WO2022134014A1 - 一种靶向AHRR基因的sgRNA组合及其应用 - Google Patents

一种靶向AHRR基因的sgRNA组合及其应用 Download PDF

Info

Publication number
WO2022134014A1
WO2022134014A1 PCT/CN2020/139467 CN2020139467W WO2022134014A1 WO 2022134014 A1 WO2022134014 A1 WO 2022134014A1 CN 2020139467 W CN2020139467 W CN 2020139467W WO 2022134014 A1 WO2022134014 A1 WO 2022134014A1
Authority
WO
WIPO (PCT)
Prior art keywords
ahrr
seq
gene
mice
ahrr gene
Prior art date
Application number
PCT/CN2020/139467
Other languages
English (en)
French (fr)
Inventor
钟诗龙
汪静
朱茜
陈爽
吴圆圆
汤雅男
Original Assignee
百迈康生物医药科技(广州)有限公司
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by 百迈康生物医药科技(广州)有限公司 filed Critical 百迈康生物医药科技(广州)有限公司
Publication of WO2022134014A1 publication Critical patent/WO2022134014A1/zh

Links

Images

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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • 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
    • C12N15/873—Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0604—Whole embryos; Culture medium therefor
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • 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
    • A01K2227/105—Murine
    • 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
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0362—Animal model for lipid/glucose metabolism, e.g. obesity, type-2 diabetes
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • 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
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
    • Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
    • Y02P60/87—Re-use of by-products of food processing for fodder production

Definitions

  • the present application belongs to the technical field of gene editing, and in particular relates to a combination of sgRNAs targeting AHRR gene and applications thereof.
  • Obesity is a complex metabolic system disease, which can cause related metabolic disorders and diseases, such as cardiovascular and cerebrovascular diseases, lipid metabolism disorders, insulin resistance, type II diabetes and metabolic syndrome.
  • cardiovascular and cerebrovascular diseases such as cardiovascular and cerebrovascular diseases, lipid metabolism disorders, insulin resistance, type II diabetes and metabolic syndrome.
  • simple obesity is the main disease, which is characterized by no obvious changes in the morphology and function of the nervous and endocrine systems, but accompanied by disorders of lipid and glucose metabolism.
  • CN107156505A discloses a rat obesity model feed, including 25-30 parts of corn, 15-20 parts of wheat, 20-25 parts of soybean meal, 5-12 parts of wheat gluten protein, 3-10 parts of fish meal, and 10-25 parts of lard. 2.9-9 parts of yeast hydrolyzate, 0.8-1.2 parts of stone powder, 1-2 parts of calcium hydrogen phosphate, 0.5-1 part of complex minerals, 0.1-0.5 parts of multivitamins, 0.1-0.2 parts of methionine and 0.1-1 part of lysine 0.2 servings.
  • the feed is rich in nutrients and has good palatability, and by regulating the state of microecological flora in the body, it can promote the absorption of nutrients such as protein by rats, so that the body weight of rats can be increased. Success rate of obesity model construction. However, the weight gain of rats by eating is greatly affected by seasons, environments and individual differences, and the repeatability is poor.
  • the construction methods of obese animal models generally suffer from cumbersome modeling conditions, long cycle, poor stability of obesity phenotype, lack of unified modeling standards, and difficulty in meeting the research needs of different pathogenesis of obesity.
  • the construction method of obesity model with short, stable and reproducible obesity phenotype has become an urgent problem to be solved.
  • the present application provides an sgRNA combination targeting AHRR gene and its application.
  • the sgRNA combination targeting AHRR gene has strong specificity and low off-target rate, and can achieve large fragment knockout of AHRR gene in combination with Cas9 nuclease mRNA , the mutation efficiency is high, the genotype can exist stably, and the application value is high.
  • the present application provides a combination of sgRNA targeting AHRR gene
  • the combination of sgRNA targeting AHRR gene includes sgRNA1 and/or sgRNA2
  • the sgRNA1 includes the nucleic acid sequence shown in SEQ ID No.1, so
  • the sgRNA2 includes the nucleic acid sequence shown in SEQ ID No.2.
  • SEQ ID No. 1 GGAGATNTCGCCAAGTNCATGGG;
  • SEQ ID No. 2 GGCACACTANCGTNATTATTGG;
  • N represents any one of A, T, C or G.
  • sgRNA1 and sgRNA2 specifically target the intron sequence in the AHRR gene, wherein sgRNA1 targets intron 5 of the AHRR gene, and sgRNA2 targets intron 8 of the AHRR gene. Off-target rate, high specificity.
  • the present application provides an AHRR gene editing system, the AHRR gene editing system comprising the sgRNA combination targeting the AHRR gene described in the first aspect.
  • the AHRR gene editing system only edits the AHRR gene, and the off-target rate is low, which can avoid non-specific gene editing; the targeting AHRR
  • the sgRNA combination of the gene is far away in the genome, which can achieve large fragment knockout of the AHRR gene, and the gene editing efficiency is extremely high.
  • the AHRR gene editing system further comprises Cas9 nuclease and/or Cas9 nuclease mRNA, preferably Cas9 nuclease mRNA.
  • the present application provides a recombinant cell containing the sgRNA combination targeting the AHRR gene described in the first aspect.
  • the recombinant cell can transmit the mutated genotype to the progeny cells by cell division, thereby realizing the stability and heritability of the gene mutation, and reducing the screening efficiency.
  • the workload and the application value are more extensive.
  • the recombinant cell contains the AHRR gene editing system described in the second aspect.
  • the recombinant cell is a fertilized egg cell with the AHRR gene knocked out in the genome after being edited by the AHRR gene editing system described in the second aspect.
  • the application provides a construction method of the recombinant cell described in the third aspect, the construction method comprising:
  • the AHRR gene editing system described in the second aspect is introduced into the nucleus of a fertilized egg cell to obtain the recombinant cell.
  • the AHRR gene editing system is directly introduced into the nucleus of the fertilized egg cell, avoiding the disadvantage that the mRNA of sgRNA1, sgRNA2 and Cas9 nucleases can only edit the genome when the cell divides and the nuclear membrane disappears, and improves the The efficiency of gene editing; the selection of fertilized egg cells for the construction of recombinant cells can reduce the probability of chimeras, ensure that sense mutations can be passed on to offspring, and reduce the workload of later screening.
  • the introducing comprises microinjection.
  • the application provides a method for constructing an obese animal model, and the method for constructing the obese animal model includes:
  • the recombinant cells are transplanted into a surrogate mother, and the obtained F0 generation is mated with the wild type to obtain an F1 generation heterozygote;
  • the F1 generation heterozygote is selfed, and the obtained F2 generation homozygote is the obese animal model.
  • the construction method of the obese animal model is simple to operate, has a high success rate, and can be easily mastered by those skilled in the relevant fields, and can be completed in a relevant laboratory; after screening for homozygotes, only the homozygotes need to be continuously
  • the self-crossing can make the mutated genotype stably inherited without re-screening, which is very convenient, and also creates convenient conditions for related research.
  • the preparation method of the AHRR gene editing system comprises:
  • the Cas9 nuclease mRNA is combined with the sgRNA targeting the AHRR gene described in the first aspect to obtain the AHRR gene editing system.
  • the mammal includes any one of mice, rats, rabbits, pigs or cynomolgus monkeys.
  • the mammal is a mouse, preferably a C57BL/6 mouse.
  • the F0 generation, F1 generation heterozygotes and F2 generation homozygotes are identified by PCR amplification and sequencing.
  • the sequence of the forward primer amplified by the PCR comprises the nucleic acid sequence shown in SEQ ID No.3;
  • the sequence of the PCR-amplified reverse primer includes the nucleic acid sequence shown in one of SEQ ID No. 4-5.
  • SEQ ID No. 3 CAGAGCTTATCCACAAAGTCACC
  • SEQ ID No. 4 GCACCTCCAAAGAATATACAAGCAG;
  • SEQ ID No. 5 CTGCAAAGGCTGACATGAAGG.
  • the construction method of the obese animal model described in this application specifically includes the following steps:
  • Ovulation induction was performed on C57BL/6 mice, and fertilized eggs were cultured after in vitro fertilization;
  • the present application provides the sgRNA combination targeting the AHRR gene described in the first aspect, the AHRR gene editing system described in the second aspect, the recombinant cell described in the third aspect, and the recombinant cell described in the fourth aspect.
  • the sgRNA combination and gene editing system targeting the AHRR gene have high specificity and low off-target rate; the recombinant cells and the construction method of the recombinant cells improve the efficiency of gene editing and reduce the workload of screening;
  • the construction method of the above-mentioned obese animal model has mature technology, simple operation, stable genotype after mutation, strong repeatability, and is less affected by external environmental factors, and is suitable for relevant mechanism research and drug screening. The value is extremely broad.
  • the sgRNA combination targeting the AHRR gene and the AHRR gene editing system described in this application have good specificity and low off-target rate, and can achieve large fragment knockout of the AHRR gene, and the gene editing efficiency is high; Introduced into the nucleus of the fertilized egg cell, the constructed recombinant cell can transmit the mutated genotype to the progeny cells through cell division, which realizes the stability and heritability of gene mutation and reduces the workload of screening;
  • Figure 2A is a picture of agarose gel electrophoresis of the amplified product of SEQ ID No.3/SEQ ID No.4 of wild-type mice in Example 5 of the application, lane 1 is DS10000 DNA marker, and lane 2 is wild-type mouse The amplification product of the AHRR gene;
  • lane 1 is the DL2000 DNA marker
  • lane 2 is the wild-type mouse The amplification product of the AHRR gene
  • 3A is a picture of agarose gel electrophoresis of the amplified product of SEQ ID No.3/SEQ ID No.4 of the AHRR gene knockout homozygous mouse in Example 5 of the application, and lanes 1 to 4 are the AHRR gene knockout pure
  • the amplification product of the AHRR gene of zygotic mice, lane 5 is the DL2000 DNA marker;
  • 3B is a picture of agarose gel electrophoresis of the amplified product of SEQ ID No.3/SEQ ID No.5 of the AHRR gene knockout homozygous mouse in Example 5 of the application, and lanes 1 to 4 are the AHRR gene knockout pure
  • the amplification product of the AHRR gene of zygotic mice, lane 5 is the DL2000 DNA marker;
  • 4B is a picture of statistical results of body weights of AHRR gene knockout homozygous mice and wild-type mice in Example 6 of the application;
  • FIG. 4C is a picture of the statistical results of the brown fat weight of AHRR gene knockout homozygous mice and wild-type mice in Example 6 of the present application.
  • the in vitro transcription kit was purchased from Inovogen Tech;
  • PCR amplification reagents were purchased from TOYOBO Company;
  • Genomic DNA extraction kit was purchased from TaKaRa company;
  • Anhydrous ethanol was purchased from Guangzhou Chemical Reagent Factory;
  • mice were from Saiye Model Biology Research Center (Taicang) Co., Ltd.;
  • Mouse feed was purchased from Wuhan Wanqianjiaxing Biotechnology Co., Ltd.;
  • mice were reared as follows:
  • This embodiment provides an sgRNA combination targeting the AHRR gene
  • the sgRNA combination targeting the AHRR gene includes sgRNA1 and sgRNA2
  • the sgRNA1 includes the nucleic acid sequence shown in SEQ ID No. 1
  • the sgRNA2 includes SEQ ID No. 1 .2 the nucleic acid sequence shown.
  • SEQ ID No. 1 GGAGATNTCGCCAAGTNCATGGG;
  • SEQ ID No. 2 GGCACATCTANCGTNATTATTGG;
  • N represents any one of A, T, C or G.
  • the sgRNA1 specifically targets intron 5 of the AHRR gene
  • the sgRNA2 specifically targets intron 8 of the AHRR gene, with good specificity, low off-target rate, and wide application value.
  • This embodiment provides an AHRR gene editing system
  • the AHRR gene editing system includes sgRNA targeting AHRR gene in combination of sgRNA1 and sgRNA2 and mRNA of Cas9 nuclease.
  • the AHRR gene editing system has the ability to knock out the AHRR gene in the genome. Through the cooperation of the sgRNA combination, the off-target rate and the probability of non-specific editing can be reduced, and it has the ability to knock out large fragments, and the editing efficiency is higher; Cas9 is selected.
  • the mRNA of nuclease is less toxic to cells, and the edited individuals are more likely to survive, reducing the difficulty of screening.
  • This example provides a recombinant cell, which is a fertilized egg cell of a C57BL/6 mouse whose genome has been mutated in the AHRR gene after being edited by the AHRR gene editing system in Example 2.
  • the recombinant cells are constructed by the following methods:
  • mRNA of Cas9 nuclease gene is transcribed in vitro, mixed with sgRNA targeting AHRR gene in combination to obtain an AHRR gene editing system;
  • Ovulation induction was performed on C57BL/6 mice, and fertilized eggs were cultured after in vitro fertilization;
  • sgRNA1, sgRNA2 and Cas9 nucleases By directly injecting the mRNAs of sgRNA1, sgRNA2 and Cas9 nucleases into the nucleus of fertilized egg cells, the efficiency of gene editing is improved; fertilized eggs are selected to construct recombinant cells, and sense mutations can be inherited through cell division, reducing the occurrence of chimeras. Probability, the edited genotype can be passed on to offspring, reducing the workload of screening.
  • Example 3 the recombinant cells constructed in Example 3 were transplanted into the body of a surrogate mother mouse to construct an obese animal model.
  • the specific steps are as follows:
  • Example 3 The recombinant cells obtained in Example 3 are cultured in vitro and transferred to the body of a surrogate mother mouse;
  • the PCR amplification system is shown in Table 1:
  • sequence of the forward primer is the nucleic acid sequence shown in SEQ ID No.3
  • sequence of the reverse primer is the nucleic acid sequence shown in one of SEQ ID No.4-5.
  • SEQ ID No. 3 CAGAGCTTATCCACAAAGTCACC
  • SEQ ID No. 4 GCACCTCCAAAGAATATACAAGCAG;
  • SEQ ID No. 5 CTGCAAAGGCTGACATGAAGG.
  • Pre-denaturation incubate at 95°C for 3min;
  • Cyclic amplification 95°C incubation for 15s; 55°C incubation for 15s; 68°C incubation for 420s;
  • the number of cycles is 35 times.
  • Amplification products were stored at 4°C.
  • Pre-denaturation incubate at 95°C for 3min;
  • Cyclic amplification incubation at 95°C for 15s; incubation at 55°C for 15s; incubation at 68°C for 30s;
  • the number of cycles is 35 times.
  • Amplification products were stored at 4°C.
  • the size of the amplified product of SEQ ID No.3/SEQ ID No.4 of wild-type mouse is 6425bp. If part of the gene fragment is deleted after editing, the size of the amplified product fragment will be reduced; therefore, the size of the amplified product of wild-type mouse is 6425bp.
  • the size of the amplified product of ID No.3/SEQ ID No.4 is 6425bp, the amplified product of SEQ ID No.3/SEQ ID No.4 of knockout homozygous mice is less than 6425bp, and the amplified product of SEQ ID No.4 of heterozygous mice is less than 6425bp.
  • ID No.3/SEQ ID No.4 amplification product contains both bands; and
  • the size of the amplification product of SEQ ID No.3/SEQ ID No.5 of wild-type mice is 400bp. If part of the gene fragment is deleted after editing, there is no corresponding gene knockout homozygous mouse for which part of the gene fragment is deleted. Amplification product; therefore, the size of the SEQ ID No.3/SEQ ID No.5 amplification product of the wild-type mouse is 400bp, and the gene knockout homozygous mouse uses SEQ ID No.3/SEQ ID No.5 without amplification
  • the amplified product, the size of the SEQ ID No.3/SEQ ID No.5 amplified product of heterozygous mice is 400bp, but the band brightness is weaker than that of wild-type mice.
  • a homozygous C57BL/6 mouse with AHRR gene knockout was successfully constructed, that is, the above-mentioned obesity animal model. Sequencing confirmed that the knockout fragment was the 74278006-74283456 region on chromosome 13 with a length of 5451 bp, and a 7 bp fragment was inserted after the knocked out fragment.
  • the specific knockout sequence is shown in SEQ ID No.6.
  • SEQ ID No. 7 CCTAACG.
  • Example 4 the AHRR gene knockout homozygous C57BL/6 mice screened in Example 4 were verified by PCR.
  • the amplification system and method were the same as those in Example 4, and wild-type mice were used for the same detection as a control. .
  • the size of the amplified product of SEQ ID No.3/SEQ ID No.4 of wild-type mouse is 6425bp. If part of the gene fragment is deleted after editing, the size of the amplified product fragment will be reduced; therefore, the size of the amplified product of wild-type mouse is 6425bp.
  • the size of the amplified product of ID No.3/SEQ ID No.4 is 6425bp, and the size of the amplified product of SEQ ID No.3/SEQ ID No.4 of knockout homozygous mice is 981bp;
  • the size of the amplified product of SEQ ID No.3/SEQ ID No.5 of wild-type mice is 400 bp. If part of the gene fragment is deleted after editing, there is no corresponding gene knockout homozygous mouse for which part of the gene fragment is deleted. Amplification product; therefore, the size of the SEQ ID No.3/SEQ ID No.5 amplification product of wild-type mice is 400bp, and the gene knockout homozygous mice use SEQ ID No.3/SEQ ID No.5 without amplification increase product.
  • the agarose gel electrophoresis picture of the SEQ ID No.3/SEQ ID No.4 amplification product of wild-type mice is shown in Figure 2A, and the amplification products of SEQ ID No.3/SEQ ID No.5 of wild-type mice are shown in Figure 2A.
  • the agarose gel electrophoresis picture of the product is shown in Figure 2B;
  • the agarose gel electrophoresis picture of the amplified product of SEQ ID No.3/SEQ ID No.4 of AHRR gene knockout homozygous mice is shown in Figure 3A
  • AHRR gene knockout homozygous mice are SEQ ID No.3
  • the agarose gel electrophoresis picture of the amplified product of /SEQ ID No.5 is shown in Figure 3B.
  • the size of the amplification product of SEQ ID No.3/SEQ ID No.4 of wild-type mice is 6425bp, while the size of SEQ ID No.
  • the size of the amplified product of .3/SEQ ID No.4 is 981bp; the size of the amplified product of SEQ ID No.3/SEQ ID No.5 of wild-type mice is 400bp, while the size of the homozygous knockout mouse uses SEQ ID No.3/SEQ ID No.5.
  • ID No.3/SEQ ID No.5 has no amplification product, and the experimental results are in line with expectations, indicating that the obtained homozygous mutant mice are indeed AHRR gene knockout homozygous mice.
  • the AHRR gene knockout homozygous C57BL/6 mice screened in Example 4 were used to identify the obesity phenotype.
  • Wild-type C57BL/6 mice and AHRR knockout homozygous C57BL/6 mice were reared under the same conditions, and after 8 months, the body weight and fat weight of the mice were statistically analyzed.
  • Figure 4A is a comparison picture of the appearance of AHRR gene knockout homozygous mice and wild-type mice. It can be clearly seen from the picture that the mutant homozygous mice with AHRR gene knockout are larger in size and have more body fat. more obese.
  • the present application forms an AHRR gene editing system by synthesizing a combination of sgRNA targeting the AHRR gene and the mRNA of Cas9 nuclease; the AHRR gene editing system is microinjected into the nucleus of C57BL/6 mouse fertilized eggs
  • the recombinant cells were successfully constructed in 1999; the recombinant cells were then transferred into the body of surrogate mother mice, and the born mice were identified and screened, and the AHRR gene knockout homozygous mice were successfully screened.
  • the appearance, body weight and fat weight of the gene mutation homozygous mice are significantly higher than those of the wild-type mice, and can be used for the screening of hypoglycemic and lipid-lowering related drugs; the construction method of the obese animal model is mature in technology and extremely efficient. High, short time, and reduce the workload of screening, has a wide range of application value.
  • the present application illustrates the detailed method of the present application through the above-mentioned embodiments, but the present application is not limited to the above-mentioned detailed method, which does not mean that the present application must rely on the above-mentioned detailed method for implementation.
  • Those skilled in the art should understand that any improvement to the application, the equivalent replacement of each raw material of the application product, the addition of auxiliary components, the selection of specific methods, etc., all fall within the protection scope and disclosure scope of the application.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Developmental Biology & Embryology (AREA)
  • Plant Pathology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Reproductive Health (AREA)
  • Gynecology & Obstetrics (AREA)
  • Toxicology (AREA)
  • Endocrinology (AREA)
  • Pathology (AREA)
  • Rheumatology (AREA)
  • Veterinary Medicine (AREA)
  • Diabetes (AREA)
  • Urology & Nephrology (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Cell Biology (AREA)
  • Public Health (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

提供了一种靶向AHRR基因的sgRNA组合及其应用，所述靶向AHRR基因的sgRNA组合包括sgRNA1和/或sgRNA2，所述sgRNA1包括SEQ ID No.1所示的核酸序列，所述sgRNA2包括SEQ ID No.2所示的核酸序列。还提供了一种AHRR基因编辑系统、一种重组细胞及其构建方法和一种肥胖动物模型的构建方法，通过基因编辑及筛选，得到的敲除了AHRR基因的纯合子小鼠具有典型的肥胖体征，体型更大、体重更重，体内脂肪含量也更高，可以用于降糖、降脂相关药物的筛选中，应用价值极其广泛。

Description

一种靶向AHRR基因的sgRNA组合及其应用 技术领域
本申请属于基因编辑技术领域，尤其涉及一种靶向AHRR基因的sgRNA组合及其应用。
背景技术
随着人们饮食结构和生活方式的改变，肥胖已成为当下全球共同面临的重要健康问题。肥胖是一种复杂的代谢系统疾病，可以引起相关的代谢紊乱及病症，如心脑血管疾病、脂代谢紊乱、胰岛素抵抗、II型糖尿病和代谢综合征等。临床中以单纯性肥胖为主，其特征为没有明显神经、内分泌系统形态及功能的改变，但伴有脂质、糖代谢过程障碍。
研究肥胖的发展机制、探索其治疗方法是指导制定肥胖预防和治疗策略的前提。肥胖的发病原因有多种，其机制的研究涉及许多生物学途径，因此，建立基于不同生物学机制的肥胖动物模型的方法，对阐明肥胖发病机制有重要的临床意义。目前，常见的动物肥胖模型的构建方法包括通过食物(高脂饲料)、内分泌(去卵巢或注射胰岛素)、下丘脑(谷氨酸钠或金硫葡萄糖)和遗传等因素进行诱导。CN107156505A公开了一种大鼠肥胖模型饲料，包括玉米25～30份、小麦15～20份、黄豆粕20～25份、小麦面筋蛋白5～12份、鱼粉3～10份、猪油10～25份、酵母水解物2.9～9份、石粉0.8～1.2份、磷酸氢钙1～2份、复合矿物质0.5～1份、复合维生素0.1～0.5份、蛋氨酸0.1～0.2份和赖氨酸0.1～0.2份。通过对原料进行合理的配比，该饲料营养物质丰富，适口性较好，且通过调节体内微生态菌群的状态，促进大鼠对蛋白等营养物质的吸收，使大鼠体重增加，提高了肥胖模型构建的成功率。然而，通过进食来使大鼠的体重增加，受季节、环境及个体差异的影响较大，重复性较差，肥胖表型也并非稳定存在且无法遗传给后代，局限性较大。
目前，肥胖动物模型的构建方法普遍存在造模条件繁琐、周期长、肥胖表型稳定性差、缺乏统一的造模标准且难以满足肥胖不同发病机制的研究需求，如何提供一种构建条件简单、周期短、肥胖表型稳定且可复制性强的肥胖模型的构建方法，已成为亟待解决的问题。
发明内容
本申请提供了一种靶向AHRR基因的sgRNA组合及其应用，所述靶向AHRR基因的sgRNA组合特异性强，脱靶率低，配合Cas9核酸酶的mRNA，可实现AHRR基因的大片段敲除，突变效率高，基因型可稳定存在，应用价值高。
第一方面，本申请提供了一种靶向AHRR基因的sgRNA组合，所述靶向AHRR基因的sgRNA组合包括sgRNA1和/或sgRNA2，所述sgRNA1包括SEQ ID No.1所示的核酸序列，所述sgRNA2包括SEQ ID No.2所示的核酸序列。
SEQ ID No.1：GGAGATNTCGCCAAGTNCATGGG；
SEQ ID No.2：GGCACACTANCGTNATTATTGG；
其中，N代表A、T、C或G中的任意一种。
本申请中，sgRNA1和sgRNA2特异性靶向AHRR基因中的内含子序列，其中sgRNA1靶向AHRR基因的内含子5，sgRNA2靶向AHRR基因的内含子8，二者相互配合，可以降低脱靶率，特异性较高。
第二方面，本申请提供了一种AHRR基因编辑系统，所述AHRR基因编辑系统包括第一方面所述的靶向AHRR基因的sgRNA组合。
本申请中，通过将靶向AHRR基因的sgRNA组合结合使用，配合Cas9，所述AHRR基因编辑系统仅对AHRR基因进行编辑，脱靶率较低，可以避免非特异性的基因编辑；所述靶向AHRR基因的sgRNA组合在基因组中的距离较远，可以实现对AHRR基因的大片段敲除，基因编辑效率极高。
优选地，所述AHRR基因编辑系统还包括Cas9核酸酶和/或Cas9核酸酶的mRNA，优选为Cas9核酸酶的mRNA。
第三方面，本申请提供了一种重组细胞，所述重组细胞含有第一方面所述的靶向AHRR基因的sgRNA组合。
本申请中，通过对细胞的AHRR基因进行编辑，所述重组细胞可以通过细胞分裂的方式将突变的基因型传递给子代细胞，实现了基因突变的稳定性及可遗传性，降低了筛选的工作量，应用价值更为广泛。
优选地，所述重组细胞含有第二方面所述的AHRR基因编辑系统。
优选地，所述重组细胞为经过第二方面所述的AHRR基因编辑系统编辑后，基因组中敲除了AHRR基因的受精卵细胞。
第四方面，本申请提供了一种第三方面所述的重组细胞的构建方法，所述构建方法包括：
将第二方面所述的AHRR基因编辑系统导入受精卵细胞的细胞核内，得到所述重组细胞。
本申请中，将所述的AHRR基因编辑系统直接导入受精卵细胞的细胞核内，避免了sgRNA1、sgRNA2和Cas9核酸酶的mRNA只能在细胞分裂、核膜消失时对基因组进行编辑的弊端，提高了基因编辑的效率；选取受精卵细胞用于构建重组细胞，可以降低嵌合体出现的概率，保证有义突变可以传递给子代，降低后期筛选的工作量。
优选地，所述导入包括显微注射。
第五方面，本申请提供了一种肥胖动物模型的构建方法，所述肥胖动物模型的构建方法包括：
将第二方面所述的AHRR基因编辑系统导入哺乳动物受精卵细胞的细胞核内，得到重组细胞；
将所述重组细胞移植入代孕母体，获得的F0代与野生型交配，得到F1代杂合子；以及
将所述F1代杂合子自交，获得的F2代纯合子即为所述肥胖动物模型。
本申请中，所述肥胖动物模型的构建方法操作简单，成功率高，相关领域的技术人员很容易掌握，可以在相关的实验室中完成；筛选得到纯合子后，仅需对纯合子进行持续的自交，即可使突变的基因型稳定遗传，无需再次筛选，十分方便，也为相关的研究创造了便利的条件。
优选地，所述AHRR基因编辑系统的制备方法包括：
对Cas9核酸酶基因进行体外转录，得到所述Cas9核酸酶的mRNA；以及
将所述Cas9核酸酶的mRNA与第一方面所述的靶向AHRR基因的sgRNA组合混合，得到所述AHRR基因编辑系统。
优选地，所述哺乳动物包括小鼠、大鼠、兔、猪或食蟹猴中的任意一种。
优选地，所述哺乳动物为小鼠，优选为C57BL/6小鼠。
优选地，所述F0代、F1代杂合子和F2代纯合子利用PCR扩增和测序进行鉴定。
优选地，所述PCR扩增的正向引物的序列包括SEQ ID No.3所示的核酸序 列；
优选地，所述PCR扩增的反向引物的序列包括SEQ ID No.4～5之一所示的核酸序列。
SEQ ID No.3：CAGAGCTTATCCACAAAGTCACC；
SEQ ID No.4：GCACCTCCAAAGAATATACAAGCAG；
SEQ ID No.5：CTGCAAAGGCTGACATGAAGG。
作为优选技术方案，本申请所述肥胖动物模型的构建方法，具体包括以下步骤：
(1)体外转录Cas9核酸酶基因的mRNA，与靶向AHRR基因的sgRNA组合混合，得到AHRR基因编辑系统；
对C57BL/6小鼠进行促排卵，体外受精后，培养受精卵；
(2)将所述AHRR基因编辑系统显微注射入C57BL/6小鼠受精卵细胞的细胞核内，得到重组细胞；
(3)对所述重组细胞进行体外培养，并转移到代孕母鼠体内；
(4)提取出生小鼠尾部组织的DNA，使用SEQ ID No.3～5进行PCR扩增，并对扩增产物进行测序鉴定，挑选AHRR基因缺失的小鼠作为F0代；
(5)将所述F0代与野生型C57BL/6小鼠交配，得到F1代杂合子小鼠；
(6)将所得F1代杂合子小鼠自交，使用SEQ ID No.3～5进行PCR扩增，并对扩增产物测序鉴定，筛选出F2代AHRR基因敲除的纯合子C57BL/6小鼠，即所述的肥胖动物模型。
第六方面，本申请提供了第一方面所述的靶向AHRR基因的sgRNA组合、第二方面所述的AHRR基因编辑系统、第三方面所述的重组细胞、第四方面所述的重组细胞的构建方法或第五方面所述的肥胖动物模型的构建方法中的任意一种或至少两种的组合在筛选降脂降糖药物和/或评价降脂降糖药物效果中的应用。
本申请中，所述靶向AHRR基因的sgRNA组合和基因编辑系统特异性高，脱靶率低；所述重组细胞和重组细胞的构建方法提高了基因编辑的效率，降低了筛选的工作量；所述肥胖动物模型的构建方法技术成熟，操作简单，突变后的基因型可稳定存在，重复性较强，且受外界环境因素的影响较小，适合应用于相关的机制研究及药物筛选中，应用价值极其广泛。
相比于现有技术，本申请具有如下有益效果：
(1)本申请所述靶向AHRR基因的sgRNA组合和AHRR基因编辑系统特异性好，脱靶率低，可以实现对AHRR基因的大片段敲除，基因编辑效率高；通过将AHRR基因编辑系统直接导入受精卵细胞的细胞核内，构建的重组细胞可以通过细胞分裂的方式将突变的基因型传递给子代细胞，实现了基因突变的稳定性及可遗传性，降低了筛选的工作量；
(2)本申请通过敲除AHRR基因，建立了一种肥胖动物模型的构建方法，通过CRISPR/Cas9系统对AHRR基因进行编辑，实现了大片段的敲除，提高了基因编辑的效率；所采用的技术十分成熟，操作简单，成功率高，具有较好的重复性；构建得到的AHRR基因敲除的纯合子小鼠缺失了基因组中5451bp的片段，体型、体重和脂肪重量明显高于野生型小鼠，具有明显的肥胖表型，且受外界环境因素的影响较小，表型稳定，耗时较短，在相关药物筛选及科学研究中具有广泛的应用价值。
附图说明
图1为本申请实施例4中基因敲除小鼠的筛选的原理图；
图2A为本申请实施例5中野生型小鼠的SEQ ID No.3/SEQ ID No.4扩增产物的琼脂糖凝胶电泳图片，泳道1为DS10000 DNA marker，泳道2为野生型小鼠的AHRR基因的扩增产物；
图2B为本申请实施例5中野生型小鼠的SEQ ID No.3/SEQ ID No.5扩增产物的琼脂糖凝胶电泳图片，泳道1为DL2000 DNA marker，泳道2为野生型小鼠的AHRR基因的扩增产物；
图3A为本申请实施例5中AHRR基因敲除纯合子小鼠的SEQ ID No.3/SEQ ID No.4扩增产物的琼脂糖凝胶电泳图片，泳道1～4为AHRR基因敲除纯合子小鼠的AHRR基因的扩增产物，泳道5为DL2000 DNA marker；
图3B为本申请实施例5中AHRR基因敲除纯合子小鼠的SEQ ID No.3/SEQ ID No.5扩增产物的琼脂糖凝胶电泳图片，泳道1～4为AHRR基因敲除纯合子小鼠的AHRR基因的扩增产物，泳道5为DL2000 DNA marker；
图4A为本申请实施例6中AHRR基因敲除纯合子小鼠和野生型小鼠的外观对比图片(比例尺＝4cm)；
图4B为本申请实施例6中AHRR基因敲除纯合子小鼠和野生型小鼠的体重的统计结果图片；
图4C为本申请实施例6中AHRR基因敲除纯合子小鼠和野生型小鼠的棕色脂肪重量的统计结果图片。
具体实施方式
为进一步阐述本申请所采取的技术手段及其效果，以下结合实施例和附图对本申请作进一步地说明。可以理解的是，此处所描述的具体实施方式仅仅用于解释本申请，而非对本申请的限定。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。
材料：
体外转录试剂盒购自英茂盛业生物科技有限公司(Inovogen Tech)；
PCR扩增试剂购自TOYOBO公司；
基因组DNA提取试剂盒购自TaKaRa公司；
无水乙醇购自广州化学试剂厂；
C57BL/6小鼠来自赛业模式生物研究中心(太仓)有限公司；
小鼠饲料购自武汉市万千佳兴生物科技有限公司；
小鼠的饲养条件为：
在温度为25℃、湿度为50％、气流速度为0.2m/s、噪音为50dB(A)、无菌等级为7级的无菌环境中进行饲养，光照时间为10h/天，每2天喂食一次，提供足够的饲料供小鼠自由取食，平均每只小鼠的进食量为3～7g/天。
实施例1
本实施例提供一种靶向AHRR基因的sgRNA组合，所述靶向AHRR基因的sgRNA组合包括sgRNA1和sgRNA2，所述sgRNA1包括SEQ ID No.1所示的核酸序列，所述sgRNA2包括SEQ ID No.2所示的核酸序列。
SEQ ID No.1：GGAGATNTCGCCAAGTNCATGGG；
SEQ ID No.2：GGCACATCTANCGTNATTATTGG；
其中，N代表A、T、C或G中的任意一种。
所述sgRNA1特异性靶向AHRR基因的内含子5，所述sgRNA2特异性靶向AHRR基因的内含子8，特异性好，脱靶率低，应用价值广泛。
实施例2
本实施例提供一种AHRR基因编辑系统，所述AHRR基因编辑系统包括靶向AHRR基因的sgRNA组合sgRNA1和sgRNA2以及Cas9核酸酶的mRNA。
所述AHRR基因编辑系统具有敲除基因组中AHRR基因的能力，通过sgRNA组合的配合，可以降低脱靶率，降低非特异性编辑的概率，且具有大片段敲除的能力，编辑效率更高；选用Cas9核酸酶的mRNA，对细胞的毒害作用较小，编辑后的个体更容易存活，降低筛选工作的难度。
实施例3
本实施例提供一种重组细胞，所述重组细胞为经过实施例2中的AHRR基因编辑系统编辑后，基因组中AHRR基因发生突变的C57BL/6小鼠的受精卵细胞。
所述重组细胞通过如下方法进行构建：
(1)体外转录Cas9核酸酶基因的mRNA，与靶向AHRR基因的sgRNA组合混合，得到AHRR基因编辑系统；和
对C57BL/6小鼠进行促排卵，体外受精后，培养受精卵；
(2)将所述AHRR基因编辑系统显微注射入C57BL/6小鼠受精卵细胞的细胞核内，得到重组细胞。
通过将sgRNA1、sgRNA2和Cas9核酸酶的mRNA直接注射入受精卵细胞的细胞核中，提高了基因编辑的效率；选取受精卵构建重组细胞，有义突变可以通过细胞分裂进行遗传，降低了嵌合体出现的概率，编辑后的基因型可以遗传给子代，减小了筛选的工作量。
实施例4
本实施例将实施例3中构建的重组细胞移植到代孕母鼠的体内，构建肥胖动物模型，具体步骤如下：
(1)将实施例3所得的重组细胞进行体外培养，并转移到代孕母鼠的体内；
(2)提取出生小鼠尾部组织的DNA，使用SEQ ID No.3～5进行PCR扩增，并对扩增产物进行测序鉴定，挑选AHRR基因缺失的小鼠作为F0代；
DNA提取的具体步骤如下：
a.剪取3mm小鼠尾巴置于离心管中，加入180μL缓冲液GL、20μL蛋白酶K和10μL RNase A；
b.将离心管在56℃孵育过夜；
c.将离心管在12000rpm下离心2min，去除杂质；
d.混合，加入200μL Buffer GB和200μL无水乙醇；
e.将离心柱放在收集管中，将样品转移至离心柱中，在12000rpm下离心，弃滤液；
f.向离心柱中加入500μL Buffer WA，在12000rpm下离心1min，弃滤液；
g.向离心柱中加入700μL Buffer WB，在12000rpm下离心1min，弃滤液；
h.重复步骤g1次；
i.将离心柱放在收集管中，在12000rpm下离心2min；
j.将离心柱放入一个新的1.5mL离心管中，加入100μL无菌水于柱膜中心，静置5min；
k.在12000rpm下离心2min，洗脱DNA；
l.收集DNA并进行检测。
PCR扩增的体系如表1所示：
表1
组分 体积(μL)
正向引物 1
反向引物 1
PCR酶 1
模板 1
2×缓冲液 25
dNTPs(浓度为2mM) 10
水 11
总体积 50
其中，正向引物的序列为SEQ ID No.3所示的核酸序列，反向引物的序列为SEQ ID No.4～5之一所示的核酸序列。
SEQ ID No.3：CAGAGCTTATCCACAAAGTCACC；
SEQ ID No.4：GCACCTCCAAAGAATATACAAGCAG；
SEQ ID No.5：CTGCAAAGGCTGACATGAAGG。
正向引物的序列为SEQ ID No.3、反向引物的序列为SEQ ID No.4时扩增的程序如下：
预变性：95℃孵育3min；
循环扩增：95℃孵育15s；55℃孵育15s；68℃孵育420s；
延伸：68℃孵育5min；
循环的次数为35次。
扩增产物在4℃下保存。
正向引物的序列为SEQ ID No.3、反向引物的序列为SEQ ID No.5时扩增的程序如下：
预变性：95℃孵育3min；
循环扩增：95℃孵育15s；55℃孵育15s；68℃孵育30s；
延伸：68℃孵育5min；
循环的次数为35次。
扩增产物在4℃下保存。
(3)将所述F0代与野生型C57BL/6小鼠交配，得到F1代杂合子小鼠；以及
(4)将所得F1代杂合子小鼠自交，采用步骤(2)中的体系与程序，使用SEQ ID No.3～5进行PCR扩增，并对扩增产物测序鉴定，筛选出F2代AHRR基因敲除的纯合子C57BL/6小鼠，即所述的肥胖动物模型。
所述基因敲除小鼠的筛选的原理如图1所示：其中
野生型小鼠的SEQ ID No.3/SEQ ID No.4扩增产物的大小为6425bp，若编辑后缺失了部分基因片段，则扩增产物片段的大小减小；因此野生型小鼠的SEQ ID No.3/SEQ ID No.4扩增产物的大小为6425bp，基因敲除纯合子小鼠的SEQ ID No.3/SEQ ID No.4的扩增产物小于6425bp，杂合子小鼠的SEQ ID No.3/SEQ ID No.4扩增产物中同时含有两条带；和
野生型小鼠的SEQ ID No.3/SEQ ID No.5扩增产物的大小为400bp，若编辑后缺失了部分基因片段，则缺失了部分基因片段的基因敲除纯合子小鼠无对应的扩增产物；因此野生型小鼠的SEQ ID No.3/SEQ ID No.5扩增产物的大小为400bp，基因敲除纯合子小鼠使用SEQ ID No.3/SEQ ID No.5无扩增产物，杂合 子小鼠的SEQ ID No.3/SEQ ID No.5扩增产物的大小为400bp，但条带亮度较野生型小鼠弱。
通过上述方法，本实施例成功构建了AHRR基因敲除的纯合子C57BL/6小鼠，即所述的肥胖动物模型。经测序鉴定，敲除片段为13号染色体上的74278006～74283456区域，长度为5451bp，并且在敲除的片段后插入了长度为7bp的片段。具体敲除的序列如SEQ ID No.6所示。
SEQ ID No.6：
Figure PCTCN2020139467-appb-000001
Figure PCTCN2020139467-appb-000002
Figure PCTCN2020139467-appb-000003
具体插入的序列如SEQ ID No.7所示。
SEQ ID No.7：CCTAACG。
实施例5
本实施例对实施例4筛选得到的AHRR基因敲除的纯合子C57BL/6小鼠进行PCR验证，扩增的体系和方法与实施例4相同，同时使用野生型小鼠进行相同的检测作为对照。
鉴定的原理为：
野生型小鼠的SEQ ID No.3/SEQ ID No.4扩增产物的大小为6425bp，若编辑后缺失了部分基因片段，则扩增产物片段的大小减小；因此野生型小鼠的SEQ ID No.3/SEQ ID No.4扩增产物的大小为6425bp，基因敲除纯合子小鼠的SEQ ID No.3/SEQ ID No.4扩增产物的大小为981bp；
野生型小鼠的SEQ ID No.3/SEQ ID No.5扩增产物的大小为400bp，若编辑后缺失了部分基因片段，则缺失了部分基因片段的基因敲除纯合子小鼠无对应的扩增产物；因此野生型小鼠的SEQ ID No.3/SEQ ID No.5扩增产物的大小为400bp，基因敲除纯合子小鼠使用SEQ ID No.3/SEQ ID No.5无扩增产物。
野生型小鼠的SEQ ID No.3/SEQ ID No.4扩增产物的琼脂糖凝胶电泳图片如图2A所示，野生型小鼠的SEQ ID No.3/SEQ ID No.5扩增产物的琼脂糖凝胶电泳图片如图2B所示；
AHRR基因敲除纯合子小鼠的SEQ ID No.3/SEQ ID No.4扩增产物的琼脂糖凝胶电泳图片如图3A所示，AHRR基因敲除纯合子小鼠的SEQ ID No.3/SEQ ID No.5扩增产物的琼脂糖凝胶电泳图片如图3B所示。
由图2A、图2B、图3A和图3B可知，野生型小鼠的SEQ ID No.3/SEQ ID No.4扩增产物的大小为6425bp，而基因敲除纯合子小鼠的SEQ ID No.3/SEQ ID No.4扩增产物的大小为981bp；野生型小鼠的SEQ ID No.3/SEQ ID No.5扩增产物的大小为400bp，而基因敲除纯合子小鼠使用SEQ ID No.3/SEQ ID No.5无扩增产 物，实验结果与预期相符，表明所得的纯合子突变体小鼠确实为AHRR基因敲除的纯合子小鼠。
实施例6
本实施例对实施例4筛选得到的AHRR基因敲除的纯合子C57BL/6小鼠进行肥胖表型的鉴定。
将野生型C57BL/6小鼠和AHRR基因敲除的纯合子C57BL/6小鼠在相同的条件下进行饲养，8个月后，对小鼠的体重及脂肪重量进行统计学分析。
图4A为AHRR基因敲除纯合子小鼠和野生型小鼠的外观对比图片，从图片可以明显看出，敲除AHRR基因的突变体纯合子小鼠的体型更大，体内脂肪含量较多，较为肥胖。
随后，分别称取AHRR基因敲除纯合子小鼠和野生型小鼠的体重，并进行统计，结果如图4B所示。由图4B可知，AHRR基因敲除小鼠与野生型小鼠的体重呈现显著性差异，突变体小鼠的体重更重。
断颈处死小鼠后，对小鼠进行解剖，取出小鼠体内的棕色脂肪进行称重，统计结果如图4C所示。由图4C可知，AHRR基因敲除的突变体小鼠的棕色脂肪重量明显高于野生型小鼠，并具有统计学意义。
综合图4A、图4B和图4C，可知AHRR基因敲除的纯合子C57BL/6小鼠的外观、体重及体内棕色脂肪重量均显著高于野生型小鼠，具有明显的肥胖表型，表明本申请所述的肥胖动物模型的构建方法科学合理，真实可信。
综上所述，本申请通过合成靶向AHRR基因的sgRNA组合，与Cas9核酸酶的mRNA组成了AHRR基因编辑系统；将所述AHRR基因编辑系统显微注射入C57BL/6小鼠受精卵的细胞核内，成功构建了重组细胞；再将重组细胞转移到代孕母鼠的体内，对出生的小鼠进行鉴定及筛选，成功筛选到了AHRR基因敲除的纯合子小鼠。所述基因突变纯合子小鼠的外观、体重及脂肪重量均明显高于野生型小鼠，可用于降糖、降脂相关药物的筛选中；所述肥胖动物模型的构建方法技术成熟，效率极高，用时较短，且降低了筛选的工作量，具有广泛的应用价值。
申请人声明，本申请通过上述实施例来说明本申请的详细方法，但本申请并不局限于上述详细方法，即不意味着本申请必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本申请的任何改进，对本申请产品各原 料的等效替换及辅助成分的添加、具体方式的选择等，均落在本申请的保护范围和公开范围之内。

Claims (12)

  1. 一种靶向AHRR基因的sgRNA组合，其包括sgRNA1和/或sgRNA2，所述sgRNA1包括SEQ ID No.1所示的核酸序列，所述sgRNA2包括SEQ ID No.2所示的核酸序列。
  2. 一种AHRR基因编辑系统，其包括权利要求1所述的靶向AHRR基因的sgRNA组合。
  3. 根据权利要求2所述的AHRR基因编辑系统，其中，所述AHRR基因编辑系统还包括Cas9核酸酶和/或Cas9核酸酶的mRNA，优选为Cas9核酸酶的mRNA。
  4. 一种重组细胞，其含有权利要求1所述的靶向AHRR基因的sgRNA组合。
  5. 根据权利要求4所述的重组细胞，其中，所述重组细胞含有权利要求2或3所述的AHRR基因编辑系统；
    优选地，所述重组细胞为经过权利要求2或3所述的AHRR基因编辑系统编辑后，基因组中敲除了AHRR基因的受精卵细胞。
  6. 一种权利要求4或5所述的重组细胞的构建方法，其包括：
    将权利要求2或3所述的AHRR基因编辑系统导入受精卵细胞的细胞核内，得到所述重组细胞；
    任选地所述导入包括显微注射。
  7. 一种肥胖动物模型的构建方法，其包括：
    (1)将权利要求2或3所述的AHRR基因编辑系统导入哺乳动物受精卵细胞的细胞核内，得到重组细胞；
    (2)将所述重组细胞移植入代孕母体，获得的F0代与野生型交配，得到F1代杂合子；以及
    (3)将所述F1代杂合子自交，获得的F2代纯合子即为所述肥胖动物模型。
  8. 根据权利要求7所述的肥胖动物模型的构建方法，其中，所述AHRR基因编辑系统的制备方法包括：
    (1)对Cas9核酸酶基因进行体外转录，得到所述Cas9核酸酶的mRNA；以及
    (2)将所述Cas9核酸酶的mRNA与权利要求1所述的靶向AHRR基因的sgRNA组合混合，得到所述AHRR基因编辑系统。
  9. 根据权利要求7或8所述的肥胖动物模型的构建方法，其中，所述哺乳动物包括小鼠、大鼠、兔、猪或食蟹猴中的任意一种；
    优选地，所述哺乳动物为小鼠，优选为C57BL/6小鼠。
  10. 根据权利要求7～9任一项所述的肥胖动物模型的构建方法，其中，所述F0代、F1代杂合子和F2代纯合子利用PCR扩增和测序进行鉴定；
    优选地，所述PCR扩增的正向引物的序列包括SEQ ID No.3所示的核酸序列；
    优选地，所述PCR扩增的反向引物的序列包括SEQ ID No.4～5之一所示的核酸序列。
  11. 根据权利要求7～10任一项所述的肥胖动物模型的构建方法，其中，所述肥胖动物模型的构建方法包括以下步骤：
    (1)体外转录Cas9核酸酶基因的mRNA，与靶向AHRR基因的sgRNA组合混合，得到AHRR基因编辑系统；和
    对C57BL/6小鼠进行促排卵，体外受精后，培养受精卵；
    (2)将所述AHRR基因编辑系统显微注射入C57BL/6小鼠受精卵细胞的细胞核内，得到重组细胞；
    (3)对所述重组细胞进行体外培养，并转移到代孕母鼠体内；
    (4)提取出生小鼠尾部组织的DNA，使用SEQ ID No.3～5进行PCR扩增，并对扩增产物进行测序鉴定，挑选AHRR基因缺失的小鼠作为F0代；
    (5)将所述F0代与野生型C57BL/6小鼠交配，得到F1代杂合子小鼠；以及
    (6)将所得F1代杂合子小鼠自交，使用SEQ ID No.3～5进行PCR扩增，并对扩增产物测序鉴定，筛选出F2代AHRR基因敲除的纯合子C57BL/6小鼠，即所述的肥胖动物模型。
  12. 权利要求1所述的靶向AHRR基因的sgRNA组合、权利要求2或3所述的AHRR基因编辑系统、权利要求4或5所述的重组细胞、权利要求6所述的重组细胞的构建方法或权利要求7～11任一项所述的肥胖动物模型的构建方法中的任意一种或至少两种的组合在筛选降脂降糖药物和/或评价降脂降糖药物效果中的应用。
PCT/CN2020/139467 2020-12-22 2020-12-25 一种靶向AHRR基因的sgRNA组合及其应用 WO2022134014A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202011534346.1 2020-12-22
CN202011534346.1A CN113913425B (zh) 2020-12-22 2020-12-22 一种靶向AHRR基因的sgRNA组合及其应用

Publications (1)

Publication Number Publication Date
WO2022134014A1 true WO2022134014A1 (zh) 2022-06-30

Family

ID=79231455

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/139467 WO2022134014A1 (zh) 2020-12-22 2020-12-25 一种靶向AHRR基因的sgRNA组合及其应用

Country Status (2)

Country Link
CN (1) CN113913425B (zh)
WO (1) WO2022134014A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115992178A (zh) * 2022-08-08 2023-04-21 首都医科大学附属北京天坛医院 核心蛋白聚糖转基因小鼠的制备方法及应用

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114591957B (zh) * 2022-03-22 2024-04-26 吴文书 一种重症a型血友病动物模型的构建方法及其应用

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016168685A1 (en) * 2015-04-17 2016-10-20 Mayo Foundation For Medical Education And Research Modulating the aryl hydrocarbon receptor system to treat major depressive disorder
CN107267611A (zh) * 2017-06-26 2017-10-20 广州医科大学 一种检测p.Pro189Ala位点基因型的引物组及其检测试剂盒和应用
CN109234278A (zh) * 2018-10-10 2019-01-18 河北伊维沃生物科技有限公司 构建ApoC2基因敲除仓鼠模型的试剂盒和方法
EP3449978A1 (en) * 2017-09-01 2019-03-06 Universite Paris Descartes Inhibitors of aryl hydrocarbon receptor for treating soft-tissue sarcoma and preventing neurofibroma growth and/or transformation to malignant peripheral nerve sheath tumors
US10344323B1 (en) * 2018-02-20 2019-07-09 The Florida International University Board Of Trustees CPG sites differentially methylated in smokers and non-smokers
CN111304259A (zh) * 2020-02-24 2020-06-19 内蒙古自治区人民医院 一种CRISPR/Cas9技术构建ABCC4基因敲除小鼠的方法及其应用
CN111374093A (zh) * 2018-12-28 2020-07-07 高倩 超级肥胖小鼠的构建与鉴定方法

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107475300B (zh) * 2017-09-18 2020-04-21 上海市同济医院 Ifit3-eKO1基因敲除小鼠动物模型的构建方法和应用
CN111321171A (zh) * 2018-12-14 2020-06-23 江苏集萃药康生物科技有限公司 一种应用CRISPR/Cas9介导ES打靶技术制备基因打靶动物模型的方法
CN111996215B (zh) * 2020-08-25 2022-08-02 山西医科大学 一种全身性Plin1基因敲除动物模型构建及鉴定方法

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016168685A1 (en) * 2015-04-17 2016-10-20 Mayo Foundation For Medical Education And Research Modulating the aryl hydrocarbon receptor system to treat major depressive disorder
CN107267611A (zh) * 2017-06-26 2017-10-20 广州医科大学 一种检测p.Pro189Ala位点基因型的引物组及其检测试剂盒和应用
EP3449978A1 (en) * 2017-09-01 2019-03-06 Universite Paris Descartes Inhibitors of aryl hydrocarbon receptor for treating soft-tissue sarcoma and preventing neurofibroma growth and/or transformation to malignant peripheral nerve sheath tumors
US10344323B1 (en) * 2018-02-20 2019-07-09 The Florida International University Board Of Trustees CPG sites differentially methylated in smokers and non-smokers
CN109234278A (zh) * 2018-10-10 2019-01-18 河北伊维沃生物科技有限公司 构建ApoC2基因敲除仓鼠模型的试剂盒和方法
CN111374093A (zh) * 2018-12-28 2020-07-07 高倩 超级肥胖小鼠的构建与鉴定方法
CN111304259A (zh) * 2020-02-24 2020-06-19 内蒙古自治区人民医院 一种CRISPR/Cas9技术构建ABCC4基因敲除小鼠的方法及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
PEI-CHIEN TSAI;CRAIGA. GLASTONBURY;MELISSAN. ELIOT;SAILALITHA BOLLEPALLI;IDIL YET;JUANE. CASTILLO-FERNANDEZ;ELENA CARNERO-MONTORO;: "Smoking induces coordinated DNA methylation and gene expression changes in adipose tissue with consequences for metabolic health", CLINICAL EPIGENETICS, BIOMED CENTRAL LTD, LONDON, UK, vol. 10, no. 1, 20 October 2018 (2018-10-20), London, UK, pages 1 - 21, XP021261751, ISSN: 1868-7075, DOI: 10.1186/s13148-018-0558-0 *
SCHANZ OLIVER, CHIJIIWA RIEKA, CENGIZ SEVGI CAN, MAJLESAIN YASMIN, WEIGHARDT HEIKE, TAKEYAMA HARUKO, FÖRSTER IRMGARD: "Dietary AhR Ligands Regulate AhRR Expression in Intestinal Immune Cells and Intestinal Microbiota Composition", INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, vol. 21, no. 9, pages 3189, XP055946829, DOI: 10.3390/ijms21093189 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115992178A (zh) * 2022-08-08 2023-04-21 首都医科大学附属北京天坛医院 核心蛋白聚糖转基因小鼠的制备方法及应用

Also Published As

Publication number Publication date
CN113913425A (zh) 2022-01-11
CN113913425B (zh) 2022-07-08

Similar Documents

Publication Publication Date Title
WO2022134014A1 (zh) 一种靶向AHRR基因的sgRNA组合及其应用
CN109628454B (zh) 斑马鱼糖原贮积症gys1和gys2基因突变体的构建方法
CN106222204B (zh) 一种黄鳝基因编辑的方法
WO2022111124A1 (zh) 发育正常无肌间刺鱼类新品种培育方法
CN106520792A (zh) 一种分离的中黑盲蝽基因及其编码的蛋白
WO2022227692A1 (zh) 一种无肌间刺鲫品系及其培育方法
CN113736787A (zh) 靶向小鼠Atp7b基因的gRNA及构建Wilson疾病小鼠模型的方法
Wu et al. Gut microbiota in dholes during estrus
Luo et al. Selection of growth-related genes and dominant genotypes in transgenic Yellow River carp Cyprinus carpio L.
CN108300738B (zh) 一种nod遗传背景的中性粒细胞缺失的人源化小鼠模型的制备方法
WO2021057806A1 (zh) 糖尿病疾病模型犬的建立方法
CN106521638A (zh) 一种基因突变大鼠资源库及其制备方法
WO2021042470A1 (zh) gRNA靶点组合在构建A型、B型和A＆B型血友病模型猪细胞系中的应用
CN111477272A (zh) 一种利用SNPs辅助选择高产仔数獭兔的方法
CN108251456B (zh) 一种nod遗传背景的动脉粥样硬化小鼠模型的制备方法
CN113491255B (zh) 一种肥胖性ii型糖尿病斑马鱼模型的构建方法及应用
Pan et al. Allelic diversification of the Wx and ALK Loci in indica restorer lines and their utilisation in hybrid rice breeding in China over the last 50 years
Yizhen et al. Comparative study of the digestion and metabolism related genes’ expression changes during the postnatal food change in different dietary mammals
CN106755474A (zh) 一种公猪kdm5b基因插入/缺失多态性的检测方法及其应用
CN112956453B (zh) 一种黑腹果蝇胰岛素抵抗糖尿病模型的建立方法
WO2021159741A1 (zh) 一种用于制备irs基因缺陷的糖尿病克隆猪核供体细胞的crispr系统及其应用
CN114908124B (zh) 锌结合醇脱氢酶基因在调控昆虫求偶交配行为中的应用
CN111808859B (zh) WAS基因的gRNA及其应用
CN111849977B (zh) 一种精子载体制备转基因动物的方法以及一种制备矮小型转基因鸡的sgRNA和制备方法
CN114958908B (zh) 基于CRISPR/Cas9构建Ets2基因超级增强子敲除动物模型的方法及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20966579

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20966579

Country of ref document: EP

Kind code of ref document: A1