduplex）；
4）将寡核苷酸二聚体连接到相应的质粒载体中，即可获得sgRNA的表达载体。
4.如权利要求1或权利要求2所述的能有效编辑猪ROSA26基因的sgRNA在猪ROSA26位点编辑中的应用；其特征在于：能有效编辑猪ROSA26基因的sgRNA在特异识别和靶向编辑猪ROSA26基因中的应用以及在制备ROSA26基因定点整合转基因猪中的应用。
5.如权利要求4所述的能有效编辑猪ROSA26基因的sgRNA在特异识别和靶向编辑猪ROSA26基因中的应用。
6.如权利要求4所述的能有效编辑猪ROSA26基因的sgRNA在制备ROSA26基因定点整合转基因猪中的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710341126.9A CN106916820B (zh) | 2017-05-16 | 2017-05-16 | 能有效编辑猪ROSA26基因的sgRNA及其应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710341126.9A CN106916820B (zh) | 2017-05-16 | 2017-05-16 | 能有效编辑猪ROSA26基因的sgRNA及其应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106916820A true CN106916820A (zh) | 2017-07-04 |
| CN106916820B CN106916820B (zh) | 2019-09-27 |
Family
ID=59567764
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710341126.9A Active CN106916820B (zh) | 2017-05-16 | 2017-05-16 | 能有效编辑猪ROSA26基因的sgRNA及其应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106916820B (zh) |
Cited By (37)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108018288A (zh) * | 2018-01-16 | 2018-05-11 | 中国人民解放军军事科学院军事医学研究院 | 用于突变rosa26基因的系统及其应用 |
| US9999671B2 (en) | 2013-09-06 | 2018-06-19 | President And Fellows Of Harvard College | Delivery of negatively charged proteins using cationic lipids |
| CN108192928A (zh) * | 2018-01-16 | 2018-06-22 | 中国人民解放军军事科学院军事医学研究院 | 制备rosa26基因突变及富含不饱和脂肪酸动物的方法 |
| CN108642055A (zh) * | 2018-05-17 | 2018-10-12 | 吉林大学 | 能有效编辑猪miR-17-92基因簇的sgRNA |
| US10113163B2 (en) | 2016-08-03 | 2018-10-30 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| CN108949763A (zh) * | 2018-08-15 | 2018-12-07 | 吉林大学 | 能有效抑制猪瘟病毒感染的精确突变LamR基因及应用 |
| CN109055385A (zh) * | 2018-08-21 | 2018-12-21 | 吉林大学 | 一种有效抑制ⅱ型prrsv感染的基因序列及其应用 |
| US10167457B2 (en) | 2015-10-23 | 2019-01-01 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
| CN109112161A (zh) * | 2018-08-17 | 2019-01-01 | 华中农业大学 | 制备rosa26基因突变及肌内脂肪提高的动物的方法 |
| 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 |
| CN110408621A (zh) * | 2018-04-27 | 2019-11-05 | 上海市公共卫生临床中心 | 猕猴rosa26基因及其基因修饰的方法和应用 |
| 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 |
| CN112301000A (zh) * | 2020-11-16 | 2021-02-02 | 吉林大学 | 一种能将应激信号转变为egfp荧光信号的报告细胞系 |
| US11046948B2 (en) | 2013-08-22 | 2021-06-29 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
| CN113073114A (zh) * | 2021-04-07 | 2021-07-06 | 广东海洋大学 | 一种抗非洲猪瘟克隆猪的制备方法 |
| CN113278618A (zh) * | 2020-06-29 | 2021-08-20 | 中国农业科学院北京畜牧兽医研究所 | 特异性识别猪COL1A1基因的gRNA及其生物材料、试剂盒和应用 |
| CN113684209A (zh) * | 2021-07-05 | 2021-11-23 | 吉林大学重庆研究院 | 能有效编辑猪PCBP1基因的sgRNA及其应用 |
| CN113817773A (zh) * | 2021-09-23 | 2021-12-21 | 中国科学院广州生物医药与健康研究院 | 一种在猪上诱导高效表达外源基因的方法 |
| US11268082B2 (en) | 2017-03-23 | 2022-03-08 | President And Fellows Of Harvard College | Nucleobase editors comprising nucleic acid programmable DNA binding proteins |
| CN114231533A (zh) * | 2022-02-24 | 2022-03-25 | 中国医学科学院医学实验动物研究所 | 一种在Rosa26位点定点敲入人源补体调节蛋白小型猪的制备方法 |
| 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 |
| 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 |
| CN118086394A (zh) * | 2024-04-24 | 2024-05-28 | 中国农业科学院北京畜牧兽医研究所 | 猪脂肪组织发育示踪系统及应用 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102851279A (zh) * | 2012-05-04 | 2013-01-02 | 东北农业大学 | 猪rosa26特异性整合位点及其应用 |
| CN105112449A (zh) * | 2015-09-02 | 2015-12-02 | 中国农业大学 | Cd28基因过表达载体及其应用 |
| CN105647968A (zh) * | 2016-02-02 | 2016-06-08 | 浙江大学 | 一种CRISPR/Cas9工作效率快速测试系统及其应用 |
-
2017
- 2017-05-16 CN CN201710341126.9A patent/CN106916820B/zh active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102851279A (zh) * | 2012-05-04 | 2013-01-02 | 东北农业大学 | 猪rosa26特异性整合位点及其应用 |
| CN105112449A (zh) * | 2015-09-02 | 2015-12-02 | 中国农业大学 | Cd28基因过表达载体及其应用 |
| CN105647968A (zh) * | 2016-02-02 | 2016-06-08 | 浙江大学 | 一种CRISPR/Cas9工作效率快速测试系统及其应用 |
Non-Patent Citations (2)
| Title |
|---|
| ROLEN M. QUADROS 等: "Insertion of sequences at the original provirus integration site of mouse ROSA26 locus using the CRISPR_Cas9 system", 《FEBS OPEN BIO》 * |
| ZICONGXIE 等: "Optimization of a CRISPR Cas9 mediated Knock in Strategy at the Porcine Rosa26 Locus in Porcine Foetal Fibroblasts", 《SCIENTIFIC REPORTS》 * |
Cited By (63)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10323236B2 (en) | 2011-07-22 | 2019-06-18 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
| US12006520B2 (en) | 2011-07-22 | 2024-06-11 | 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 |
| US10954548B2 (en) | 2013-08-09 | 2021-03-23 | President And Fellows Of Harvard College | Nuclease profiling system |
| US11920181B2 (en) | 2013-08-09 | 2024-03-05 | 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 |
| US10912833B2 (en) | 2013-09-06 | 2021-02-09 | President And Fellows Of Harvard College | Delivery of negatively charged proteins using cationic lipids |
| US11299755B2 (en) | 2013-09-06 | 2022-04-12 | President And Fellows Of Harvard College | Switchable CAS9 nucleases and uses thereof |
| US10858639B2 (en) | 2013-09-06 | 2020-12-08 | President And Fellows Of Harvard College | CAS9 variants 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 |
| US10682410B2 (en) | 2013-09-06 | 2020-06-16 | President And Fellows Of Harvard College | Delivery system for functional nucleases |
| US10597679B2 (en) | 2013-09-06 | 2020-03-24 | President And Fellows Of Harvard College | Switchable Cas9 nucleases and uses thereof |
| US11124782B2 (en) | 2013-12-12 | 2021-09-21 | President And Fellows Of Harvard College | Cas variants for gene editing |
| US10465176B2 (en) | 2013-12-12 | 2019-11-05 | 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 |
| 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 |
| US12043852B2 (en) | 2015-10-23 | 2024-07-23 | President And Fellows Of Harvard College | Evolved Cas9 proteins for gene editing |
| US11214780B2 (en) | 2015-10-23 | 2022-01-04 | President And Fellows Of Harvard College | 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 |
| US11702651B2 (en) | 2016-08-03 | 2023-07-18 | President And Fellows Of Harvard College | Adenosine 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 |
| 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 |
| US12084663B2 (en) | 2016-08-24 | 2024-09-10 | 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 |
| US11820969B2 (en) | 2016-12-23 | 2023-11-21 | President And Fellows Of Harvard College | Editing of CCR2 receptor gene to protect against HIV infection |
| US10745677B2 (en) | 2016-12-23 | 2020-08-18 | President And Fellows Of Harvard College | Editing of CCR5 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 |
| CN108018288A (zh) * | 2018-01-16 | 2018-05-11 | 中国人民解放军军事科学院军事医学研究院 | 用于突变rosa26基因的系统及其应用 |
| CN108192928A (zh) * | 2018-01-16 | 2018-06-22 | 中国人民解放军军事科学院军事医学研究院 | 制备rosa26基因突变及富含不饱和脂肪酸动物的方法 |
| CN110408621A (zh) * | 2018-04-27 | 2019-11-05 | 上海市公共卫生临床中心 | 猕猴rosa26基因及其基因修饰的方法和应用 |
| CN108642055B (zh) * | 2018-05-17 | 2021-12-03 | 吉林大学 | 能有效编辑猪miR-17-92基因簇的sgRNA |
| CN108642055A (zh) * | 2018-05-17 | 2018-10-12 | 吉林大学 | 能有效编辑猪miR-17-92基因簇的sgRNA |
| CN108949763A (zh) * | 2018-08-15 | 2018-12-07 | 吉林大学 | 能有效抑制猪瘟病毒感染的精确突变LamR基因及应用 |
| CN109112161A (zh) * | 2018-08-17 | 2019-01-01 | 华中农业大学 | 制备rosa26基因突变及肌内脂肪提高的动物的方法 |
| CN109055385A (zh) * | 2018-08-21 | 2018-12-21 | 吉林大学 | 一种有效抑制ⅱ型prrsv感染的基因序列及其应用 |
| CN109055385B (zh) * | 2018-08-21 | 2021-08-27 | 吉林大学 | 一种有效抑制ⅱ型prrsv感染的基因序列及其应用 |
| US11447770B1 (en) | 2019-03-19 | 2022-09-20 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| 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 |
| 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) | 2020-05-08 | 2024-07-09 | The Broad Institute, Inc. | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
| CN113278618A (zh) * | 2020-06-29 | 2021-08-20 | 中国农业科学院北京畜牧兽医研究所 | 特异性识别猪COL1A1基因的gRNA及其生物材料、试剂盒和应用 |
| CN112301000A (zh) * | 2020-11-16 | 2021-02-02 | 吉林大学 | 一种能将应激信号转变为egfp荧光信号的报告细胞系 |
| CN112301000B (zh) * | 2020-11-16 | 2023-04-25 | 吉林大学 | 一种能将应激信号转变为egfp荧光信号的报告细胞系 |
| CN113073114B (zh) * | 2021-04-07 | 2022-11-15 | 广东海洋大学 | 一种抗非洲猪瘟克隆猪的制备方法 |
| CN113073114A (zh) * | 2021-04-07 | 2021-07-06 | 广东海洋大学 | 一种抗非洲猪瘟克隆猪的制备方法 |
| CN113684209A (zh) * | 2021-07-05 | 2021-11-23 | 吉林大学重庆研究院 | 能有效编辑猪PCBP1基因的sgRNA及其应用 |
| CN113817773A (zh) * | 2021-09-23 | 2021-12-21 | 中国科学院广州生物医药与健康研究院 | 一种在猪上诱导高效表达外源基因的方法 |
| CN114231533A (zh) * | 2022-02-24 | 2022-03-25 | 中国医学科学院医学实验动物研究所 | 一种在Rosa26位点定点敲入人源补体调节蛋白小型猪的制备方法 |
| CN114231533B (zh) * | 2022-02-24 | 2022-05-06 | 中国医学科学院医学实验动物研究所 | 一种在Rosa26位点定点敲入人源补体调节蛋白小型猪的制备方法 |
| CN118086394A (zh) * | 2024-04-24 | 2024-05-28 | 中国农业科学院北京畜牧兽医研究所 | 猪脂肪组织发育示踪系统及应用 |
| CN118086394B (zh) * | 2024-04-24 | 2024-07-12 | 中国农业科学院北京畜牧兽医研究所 | 猪脂肪组织发育示踪系统及应用 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106916820B (zh) | 2019-09-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106916820B (zh) | 能有效编辑猪ROSA26基因的sgRNA及其应用 | |
| CN108642055B (zh) | 能有效编辑猪miR-17-92基因簇的sgRNA | |
| CN108285906B (zh) | 一种定点整合外源dna转基因猪的构建方法 | |
| CN104404036B (zh) | 基于CRISPR/Cas9技术的条件性基因敲除方法 | |
| JP2020062044A5 (zh) | ||
| CN107043782B (zh) | 一种基因敲除方法及其sgRNA片段与应用 | |
| CN105671080B (zh) | CRISPR-Cas9系统介导的羊MSTN基因敲除和定点整合外源基因的方法 | |
| CN107988256A (zh) | 人亨廷顿基因敲入用重组载体及其构建方法和在模型猪构建中的应用 | |
| CN105907785A (zh) | 化学合成的crRNA用于CRISPR/Cpf1系统在基因编辑中的应用 | |
| CN106755091A (zh) | 基因敲除载体，mh7a细胞nlrp1基因敲除方法 | |
| CN109136248A (zh) | 多靶点编辑载体及其构建方法和应用 | |
| CN106086031B (zh) | 猪肌抑素基因编辑位点及其应用 | |
| CN111778252B (zh) | 一种靶向敲除SST基因的sgRNA及其CRISPR/Cas9系统和应用 | |
| CN109112159A (zh) | 基于Cas9介导的定点整合FABP4基因和MSTN基因点突变打靶载体及重组细胞 | |
| WO2018030208A1 (ja) | 遺伝子ノックイン細胞の作製方法 | |
| CN109456994B (zh) | 一种厚壳贻贝眼点幼虫的电穿孔转染方法 | |
| CN104293833B (zh) | 一种基于TALEN介导的Sp110巨噬细胞特异打靶载体及重组细胞 | |
| CN106754949B (zh) | 猪肌抑素基因编辑位点864-883及其应用 | |
| CN110643636A (zh) | 一种团头鲂MSTNa&b基因敲除方法与应用 | |
| CN111647604A (zh) | 特异性识别猪COL1A1基因的gRNA及其生物材料、试剂盒和应用 | |
| CN104059877B (zh) | 一种“仿比利时蓝牛”mstn基因型的基因编辑猪的制备方法 | |
| CN103088046A (zh) | ZFNs介导的牛MSTN基因敲除和定点整合外源基因的方法 | |
| CN103952405A (zh) | 一种山羊mstn基因定点修饰系统及其应用 | |
| CN108753814B (zh) | 一种加速物种突变的新育种方法 | |
| CN115807038A (zh) | 一种视网膜分化潜能细胞系CRX-Shen001及其构建方法 |
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 | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20211214 Address after: 401120 building 11, No. 16, Huashan South Road, Dazhulin street, Yubei District, Chongqing Patentee after: Chongqing Research Institute of Jilin University Address before: No.2699, Qianjin Street, Changchun City, Jilin Province, 130011 Patentee before: Jilin University |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20230803 Address after: 618 Liangjiang Avenue, Longxing Town, Yubei District, Chongqing Patentee after: Chongqing Jiyu Technology Co.,Ltd. Address before: 401120 building 11, No. 16, Huashan South Road, Dazhulin street, Yubei District, Chongqing Patentee before: Chongqing Research Institute of Jilin University |
|
| TR01 | Transfer of patent right |