CN106434748A - 一种热激诱导型 Cas9 酶转基因斑马鱼的研制及应用 - Google Patents
一种热激诱导型 Cas9 酶转基因斑马鱼的研制及应用 Download PDFInfo
- Publication number
- CN106434748A CN106434748A CN201610614071.XA CN201610614071A CN106434748A CN 106434748 A CN106434748 A CN 106434748A CN 201610614071 A CN201610614071 A CN 201610614071A CN 106434748 A CN106434748 A CN 106434748A
- Authority
- CN
- China
- Prior art keywords
- cas9
- enzymes
- gene
- cas9 expression
- expression
- 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
- 108091033409 CRISPR Proteins 0.000 title claims abstract description 93
- 241000252212 Danio rerio Species 0.000 title claims abstract description 60
- 230000035939 shock Effects 0.000 title abstract description 6
- 238000011161 development Methods 0.000 title abstract description 4
- 108700019146 Transgenes Proteins 0.000 title abstract 4
- 230000014509 gene expression Effects 0.000 claims abstract description 51
- 241000251468 Actinopterygii Species 0.000 claims abstract description 36
- 101710163595 Chaperone protein DnaK Proteins 0.000 claims abstract description 7
- 101710178376 Heat shock 70 kDa protein Proteins 0.000 claims abstract description 7
- 101710152018 Heat shock cognate 70 kDa protein Proteins 0.000 claims abstract description 7
- 238000011160 research Methods 0.000 claims abstract description 6
- 102000004190 Enzymes Human genes 0.000 claims description 52
- 108090000790 Enzymes Proteins 0.000 claims description 52
- 230000009261 transgenic effect Effects 0.000 claims description 30
- 238000000034 method Methods 0.000 claims description 27
- 108090000623 proteins and genes Proteins 0.000 claims description 27
- 238000002347 injection Methods 0.000 claims description 18
- 239000007924 injection Substances 0.000 claims description 18
- 210000004602 germ cell Anatomy 0.000 claims description 16
- 238000011534 incubation Methods 0.000 claims description 13
- 238000012216 screening Methods 0.000 claims description 13
- 230000001939 inductive effect Effects 0.000 claims description 11
- 238000012360 testing method Methods 0.000 claims description 9
- 239000003550 marker Substances 0.000 claims description 7
- 102000004169 proteins and genes Human genes 0.000 claims description 7
- 241001465754 Metazoa Species 0.000 claims description 5
- 102000002322 Egg Proteins Human genes 0.000 claims description 4
- 108010000912 Egg Proteins Proteins 0.000 claims description 4
- 238000009395 breeding Methods 0.000 claims description 4
- 210000004681 ovum Anatomy 0.000 claims description 4
- 108091027544 Subgenomic mRNA Proteins 0.000 claims description 3
- 230000004720 fertilization Effects 0.000 claims description 2
- 238000009396 hybridization Methods 0.000 claims description 2
- 241000448255 Congiopodus peruvianus Species 0.000 claims 1
- 101150110867 MC4R gene Proteins 0.000 abstract description 12
- 239000013604 expression vector Substances 0.000 abstract description 6
- 238000010356 CRISPR-Cas9 genome editing Methods 0.000 abstract description 4
- 238000003209 gene knockout Methods 0.000 abstract description 3
- 238000003198 gene knock in Methods 0.000 abstract description 2
- 238000010362 genome editing Methods 0.000 abstract description 2
- 230000004048 modification Effects 0.000 abstract description 2
- 238000012986 modification Methods 0.000 abstract description 2
- 108020004999 messenger RNA Proteins 0.000 abstract 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 11
- 238000005516 engineering process Methods 0.000 description 9
- 108020005004 Guide RNA Proteins 0.000 description 8
- 239000012634 fragment Substances 0.000 description 8
- 238000000520 microinjection Methods 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 235000013601 eggs Nutrition 0.000 description 6
- 230000000366 juvenile effect Effects 0.000 description 6
- 108020004414 DNA Proteins 0.000 description 5
- 239000011543 agarose gel Substances 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 4
- 108010077544 Chromatin Proteins 0.000 description 4
- 210000003483 chromatin Anatomy 0.000 description 4
- 230000035772 mutation Effects 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 239000013600 plasmid vector Substances 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 238000013518 transcription Methods 0.000 description 4
- 230000035897 transcription Effects 0.000 description 4
- 241000595940 Notostraca Species 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 210000002969 egg yolk Anatomy 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 238000003780 insertion Methods 0.000 description 3
- 230000037431 insertion Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 230000001568 sexual effect Effects 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 239000013598 vector Substances 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 241000252210 Cyprinidae Species 0.000 description 2
- BELBBZDIHDAJOR-UHFFFAOYSA-N Phenolsulfonephthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2S(=O)(=O)O1 BELBBZDIHDAJOR-UHFFFAOYSA-N 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 230000004087 circulation Effects 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 108010087236 cobra venom endonuclease Proteins 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 230000008034 disappearance Effects 0.000 description 2
- 238000010363 gene targeting Methods 0.000 description 2
- 210000001161 mammalian embryo Anatomy 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229960003531 phenolsulfonphthalein Drugs 0.000 description 2
- 238000003259 recombinant expression Methods 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 210000000145 septum pellucidum Anatomy 0.000 description 2
- 239000013589 supplement Substances 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 102000005869 Activating Transcription Factors Human genes 0.000 description 1
- 108010005254 Activating Transcription Factors Proteins 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 108091006027 G proteins Proteins 0.000 description 1
- 102000030782 GTP binding Human genes 0.000 description 1
- 108091000058 GTP-Binding Proteins 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 1
- 108091027305 Heteroduplex Proteins 0.000 description 1
- 101000978418 Homo sapiens Melanocortin receptor 4 Proteins 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 102100023724 Melanocortin receptor 4 Human genes 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 208000008589 Obesity Diseases 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 230000027326 copulation Effects 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000011559 double-strand break repair via nonhomologous end joining Effects 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 231100000221 frame shift mutation induction Toxicity 0.000 description 1
- 230000037433 frameshift Effects 0.000 description 1
- 239000005090 green fluorescent protein Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000013632 homeostatic process Effects 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 210000003016 hypothalamus Anatomy 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 235000020824 obesity Nutrition 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000009182 swimming Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 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
- C12N15/63—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
-
- 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
- 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
- C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
- C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
-
- 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
- C12N15/89—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microinjection
-
- 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
- A01K2227/40—Fish
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/60—Vectors containing traps for, e.g. exons, promoters
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/90—Vectors containing a transposable element
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Veterinary Medicine (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Environmental Sciences (AREA)
- Plant Pathology (AREA)
- Cell Biology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Animal Behavior & Ethology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Endocrinology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明属于生物技术领域，具体涉及一种热激诱导型Cas9酶转基因斑马鱼的研制及应用。本发明首先提供了一种Cas9酶表达载体，所述载体利用热激诱导型启动子HSP70来驱动下游Cas9基因表达。采用所述Cas9酶表达载体和Tol2 mRNA共同注射入野生型斑马鱼单细胞受精卵，选育得到热激诱导型Cas9酶转基因斑马鱼，成功实现了在斑马鱼中进行CRISPR‑Cas9***的基因编辑研究，并首次实现了MC4R基因在此转基因斑马鱼中的敲除。所述Cas9酶表达载体同样适用于其他鱼类的热激诱导型基因敲除、基因敲入、基因表达修饰等应用。
Description
技术领域
本发明属于生物技术领域，具体涉及一种热激诱导型Cas9酶转基因斑马鱼的研制及应用。
背景技术
斑马鱼(Danio rerio)是属于辐鳍亚纲(Actinopterygii)鲤科(Cyprinidae)短担尼鱼属(Danio)的一种硬骨鱼，因其体侧具有像斑马一样纵向的暗蓝与银色相间的条纹而得名。斑马鱼与人类基因有着87％的高度同源性，这意味着以斑马鱼为实验动物，得到的结果大多数情况下可以适用于人体，因此其作为模式生物的优势很突出。其幼鱼具有繁殖快，卵及幼鱼全身透明的特点，常被用于水环境污染物的监测，相关疾病模型的制作等等。其中转基因技术的成熟也给斑马鱼的应用带来的更大的前景。转基因斑马鱼的制备通常包括以下几个步骤：首先构建好转基因重组表达载体,利用经典的显微注射技术将带有目的基因或荧光基因的重组表达载体导入斑马鱼单细胞受精卵中，使外源目的基因或荧光基因能够整合到斑马鱼胚胎染色体上,并使目的基因或荧光标记基因表达，通过PCR或者荧光显微镜来检测外源基因是否整合和表达成功,从而筛选和鉴定转基因后代或转基因的嵌合体。
CRISPR-Cas9***成功被改造为第三代人工核酸内切酶，与锌指核酸内切酶(ZFN)和类转录激活因子效应物核酸酶(TALEN)一样可用于各种复杂基因组的编辑。其主要功能部件包括识别靶位点的gRNA和Cas9蛋白，gRNA负责识别靶位点，Cas9蛋白行使剪切功能，再利用机体自身的非同源末端修复(NHEJ)，在修复的过程中给基因组引入突变。目前该技术已成功应用于人类细胞，斑马鱼和小鼠以及细菌的基因组精确修饰，修饰类型包括***缺失突变、基因定点敲入、大片段的缺失。由于其突变效率高、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分子工具。MC4R属于G-蛋白偶联受体的家族，是下丘脑腹内侧核分泌的一类肽类物质，在调节能量动态平衡和肥胖症发生上具有重要作用。通过十几年的研究，MC4R基因在哺乳动物中的功能研究已经比较透彻。然而，因为鱼类中缺乏有效的基因编辑手段，鱼类的MC4R基因功能的研究才刚刚起步。
发明内容
为了克服现有技术中所存在的问题，本发明的目的在于提供一种热激诱导型Cas9酶转基因斑马鱼的制作方法及应用。
为了实现上述目的以及其他相关目的，本发明采用如下技术方案：
本发明的第一方面，提供一种Cas9酶表达载体，包括：Cas9酶表达元件以及位于所述Cas9酶表达元件两端的转座子Tol2，所述Cas9酶表达元件包括热激诱导型启动子HSP70和Cas9酶基因编码序列。
优选地，所述热激诱导型启动子HSP70含有如SEQ ID NO.7所示的序列。
优选地，所述Cas9酶基因编码序列含有如SEQ ID NO.8所示的序列。
优选地，位于所述Cas9酶表达元件两端的转座子Tol2包括位于所述Cas9酶表达元件5’端的转座子Tol2和位于所述Cas9酶表达元件3’端的转座子Tol2。
优选地，位于所述Cas9酶表达元件5’端的转座子Tol2含有如SEQ ID NO.9所示的序列。
优选地，位于所述Cas9酶表达元件3’端的转座子Tol2含有如SEQ ID NO.10所示的序列。
优选地，所述Cas9酶表达元件还包括荧光标记蛋白序列。
优选地，所述荧光标记蛋白序列通过2A序列与Cas9酶基因编码序列连接。
进一步优选地，所述2A序列含有如SEQ ID NO.11所示的序列。
优选地，所述荧光标记蛋白为绿色荧光蛋白eGFP。
优选地，所述Cas9酶表达载体含有如SEQ ID NO.12所示的序列。
本发明的第二方面，提供前述Cas9酶表达载体在培育Cas9酶转基因动物中的用途。
本发明的第三方面，提供一种Cas9酶转基因斑马鱼培养方法，包括：将前述Cas9酶表达载体和Tol2mRNA共同注射入野生型斑马鱼单细胞受精卵，选育得到Cas9酶转基因斑马鱼。
优选地，所述Tol2mRNA含有如SEQ ID NO.13所示的序列。
优选地，所述注射采用显微注射法。
优选地，所述选育方法包括：对注射后的受精卵进行热激诱导。
进一步优选地，所述选育方法具体包括：注射后的受精卵于28℃条件下培育24h，然后在37℃条件下孵育1h，筛选带有荧光标记的鱼卵孵育培养至成鱼。
优选地，所述选育方法还包括纯系Cas9酶转基因斑马鱼的筛选，
首先选择受精卵中表达荧光的嵌合体P0与野生型斑马鱼进行杂交产生杂合体F1代，筛选后代中发荧光的个体；将所述F1代中发荧光的单个个体和野生型进行测交得到杂合体F2代，筛选后代中发荧光个体；将杂合体F2代中发荧光的雌鱼和雄鱼自交，得到F3代；将所得F3代单个个体继续与野生型测交并统计F4鱼卵孵育后发荧光情况；若F4鱼卵全部为发荧光，则确定其亲本F3为纯系Cas9酶转基因斑马鱼。
本发明的第四方面，提供一种由前述方法获得的Cas9酶转基因斑马鱼。
本发明的第五方面，提供了所述Cas9酶转基因斑马鱼用于鱼类基因功能的研究中的用途。
所述鱼类基因包括但不限于MC4R基因。
本发明的第六方面，提供了一种研究鱼类基因功能的方法，包括：将针对鱼类基因的sgRNA注射到纯系Cas9酶转基因斑马鱼的单细胞受精卵中，以敲除鱼类基因。
本发明的第七方面，提供一种CRISPR-Cas9基因敲除试剂盒，所述试剂盒包括：前述Cas9酶表达载体和sgRNA。
与现有技术相比，本发明具有如下有益效果：
本发明首先构建了一种Cas9酶表达载体，所述载体利用热激诱导型启动子HSP70来驱动下游Cas9基因表达。采用所述Cas9酶表达载体和Tol2mRNA共同注射入野生型斑马鱼单细胞受精卵，选育得到热激诱导型Cas9酶转基因斑马鱼，成功实现了在斑马鱼中进行CRISPR-Cas9***的基因编辑研究，并首次实现了MC4R基因在此转基因斑马鱼中的敲除。所述Cas9酶表达载体同样适用于其他鱼类的热激诱导型基因敲除、基因敲入、基因表达修饰等应用。
附图说明
图1是本发明的Cas9表达载体pTol2-HSP70-Cas9-2A-eGFP图谱。
图2是质粒pTol2-LoxP-CMV-eCFP图谱。
图3是质粒pISceI-HSP70-Cas9-2A-eGFP图谱。
图4是MC4R基因***缺失测序图，图4中所涉及各序列分别如SEQ ID NO.14～17所示。
图5是MC4R基因T7E1检测结果电泳图。
具体实施方式
在进一步描述本发明具体实施方式之前，应理解，本发明的保护范围不局限于下述特定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围。下列实施例中未注明具体条件的试验方法，通常按照常规条件，或者按照各制造商所建议的条件。
当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
除非另外说明，本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。这些技术在现有文献中已有完善说明，具体可参见Sambrook等MOLECULAR CLONING：A LABORATORY MANUAL，Second edition，Cold Spring HarborLaboratory Press，1989and Third edition，2001；Ausubel等，CURRENT PROTOCOLS INMOLECULAR BIOLOGY，John Wiley&Sons，New York，1987and periodic updates；theseries METHODS IN ENZYMOLOGY，Academic Press，San Diego；Wolffe，CHROMATINSTRUCTURE AND FUNCTION，Third edition，Academic Press，San Diego，1998；METHODS INENZYMOLOGY，Vol.304，Chromatin(P.M.Wassarman and A.P.Wolffe，eds.)，AcademicPress，San Diego，1999；和METHODS IN MOLECULAR BIOLOGY，Vol.119，ChromatinProtocols(P.B.Becker，ed.)Humana Press，Totowa，1999等。
实施例1获得Cas9酶表达载体pTol2-HSP70-Cas9-2A-eGFP
利用BamHI与SalI双酶切质粒载体pTol2-CMV-eGFP(如图2所示)，酶切体系：10xGreen Buffer 5ul、质粒载体pTol2-CMV-eGFP 8.8ul(0.284ug/ul)、BamHI 2.5ul、SalI2.5ul、加水补足50ul。通过琼脂糖凝胶电泳回收7993bp的片段，得到骨架片段pTol2。
通过BamHI与SalI双酶切质粒载体pISceI-HSP70-Cas9-2A-eGFP(如图3所示)，酶切体系：10x Green Buffer 5ul、质粒载体pISceI-HSP70-Cas9-2A-eGFP 7.7ul(0.321ug/ul)、BamHI2.5ul、SalI 2.5ul、加水补足50ul。酶切完成后，将酶切产物进行琼脂糖凝胶电泳回收6969bp片段，得到***片段HSP70-Cas9-2A-eGFP。
将骨架片段pTol2和***片段HSP70-Cas9-2A-eGFP两片段进行连接，连接反应体系：HSP70-Cas9-2A-eGFP 3ul、pTol2 2ul、10xBuffer 2ul、T4连接酶0.2ul、灭菌水12.8ul。用T4DNA连接酶16℃连接过夜。
取连接产物10ul转化90ul大肠杆菌感受态细胞DH5α，用移液器轻轻吸打混匀，冰上孵育30min后，42℃热击90s，立即置于冰上2min，加入37℃LB培养基900ul，于37℃180rpm摇床活化1h，活化菌液5000rpm离心3min富集，吸掉900ul上清液，100ul菌液混匀后涂于氨苄抗性培养皿上，于37℃温箱中倒置培养过夜。随机挑选10个单菌落利用PCR方法进行检测，将阳性克隆扩大培养于6ml LB液体培养基，37℃250rpm培养过夜，收集菌液并提取质粒。将得到质粒测序验证，测序结果与预期一致，得到Cas9酶表达载体pTol2-HSP70-Cas9-2A-eGFP。
如图1所示，Cas9酶表达载体pTol2-HSP70-Cas9-2A-eGFP，包括：Cas9酶表达元件以及位于所述Cas9酶表达元件两端的转座子Tol2，所述Cas9酶表达元件包括热激诱导型启动子HSP70、和Cas9酶基因编码序列。
其中，热激诱导型启动子HSP70含有如SEQ ID NO.7所示的序列。如SEQ ID NO.7所示的序列具体详见序列表部分。
其中，Cas9酶基因编码序列含有如SEQ ID NO.8所示的序列。如SEQ ID NO.8所示的序列具体详见序列表部分。
位于所述Cas9酶表达元件两端的转座子Tol2包括位于所述Cas9酶表达元件5’端的转座子Tol2和位于所述Cas9酶表达元件3’端的转座子Tol2。
其中，位于所述Cas9酶表达元件5’端的转座子Tol2含有如SEQ ID NO.9所示的序列。如SEQ ID NO.9所示的序列具体详见序列表部分。
位于所述Cas9酶表达元件3’端的转座子Tol2含有如SEQ ID NO.10所示的序列。如SEQ ID NO.10所示的序列具体详见序列表部分。
所述Cas9酶表达元件还包括荧光标记蛋白序列。所述荧光标记蛋白序列通过2A序列与Cas9酶基因编码序列连接。所述2A序列含有如SEQ ID NO.11所示的序列。如SEQ IDNO.11所示的序列具体详见序列表部分。
Cas9酶表达载体pTol2-HSP70-Cas9-2A-eGFP含有如SEQ ID NO.12所示的序列。如SEQ ID NO.12所示的序列具体详见序列表部分。
实施例2将Cas9酶表达载体注射入斑马鱼受精卵
(1)斑马鱼受精卵的获取
受精前将雌雄亲鱼分开按1:1-2比例放入产卵盒中进行隔离培养。产卵盒置于26℃-29℃恒温环境中进行黑暗过夜培养，光周期为白昼14h、黑暗10h。培养结束，抽离隔板并将底部有栅栏的内层倾斜放置在外层交配盒上，并放置在恒温环境中，得到斑马鱼受精卵。
(2)将Cas9酶表达载体注射入斑马鱼受精卵
利用显微注射仪并参照显微注射法将实施例1构建好的Cas9酶表达载体pTol2-HSP70-Cas9-2A-eGFP(如图1所示)和Tol2mRNA共同注射入获得的斑马鱼单细胞受精卵。注射体系为10ul，其中Cas9表达载体50ng/ul，Tol2mRNA 1ul、酚红2ul，加水补足10ul。取0.6ul注射液注入毛细管中，将其装入注射仪中，按次序注射入斑马鱼受精卵的动物极。整个操作过程在45min内完成，以确保在细胞的第一次卵裂之前注射完成。
其中，所述Tol2mRNA含有如SEQ ID NO.13所示的序列。如SEQ ID NO.13所示的序列具体详见序列表部分。
(3)荧光筛选和子代培育
注射后的受精卵培养于28℃，24h后在37℃恒温水浴锅孵育1h，然后利用体视显微镜进行观察，筛选带有绿色荧光的鱼卵进行孵育培养至成鱼。筛选后第1d-4d，所述斑马鱼胚胎在26℃-29℃恒温环境培育，获得幼鱼；第5d-9d幼鱼喂食卵黄水，每日喂食2次；第10d-16d，幼鱼2喂食丰年虫与滤网磨碎的卵黄水，具体方法是先向饲养容器中投入少量丰年虫，25min-35min后再加入滤网磨碎的卵黄水，每日喂食2次；第17d以后，幼鱼喂食丰年虫，每日喂食2次，每日更换一次养鱼水。
(4)筛选稳定遗传的纯系Cas9酶转基因斑马鱼
首先剪取2月龄P0嵌合体个体尾鳍提取基因组DNA，PCR方法检测Cas9基因序列，PCR程序：94℃5min；94℃5s，55℃15s，72℃20s共35个循环，72℃5min。筛选PCR结果为阳性的P0斑马鱼与野生型斑马鱼进行杂交，将鱼卵置于37℃水浴锅孵育1h，然后在体视荧光显微镜下观察荧光，挑选出有绿色荧光的卵培育至性成熟，得到杂合体F1代。PCR检测F1代，筛选PCR结果为阳性的斑马鱼培育至性成熟，将此杂合体F1中的单个个体和野生型进行测交，二月龄后PCR筛选出阳性斑马鱼，培养至性成熟，此为杂合体F2。由于杂合体F2来源于同一亲本(杂合体F1和野生型)，因此所有F2杂合子基因型一致。将杂合体F2中的雌鱼与雄鱼自交，其后代F3中，有1/4概率得到含Cas9转基因斑马鱼的纯合子F3。二月龄后PCR剪尾检测筛选所有的纯合子和杂合子，为验证其中的纯合子F3，将筛选出的F3继续与野生型测交并PCR检测F4鱼卵DNA，若F4鱼卵全部为PCR阳性，鱼卵37℃孵育后然后体视荧光显微镜观察全有绿色荧光，则可以确定其亲本F3为纯系。
实施例3斑马鱼MC4R基因的敲除
(1)MC4R基因打靶位点的确定和gRNA的制备
设计MC4R基因打靶位点，根据网站(http://zifit.partners.org/favicon.ico)给出的结果选择合适的打靶序列，本发明选择的打靶序列如SEQ ID NO.1所示，具体为：GGGGGTGTTTGTGGTGTGCT。
本发明采用PCR的方法扩增出gRNA的转录模板。首先根据选定的打靶序列设计引物，上游引物序列如SEQ ID NO.2，具体为：
TAATACGACTCACTATAGGGGGTGTTTGTGGTGTGCTGTTTTAGAGCTAGAAATAGC；下游引物序列如SEQ ID NO.3所示，具体为：AGCACCGACTCGGTGCCAC。
以含有gRNA骨架的质粒为模板，骨架序列如SEQ ID NO.4所示，具体为：AGCTTGAAATTAATACGACTCACTATAGGGAGTCTTCAGATCTAACACACAACTCGAGGAAGACATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTC GGTGCG。
PCR扩增程序为：98℃5min；98℃10s，58℃30s，72℃20s共35个循环，72℃5min。PCR产物经1％琼脂糖凝胶电泳检测后，通过PCR产物纯化试剂盒回收产物。以纯化后的产物为模板进行体外转录，转录体系为20ul，其中纯化后的产物600ng，T7转录酶1ul，反应缓冲液2ul，dNTP混合物1ul，加无菌水补足20ul。37℃孵育3h。转录产物经1％琼脂糖凝胶电泳检测后，通过RNA纯化试剂盒纯化回收，获得gRNA，在-80℃冰箱保存。
(2)体外显微注射与敲除验证
准备受精卵：挑选性成熟的稳定遗传的纯系Cas9转基因斑马鱼(详见实施例2)，2条雌鱼1条雄鱼，放入产卵盒，中间***透明隔板将雌雄鱼分开。将产卵盒置于26℃-29℃恒温室中黑暗过夜饲养，光周期为白昼14h，黑暗10h。显微注射。次日上午，拔开透明隔板，保持安静的环境，让雌雄鱼自行追逐产卵。产卵后，立即收取受精卵，通过显微注射仪进行注射。注射体系为：gRNA 30ng/ul，酚红0.5ul，无菌水补足10ul。注射部位为受精卵的动物极，整个操作过程在45min内完成，以确保在细胞的第一次卵裂之前注射完成。敲除验证。注射后的受精卵在28℃培养。24h后，对照组取10颗，两个实验组每组取10颗斑马鱼受精卵，分别提取DNA，在gRNA打靶位点两侧选择500bp左右的序列设计引物，引物序列如SEQ ID NO.5和SEQ ID NO.6所示，
SEQ ID NO.5：GACCGCTACATCACAATCT；
SEQ ID NO.6：TTGGCTTCTGAAGGCATAT。
以此引物对斑马鱼DNA进行扩增，98℃5min；98℃10s，52℃20s，72℃20s共35个循环，72℃5min。T7核酸内切酶I(T7E1)具有识别并切割不完全配对DNA、异源双链DNA特性，因此用该酶进行突变验证，反应体系为：PCR产物8.5ul，缓冲液1ul，混合均匀后，PCR仪中退火。退火程序为：95℃5min，94℃2sec，-0.1℃/cycle,200times，75℃1sec，-0.1℃/cycle,600times，16℃2min。退火完成后，加入0.5ulT7E1酶，37℃孵育30min。孵育完成后通过2％琼脂糖凝胶电泳检测是否敲除成功，结果如图5所示。随后亚克隆到测序载体中送样测序并同野生型基因序列进行比较验证，确认靶基因的有效敲除。剪尾检测成功的鱼(F0)与野生型杂交，得到F1代杂合体，通过测序与野生型比较，分别得到缺失5个、12个和8个碱基的基因型，引起基因的移码突变。结果如图4所示。
Cas9转基因斑马鱼能实现对MC4R基因的有效敲除，说明Cas9转基因斑马鱼构建成功。与现代技术相比，本发明的有益效果是：(1)方法实施过程简单易控；(2)方法能获得Cas9转基因斑马鱼；(3)所得斑马鱼品系能为基因的敲除提供方便；(4)实现了对MC4R基因的有效敲除。
以上所述，仅为本发明的较佳实施例，并非对本发明任何形式上和实质上的限制，应当指出，对于本技术领域的普通技术人员，在不脱离本发明方法的前提下，还将可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。凡熟悉本专业的技术人员，在不脱离本发明的精神和范围的情况下，当可利用以上所揭示的技术内容而做出的些许更动、修饰与演变的等同变化，均为本发明的等效实施例；同时，凡依据本发明的实质技术对上述实施例所作的任何等同变化的更动、修饰与演变，均仍属于本发明的技术方案的范围内。
Claims (10)
1.一种Cas9酶表达载体，包括：Cas9酶表达元件以及位于所述Cas9酶表达元件两端的转座子Tol2，所述Cas9酶表达元件包括热激诱导型启动子HSP70和Cas9酶基因编码序列。
2.根据权利要求1所述的Cas9酶表达载体，其特征在于，位于所述Cas9酶表达元件两端的转座子Tol2包括位于所述Cas9酶表达元件5’端的转座子Tol2和位于所述Cas9酶表达元件3’端的转座子Tol2。
3.根据权利要求1所述的Cas9酶表达载体，其特征在于，所述Cas9酶表达元件还包括荧光标记蛋白序列，所述荧光标记蛋白序列通过2A序列与Cas9酶基因编码序列连接。
4.根据权利要求1所述的Cas9酶表达载体，其特征在于，所述Cas9酶表达载体含有如SEQ ID NO.12所示的序列。
5.如权利要求1～4任一权利要求所述Cas9酶表达载体在培育Cas9酶转基因动物中的用途。
6.一种Cas9酶转基因斑马鱼培养方法，包括：将如权利要求1～4任一权利要求所述Cas9酶表达载体和Tol2mRNA共同注射入野生型斑马鱼单细胞受精卵，选育得到Cas9酶转基因斑马鱼。
7.根据权利要求6所述的方法，其特征在于，所述选育方法包括：对注射后的受精卵进行热激诱导，具体包括：注射后的受精卵于28℃条件下培育24h，然后在37℃条件下孵育1h，筛选带有荧光标记的鱼卵孵育培养至成鱼。
8.根据权利要求6所述的方法，其特征在于，所述选育方法还包括纯系Cas9酶转基因斑马鱼的筛选，首先选择受精卵中表达荧光的嵌合体P0与野生型斑马鱼进行杂交产生杂合体F1代，筛选后代中发荧光个体；将所述F1代中发荧光的单个个体和野生型进行测交得到杂合体F2代，筛选后代中发荧光个体；将杂合体F2代中发荧光的雌鱼和雄鱼自交，得到F3代；将所得F3代单个个体继续与野生型测交并统计F4鱼卵孵育后发荧光情况，若F4鱼卵全部为发荧光，则确定其亲本F3为纯系Cas9酶转基因斑马鱼。
9.由权利要求6～8任一权利要求所述方法获得的Cas9酶转基因斑马鱼用于鱼类基因功能的研究中的用途。
10.一种研究鱼类基因功能的方法，包括：将针对鱼类基因的sgRNA注射到由权利要求6～8任一权利要求所述方法获得的Cas9酶转基因斑马鱼的单细胞受精卵中，以敲除斑马鱼对应的基因。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610614071.XA CN106434748A (zh) | 2016-07-29 | 2016-07-29 | 一种热激诱导型 Cas9 酶转基因斑马鱼的研制及应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610614071.XA CN106434748A (zh) | 2016-07-29 | 2016-07-29 | 一种热激诱导型 Cas9 酶转基因斑马鱼的研制及应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106434748A true CN106434748A (zh) | 2017-02-22 |
Family
ID=58184745
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610614071.XA Pending CN106434748A (zh) | 2016-07-29 | 2016-07-29 | 一种热激诱导型 Cas9 酶转基因斑马鱼的研制及应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106434748A (zh) |
Cited By (33)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107043782A (zh) * | 2017-04-10 | 2017-08-15 | 西南大学 | 一种基因敲除方法及其sgRNA片段与应用 |
| US9999671B2 (en) | 2013-09-06 | 2018-06-19 | President And Fellows Of Harvard College | Delivery of negatively charged proteins using cationic lipids |
| CN108192927A (zh) * | 2017-12-29 | 2018-06-22 | 上海海洋大学 | 一种瓯江彩鲤的基因编辑与过表达操作方法 |
| CN108251452A (zh) * | 2018-01-17 | 2018-07-06 | 扬州大学 | 一种表达Cas9基因的转基因斑马鱼及其构建方法和应用 |
| CN108531510A (zh) * | 2018-05-16 | 2018-09-14 | 华南理工大学 | 一种转基因斑马鱼在制备慢性粒细胞白血病的动物模型中的应用 |
| US10077453B2 (en) | 2014-07-30 | 2018-09-18 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
| US10113163B2 (en) | 2016-08-03 | 2018-10-30 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| CN108929879A (zh) * | 2017-05-24 | 2018-12-04 | 南方医科大学 | 一种抗细菌感染鱼类模型及其制备方法和用途 |
| US10167457B2 (en) | 2015-10-23 | 2019-01-01 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
| 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 |
| CN110747186A (zh) * | 2019-11-07 | 2020-02-04 | 北京大学 | 在植物中高效生成不携带转基因元件的突变体的CRISPR/Cas9***和方法 |
| US10597679B2 (en) | 2013-09-06 | 2020-03-24 | President And Fellows Of Harvard College | Switchable Cas9 nucleases and uses thereof |
| CN111149730A (zh) * | 2019-12-25 | 2020-05-15 | 湖南师范大学 | 一种快速培育多能干细胞荧光标记斑马鱼纯合个体的方法 |
| CN111386038A (zh) * | 2017-09-28 | 2020-07-07 | 国立大学法人京都大学 | 鱼类及鱼类的生产方法 |
| 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 |
| CN112955540A (zh) * | 2018-08-30 | 2021-06-11 | 因思科瑞普特公司 | 在自动化模块和仪器中对经核酸酶经编辑的序列的改进的检测 |
| US11046948B2 (en) | 2013-08-22 | 2021-06-29 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
| 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 |
| US11447770B1 (en) | 2019-03-19 | 2022-09-20 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| US11542496B2 (en) | 2017-03-10 | 2023-01-03 | President And Fellows Of Harvard College | Cytosine to guanine base editor |
| US11542509B2 (en) | 2016-08-24 | 2023-01-03 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
| 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 (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105274141A (zh) * | 2015-11-20 | 2016-01-27 | 中国科学院水生生物研究所 | 一种用于原始生殖细胞靶向突变的转基因载体及制备方法和用途 |
-
2016
- 2016-07-29 CN CN201610614071.XA patent/CN106434748A/zh active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105274141A (zh) * | 2015-11-20 | 2016-01-27 | 中国科学院水生生物研究所 | 一种用于原始生殖细胞靶向突变的转基因载体及制备方法和用途 |
Non-Patent Citations (4)
| Title |
|---|
| JULIEN ABLAIN等: "A CRISPR Cas9 Vector System for Tissue-Specific Gene Disruption in Zebrafish", 《DEVELOPMENTAL CELL》 * |
| YUKIKO KIMURA等: "Efficient generation of knock-in transgenic zebrafish carrying reporter driver genes by CRISPR Cas9-mediated genome engineering", 《SCIENTIFIC REPORTS》 * |
| 刘勇等: "GAL4/fUAS***在果蝇Tis11基因RNA干扰中的应用", 《生命科学研究》 * |
| 杜久林等: "CRISPR/Cas9技术在斑马鱼基因修饰中的应用", 《生命科学》 * |
Cited By (54)
| 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 |
| US10954548B2 (en) | 2013-08-09 | 2021-03-23 | President And Fellows Of Harvard College | Nuclease profiling system |
| 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 |
| 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 |
| US10912833B2 (en) | 2013-09-06 | 2021-02-09 | President And Fellows Of Harvard College | Delivery of negatively charged proteins using cationic lipids |
| US9999671B2 (en) | 2013-09-06 | 2018-06-19 | 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 |
| US10858639B2 (en) | 2013-09-06 | 2020-12-08 | President And Fellows Of Harvard College | CAS9 variants and uses thereof |
| US10682410B2 (en) | 2013-09-06 | 2020-06-16 | President And Fellows Of Harvard College | Delivery system for functional nucleases |
| 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 |
| US11124782B2 (en) | 2013-12-12 | 2021-09-21 | President And Fellows Of Harvard College | Cas variants for gene editing |
| US10077453B2 (en) | 2014-07-30 | 2018-09-18 | 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 |
| US10704062B2 (en) | 2014-07-30 | 2020-07-07 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
| US11214780B2 (en) | 2015-10-23 | 2022-01-04 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
| US10167457B2 (en) | 2015-10-23 | 2019-01-01 | President And Fellows Of Harvard College | 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 |
| 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 |
| 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 |
| CN107043782A (zh) * | 2017-04-10 | 2017-08-15 | 西南大学 | 一种基因敲除方法及其sgRNA片段与应用 |
| 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 |
| CN108929879B (zh) * | 2017-05-24 | 2021-08-20 | 南方医科大学 | 一种抗细菌感染鱼类模型及其制备方法和用途 |
| CN108929879A (zh) * | 2017-05-24 | 2018-12-04 | 南方医科大学 | 一种抗细菌感染鱼类模型及其制备方法和用途 |
| 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) |
| US11932884B2 (en) | 2017-08-30 | 2024-03-19 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
| US11319532B2 (en) | 2017-08-30 | 2022-05-03 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
| CN111386038A (zh) * | 2017-09-28 | 2020-07-07 | 国立大学法人京都大学 | 鱼类及鱼类的生产方法 |
| US11795443B2 (en) | 2017-10-16 | 2023-10-24 | The Broad Institute, Inc. | Uses of adenosine base editors |
| CN108192927A (zh) * | 2017-12-29 | 2018-06-22 | 上海海洋大学 | 一种瓯江彩鲤的基因编辑与过表达操作方法 |
| CN108251452A (zh) * | 2018-01-17 | 2018-07-06 | 扬州大学 | 一种表达Cas9基因的转基因斑马鱼及其构建方法和应用 |
| CN108531510A (zh) * | 2018-05-16 | 2018-09-14 | 华南理工大学 | 一种转基因斑马鱼在制备慢性粒细胞白血病的动物模型中的应用 |
| CN112955540A (zh) * | 2018-08-30 | 2021-06-11 | 因思科瑞普特公司 | 在自动化模块和仪器中对经核酸酶经编辑的序列的改进的检测 |
| US11795452B2 (en) | 2019-03-19 | 2023-10-24 | 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 |
| US11447770B1 (en) | 2019-03-19 | 2022-09-20 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| CN110747186A (zh) * | 2019-11-07 | 2020-02-04 | 北京大学 | 在植物中高效生成不携带转基因元件的突变体的CRISPR/Cas9***和方法 |
| CN110747186B (zh) * | 2019-11-07 | 2021-07-30 | 北京大学 | 在植物中高效生成不携带转基因元件的突变体的CRISPR/Cas9***和方法 |
| CN111149730A (zh) * | 2019-12-25 | 2020-05-15 | 湖南师范大学 | 一种快速培育多能干细胞荧光标记斑马鱼纯合个体的方法 |
| CN111149730B (zh) * | 2019-12-25 | 2021-04-13 | 湖南师范大学 | 一种快速培育多能干细胞荧光标记斑马鱼纯合个体的方法 |
| 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 |
| 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 |
|---|---|---|
| CN106434748A (zh) | 一种热激诱导型 Cas9 酶转基因斑马鱼的研制及应用 | |
| Dahm et al. | Learning from small fry: the zebrafish as a genetic model organism for aquaculture fish species | |
| CN104195177B (zh) | 一种显著提高鱼类基因组编辑效率的方法 | |
| CN106191114A (zh) | 利用CRISPR‑Cas9***敲除鱼类MC4R基因的育种方法 | |
| CN106222204B (zh) | 一种黄鳝基因编辑的方法 | |
| CN102653756B (zh) | 一种定向改造动物基因组特定基因的方法及其应用 | |
| CN108103108A (zh) | Cebpa基因缺失斑马鱼突变体的制备及其应用 | |
| CN105274140A (zh) | 肌细胞特异表达mCherry荧光蛋白斑马鱼家系构建方法 | |
| CN105274141A (zh) | 一种用于原始生殖细胞靶向突变的转基因载体及制备方法和用途 | |
| CN114438132A (zh) | 尼罗罗非鱼mstnb纯合敲除系的建立方法及以此获得的快速生长品系 | |
| Kontarakis et al. | Transgenesis in non-model organisms: the case of Parhyale | |
| CN105671084A (zh) | 一种基于基因组编辑的海水鲆鲽鱼类种质构建方法及应用 | |
| WO2004053119A1 (en) | Fluorescent proteins from aquatic species | |
| CN114058618B (zh) | 谷氨酸脱氢酶作为靶点在防治害虫中的应用 | |
| CN116515907A (zh) | 一种在草鱼中双gRNA位点敲除runx2b基因的方法及应用 | |
| CN110592122B (zh) | 斑马鱼naalad2基因启动子及其应用 | |
| CN105063088B (zh) | 一种生态安全型转基因荧光观赏鱼的研制方法 | |
| CN110402893A (zh) | 一种Nrf2基因缺失斑马鱼突变体的制备及其应用 | |
| CN113462692B (zh) | 一种适于翘嘴鲌受精卵Mstn基因敲除的方法及应用 | |
| CN103937824B (zh) | 一种转溶菌酶基因罗非鱼的制备方法 | |
| CN112111527B (zh) | 一种基于基因编辑的获得翅膀缺失的黑水虻的方法 | |
| CN102911937B (zh) | 肌肉增强子序列及其应用 | |
| CN105409882B (zh) | 一种筛选携带缺失型Tgf2转座子金鱼亲本以繁育高正品率后代的方法 | |
| CN107164385A (zh) | 一种羊的长绒毛及其制备方法 | |
| CN106434952A (zh) | 一种鸽性别分子鉴定方法及所用引物对 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170222 |
|
| RJ01 | Rejection of invention patent application after publication |