WO2016150336A1 - Crispr-cas9 system, and preparation method and use thereof - Google Patents

Crispr-cas9 system, and preparation method and use thereof Download PDF

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WO2016150336A1
WO2016150336A1 PCT/CN2016/076638 CN2016076638W WO2016150336A1 WO 2016150336 A1 WO2016150336 A1 WO 2016150336A1 CN 2016076638 W CN2016076638 W CN 2016076638W WO 2016150336 A1 WO2016150336 A1 WO 2016150336A1
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cas9
primer pair
crispr
ltr
vif
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French (fr)
Chinese (zh)
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蒋兴宇
刘野
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国家纳米科学中心
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • 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

Definitions

  • the present invention relates to the field of gene therapy drugs, and in particular to the field of gene therapy of HIV, and more particularly to a CRISPR-Cas9 system which can be used for the prevention and/or treatment of HIV, a preparation method and use thereof.
  • HIV Human Immunodeficiency Virus
  • the conventional means of preventing infectious diseases are human intervention and vaccines.
  • Human interventions (such as condoms) cannot truly eliminate HIV. They are only a physical coercive measure to limit HIV.
  • the related vaccines have been used for more than 30 years. Exploration and development, there are no mature products available yet. Therefore, the current primary treatment for HIV is the highly effective combination of antiretroviral methods, but it can only delay the development of HIV. Different from the above treatments, gene therapy has always been regarded as the most ideal mode of HIV treatment; because it can theoretically achieve the effect of preventing and eliminating HIV.
  • CRISPR-Cas9 technology has been rapidly promoted and applied for its efficiency and simplicity. It is an adaptive immune defense system formed by bacteria and archaea during long-term evolution and can be used against invading viruses and foreign DNA.
  • the CRISPR-Cas9 system directs the degradation of homologous sequences by integrating fragments of invading phage and plasmid DNA into CRISPR and using the corresponding CRISPR RNAs (sgRNAs).
  • the main components of the system are: (1) sgRNA sequence, responsible for targeting specific gene loci; (2) Cas9 enzyme, responsible for modification and cleavage of DNA at the target site.
  • a CRISPR-Cas9 system comprising an sgRNA that specifically targets a particular gene locus on the HIV genome, the specific gene loci on the HIV genome comprising Gag, Env, One or more of Pol, Tat, Nef, Vif, Vpr, Vpu, 5'LTR, 3'LTR and Rev.
  • the particular genetic locus on the HIV genome is selected from one or more of Gag, Env, Pol, Tat, Nef, Vif, Vpr, Vpu, 5' LTR, 3' LTR and Rev.
  • the specific genetic loci on the HIV genome are Gag, Env, Pol, Tat, Nef, Vif, Vpr, Vpu, 5' LTR, 3' LTR and Rev.
  • each of the sgRNAs specifically targeting each of the specific gene loci is independently one or more, preferably from 1 to 11, and may be, for example, one, two, or three.
  • Articles, 4, 5, 6, 7, 8, 9, 10 or 11 may be most preferably 4.
  • the length of each of the sgRNAs is independently 16-22 bp, for example, 16 bp, 17 bp, 18 bp, 19 bp, 20 bp, 21 bp or 22 bp, respectively.
  • the CRISPR-Cas9 system further includes Cas9.
  • the Cas9 and the sgRNA specific for a particular gene locus on the HIV genome can be present in a plasmid, respectively.
  • a method of making the CRISPR-Cas9 system of the first aspect of the invention comprising the step of obtaining an sgRNA fragment by PCR amplification.
  • the primer used for the PCR amplification is selected from the group consisting of a Cas9-5'LTR-1 primer pair, a Cas9-5'LTR-2 primer pair, a Cas9-5'LTR-3 primer pair, and a Cas9-5'LTR-4.
  • primer pair base sequence is in the 5'-3' direction as follows:
  • Ggaagctttagagaagtttt and TTCTCTAAAGCTTCCcggtg.
  • the annealing temperatures of the PCR amplification are respectively:
  • Gag 63 ° C
  • Vpu 60 ° C
  • the reaction conditions for the PCR amplification may further include: pre-denaturation at 95 ° C for 3 min (minutes), denaturation at 95 ° C for 30 sec (seconds), annealing for 30 sec (annealing temperature may be respectively used at the above temperature), and extension at 72 ° C for 30 sec to 1 min. 30 cycles, extending at 72 ° C for 8 min after cycling.
  • the PCR amplification reaction system can be: sterile ddH 2 O: 37.5 ⁇ L, 10 ⁇ PCR Buffer (containing MgCl 2 ): 5 ⁇ L, 2.5 mM dNTP: 4 ⁇ L, and corresponding upper and downstream primers each 1 ⁇ L (primer concentration 50 pmol/ ⁇ L) ), template DNA (50 ng/ ⁇ L): 1 ⁇ L, PyrobestTM DNA Polymerase: 0.5 ⁇ L. After gentle shaking and mixing, PCR amplification can be carried out according to the conditions described above.
  • the preparation method may further comprise the step of separately constructing each of the amplified sgRNA fragments into a plasmid vector.
  • the step of constructing the vector may include: recovering the PCR amplification product according to a conventional method using a PCR recovery kit manufactured by Quigen, and then separately digesting the PCR fragment with SalI, and double-digesting the plasmid vector p1.0 with SalI and EcoRV. Ligation, transformation of Top10 or DH5 ⁇ competent state, screening positive clones by SalI and EcoRV digestion. The positive clone can be sequenced, for example, sent to Invitrogen for sequencing.
  • kits comprising the present The CRISPR-Cas9 system of the first aspect of the invention or the CRISPR-Cas9 system prepared according to the method of the second aspect of the invention, or the expression cassette or recombinant vector expressing the CRISPR-Cas9 system.
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier, and the CRISPR-Cas9 system of the first aspect of the invention or according to the second aspect of the invention
  • the CRISPR-Cas9 system prepared by the method described.
  • the CRISPR-Cas9 system of the first aspect of the invention or the CRISPR-Cas9 system prepared according to the method of the second aspect of the invention may be in an effective amount or in a therapeutically effective amount in the pharmaceutical composition.
  • a fifth aspect of the invention there is provided an application of the CRISPR-Cas9 system according to the first aspect of the invention or the CRISPR-Cas9 system or the method prepared according to the method of the second aspect of the invention
  • a method of preventing and/or treating an HIV infection comprising administering to a subject in need thereof a therapeutically effective amount of a CRISPR-Cas9 system according to the first aspect of the invention or according to The CRISPR-Cas9 system prepared by the method of the second aspect of the invention or the pharmaceutical composition of the fourth aspect of the invention.
  • the CRISPR-Cas9 system can be administered by a highly efficient gene delivery system, for example using a gene delivery vector. More preferably, the CRISPR-Cas9 system can be administered by nanocarrier delivery.
  • a CRISPR-Cas9 system for preventing and/or treating HIV infection, the CRISPR-Cas9 system being the CRISPR-Cas9 system of the first aspect of the invention or according to the invention
  • the CRISPR-Cas9 system prepared by the method described in the second aspect.
  • the present invention has at least but not limited to the following beneficial effects:
  • the CRISPR-Cas9 system of the present invention optimizes the design of sgRNAs targeting 11 gene loci (Gag, Env, Pol, Tat, Nef, Vif, Vpr, Vpu, 5'LTR, 3'LTR, Rev) on the HIV genome.
  • sgRNAs targeting 11 gene loci Gag, Env, Pol, Tat, Nef, Vif, Vpr, Vpu, 5'LTR, 3'LTR, Rev
  • the inhibition rate is as high as 96%, and the inhibition rate is comparable to that of peptide anti-HIV drugs.
  • FIG. 1 shows the function of the CRISPR-Cas9 system of the present invention.
  • the inventors designed sgRNAs for 11 loci on the HIV genome, and then prepared the CRISPR-Cas9 system, which can effectively inhibit the production of HIV, and its inhibition rate is equivalent to that of peptide anti-HIV drugs. Effect.
  • the present invention has been completed on this basis.
  • Examples 1-11 are used to illustrate a CRISPR-Cas9 system comprising sgRNAs that specifically target specific gene loci on the HIV genome, and methods for their preparation, which are Gag, Env, Pol, Tat, Nef, respectively. , Vif, Vpr, Vpu, 5'LTR, 3'LTR and Rev (which can be respectively referred to as Examples 1 to 11).
  • Primer sequence (5'-3') Primer name serial number Gacaagatatccttggtttt Cas9-5'LTR-1 SEQ ID NO: 1 CAAGGATATCTTGTCcggtg Cas9-5'LTR-1 SEQ ID NO: 2 Cctatgagcctgcagtttt Cas9-5'LTR-2 SEQ ID NO: 3 TGCAGGCTCATAGGcggtg Cas9-5'LTR-2 SEQ ID NO: 4 Gcttttgcctgtagtttt Cas9-5'LTR-3 SEQ ID NO: 5
  • Reaction system Sterile ddH 2 O: 37.5 ⁇ L, 10 ⁇ PCR Buffer (containing MgCl 2 ): 5 ⁇ L, 2.5 mM dNTP: 4 ⁇ L, corresponding 1 ⁇ L of each of the upstream and downstream primers (primer concentration 50 pmol/ ⁇ L), template DNA (50 ng / ⁇ L): 1 ⁇ L, PyrobestTM DNA Polymerase: 0.5 ⁇ L. After gently shaking and mixing, PCR amplification was carried out under the conditions shown in Table 2.
  • Pre-denaturation transsexual annealing extend cycle extend Gag 95 ° C, 3 min 95°C, 30sec 63°C, 30sec 72 ° C, 1 min 30 72 ° C, 8 min Env 95 ° C, 3 min 95°C, 30sec 63°C, 30sec 72°C, 30sec 30 72 ° C, 8 min Pol 95 ° C, 3 min 95°C, 30sec 58°C, 30sec 72°C, 30sec 30 72 ° C, 8 min Tat 95 ° C, 3 min 95°C, 30sec 51 ° C, 30 sec 72 ° C, 1 min 30 72 ° C, 8 min Vif 95 ° C, 3 min 95°C, 30sec 51 ° C, 30 sec 72°C, 30sec 30 72 ° C, 8 min Nef 95 ° C, 3 min 95°C, 30sec 64°C, 30sec 72°C, 30sec 30 72 ° C, 8 min Vpu 95 ° C, 3 min 95°C, 30sec 60 ° C, 30 sec 72°
  • PCR amplification kits were used to recover PCR amplification products according to the conventional method. Then, the PCR fragments were digested with SalI, and the plasmid vector p1.0 was double-digested with SalI and EcoRV. The two were ligated to transform Top10 or DH5 ⁇ . Positive clones were screened by SalI and EcoRV and sent to Invitrogen for sequencing.
  • the TZM-BL cell line was selected and previously infected with HIV for 6 hours, and the CRISPR-Cas9 system of the present invention was delivered into the cells using a nanocarrier system to direct the expression of CRISPR-Cas9 in the cells.
  • the cell group in which only HIV was infected alone was set as a negative control, and the cell group in which neither HIV nor CRISPR-Cas9 was added as a blank control; the cell group to which HIV and the marketed anti-HIV drug T20 were added was set as a positive control.
  • the test results are shown in Table 3.
  • CRISPR-Cas9 System Inhibition rate CRISPR-Cas9(Env) 0.96 CRISPR-Cas9(Pol) 0.96 CRISPR-Cas9(Gag) 0.96 CRISPR-Cas9(3’LTR) 0.92 CRISPR-Cas9(5’LTR) 0.93 CRISPR-Cas9(Vif) 0.96 CRISPR-Cas9(Rev) 0.95 CRISPR-Cas9(Tat) 0.96 CRISPR-Cas9(Nef) 0.90 CRISPR-Cas9(Vpr) 0.88 CRISPR-Cas9(Vpu) 0.73 Positive control 0.90 Negative control 0 Blank control 0
  • the CRISPR-Cas9 system of the present invention has an inhibition rate of HIV of 73% to 96%, indicating that the CRISPR-Cas9 system of the present invention can inhibit the production of HIV in cells; the positive control is a marketed anti-antibody.
  • the HIV drug has an inhibition rate of 90%.
  • the CRISPR-Cas9 system of the present invention achieves comparable effects to commercially available anti-HIV drugs and peptide anti-HIV drugs.
  • the present invention also provides a kit comprising the CRISPR-Cas9 system of the present invention or the CRISPR-Cas9 system prepared according to the method of the present invention, or an expression cassette or recombinant vector expressing the CRISPR-Cas9 system.
  • reagents required for PCR amplification, vector construction, and the like including but not limited to amplification buffers, primers, template DNA, enzymes, and the like, can also be included in the kit.
  • instructions for use and/or use/analysis software may also be included in the kit.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier, and a CRISPR-Cas9 system of the invention or a CRISPR-Cas9 system prepared according to the methods of the invention.
  • the CRISPR-Cas9 system can be an effective amount or a therapeutically effective amount in the pharmaceutical composition.
  • an effective amount refers to an amount that is functional or active to a human and/or animal and that is acceptable to humans and/or animals.
  • a "pharmaceutically acceptable” ingredient is suitable for use in humans and/or animals (eg, mammals and birds) without excessive adverse side effects (eg, toxicity, irritation, and allergies), ie, has reasonable benefits / risk ratio substance.
  • “Pharmaceutically acceptable carrier” means a carrier for administration, and may include various excipients, diluents and the like.
  • the pharmaceutical composition of the present invention may contain a safe and effective amount of the CRISPR-Cas9 system of the present invention as an active ingredient together with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier can include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should be matched with the administration mode, and the dosage form of the pharmaceutical composition of the present invention can be prepared as an injection, an oral preparation (tablet, capsule, oral liquid), a transdermal agent, a diluent, and the like as needed.
  • an oral preparation tablette, oral liquid
  • transdermal agent a transdermal agent
  • a diluent a diluent
  • the aqueous solution of the material is usually prepared in a conventional manner.
  • the pharmaceutical composition is more suitably manufactured under sterile conditions.
  • the effective amount of the present invention may vary depending on the mode of administration and the severity of the disease to be treated and the like. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., by clinical trials). The factors include, but are not limited to, the pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, The route of medicine, etc.
  • the active ingredient of the present invention is administered at a dose of about 0.00001 mg to 50 mg/kg of animal body weight per day (preferably 0.0001 mg to 10 mg/kg of animal body weight), a satisfactory effect can be obtained. For example, several separate doses may be administered per day, or the dose may be proportionally reduced, as is critical to the condition of the treatment.
  • Pharmaceutically acceptable carriers of the invention include, but are not limited to, water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptide materials, cellulose, nanogels, or combinations thereof .
  • the choice of carrier should generally be matched to the mode of administration, as is well known to those of ordinary skill in the art.
  • the invention also provides the use of the pharmaceutical composition for the preparation of a medicament for the prevention and/or treatment of HIV infection.
  • the invention also provides a method of preventing and/or treating HIV infection.
  • the method comprises administering to a subject in need thereof a therapeutically effective amount of a CRISPR-Cas9 system of the invention or a CRISPR-Cas9 system prepared according to the methods of the invention, or a pharmaceutical composition of the invention.
  • the present invention illustrates the process of the present invention by the above-described embodiments, but the present invention is not limited to the above process steps, that is, it does not mean that the present invention must rely on the above process steps to be implemented. It will be apparent to those skilled in the art that any modifications of the present invention, equivalent substitutions of the materials selected for the present invention, and the addition of the auxiliary ingredients, the selection of the specific means, etc., are all within the scope of the present invention.

Abstract

Provided are a CRISPR-Cas9 system, and preparation method and use thereof. The system comprises sgRNAs at specific gene loci on a specific target HIV genome, and the specific gene loci on the HIV genome comprise one or more of Gag, Env, Pol, Tat, Nef, Vif, Vpr, Vpu, 5'LTR, 3'LTR and Rev.

Description

一种CRISPR-Cas9系统及其制备方法和应用CRISPR-Cas9 system and preparation method and application thereof 技术领域Technical field
本发明涉及基因治疗药物领域，特别涉及HIV的基因治疗领域，更具体地，涉及一种可以用于预防和/或治疗HIV的CRISPR-Cas9系统及其制备方法和用途。The present invention relates to the field of gene therapy drugs, and in particular to the field of gene therapy of HIV, and more particularly to a CRISPR-Cas9 system which can be used for the prevention and/or treatment of HIV, a preparation method and use thereof.
背景技术Background technique
人类免疫缺陷病毒(Human Immunodeficiency Virus或HIV)属逆转录病毒，是一种感染人类免疫系统细胞的慢病毒，能造成人类免疫系统缺陷，使人体失去抵抗力，易产生各种疾病甚至癌症。其导致的疾病——艾滋病至今无有效疗法，具有致命性。Human Immunodeficiency Virus (HIV) is a retrovirus that is a lentivirus that infects the cells of the human immune system. It can cause defects in the human immune system, cause the body to lose its resistance, and easily cause various diseases and even cancer. The disease it causes - AIDS has no effective therapy to date and is fatal.
预防传染病的常规手段是人为干预和疫苗，人为干预(如避孕套等)无法真正意义上消除HIV，仅作为限制HIV的一种物理性强制手段；而相关疫苗虽然经历了三十多年的探索和开发，至今尚未有成熟的产品问世。因此，现行的HIV主要疗法为高效联合抗逆转录病毒法，但其同样只能延缓HIV的发展。与以上疗法不同的是，基因治疗一直被视为HIV治疗的最理想模式；因其理论上可以达到预防和消除HIV的效果。The conventional means of preventing infectious diseases are human intervention and vaccines. Human interventions (such as condoms) cannot truly eliminate HIV. They are only a physical coercive measure to limit HIV. The related vaccines have been used for more than 30 years. Exploration and development, there are no mature products available yet. Therefore, the current primary treatment for HIV is the highly effective combination of antiretroviral methods, but it can only delay the development of HIV. Different from the above treatments, gene therapy has always been regarded as the most ideal mode of HIV treatment; because it can theoretically achieve the effect of preventing and eliminating HIV.
近年来，CRISPR-Cas9技术因其高效性和简便性被迅速推广和应用。它是细菌和古细菌在长期演化过程中形成的一种适应性免疫防御系统，可用来对抗入侵的病毒及外源DNA。CRISPR-Cas9系统通过将入侵噬菌体和质粒DNA的片段整合到CRISPR，并利用相应的CRISPR RNAs(sgRNAs)来指导同源序列的降解。该系统的主要组成部分为：(1)sgRNA序列，负责靶向特异性基因位点；(2)Cas9酶，负责对靶向位点的DNA进行修饰切割。目前，这一技术主要应用于基因修饰动物模型的构建，而将其开发为HIV预防性或治疗性的药物则很少被涉及。在公开发表的论文“RNA-directed Gene Editing Specifically Eradicates Latent and Prevents New HIV-1Infection”中，刘文辉等根据HIV基因的两个片段序列构建了一个长度为20bp的sgRNA，其能靶向位于HIV序列5’LTR的HIV-1U3位点；由于位点单一等原因，该系统仅能说明其 对于HIV的抑制具有关联性，但抑制的效果十分有限。In recent years, CRISPR-Cas9 technology has been rapidly promoted and applied for its efficiency and simplicity. It is an adaptive immune defense system formed by bacteria and archaea during long-term evolution and can be used against invading viruses and foreign DNA. The CRISPR-Cas9 system directs the degradation of homologous sequences by integrating fragments of invading phage and plasmid DNA into CRISPR and using the corresponding CRISPR RNAs (sgRNAs). The main components of the system are: (1) sgRNA sequence, responsible for targeting specific gene loci; (2) Cas9 enzyme, responsible for modification and cleavage of DNA at the target site. At present, this technology is mainly applied to the construction of genetically modified animal models, and the development of HIV preventive or therapeutic drugs is rarely involved. In the published paper "RNA-directed Gene Editing specifically Eradicates Latent and Prevents New HIV-1 Infection", Liu Wenhui et al. constructed a 20 bp sgRNA based on the two fragments of the HIV gene, which can be targeted at the HIV sequence 5 'LTR's HIV-1U3 locus; due to single location, etc., the system can only explain its There is a correlation between the inhibition of HIV, but the effect of inhibition is very limited.
发明内容Summary of the invention
本发明的目的在于提供一种可有效用于预防和/或治疗HIV感染的CRISPR-Cas9系统以及相应的制备方法和应用。It is an object of the present invention to provide a CRISPR-Cas9 system which is effective for the prevention and/or treatment of HIV infection and corresponding preparation methods and uses.
在本发明的第一方面，提供了一种CRISPR-Cas9系统，该系统包括特异性靶向HIV基因组上的特定基因位点的sgRNA，所述HIV基因组上的特定基因位点包括Gag、Env、Pol、Tat、Nef、Vif、Vpr、Vpu、5’LTR、3’LTR和Rev中的一个或多个。优选地，所述HIV基因组上的特定基因位点选自Gag、Env、Pol、Tat、Nef、Vif、Vpr、Vpu、5’LTR、3’LTR和Rev中的一个或多个。更优选地，所述HIV基因组上的特定基因位点为Gag、Env、Pol、Tat、Nef、Vif、Vpr、Vpu、5’LTR、3’LTR和Rev。In a first aspect of the invention, there is provided a CRISPR-Cas9 system comprising an sgRNA that specifically targets a particular gene locus on the HIV genome, the specific gene loci on the HIV genome comprising Gag, Env, One or more of Pol, Tat, Nef, Vif, Vpr, Vpu, 5'LTR, 3'LTR and Rev. Preferably, the particular genetic locus on the HIV genome is selected from one or more of Gag, Env, Pol, Tat, Nef, Vif, Vpr, Vpu, 5' LTR, 3' LTR and Rev. More preferably, the specific genetic loci on the HIV genome are Gag, Env, Pol, Tat, Nef, Vif, Vpr, Vpu, 5' LTR, 3' LTR and Rev.
根据本发明的CRISPR-Cas9系统，其中，特异性靶向每个所述特定基因位点的sgRNA各自独立地为1条以上，优选为1～11条，例如可以为1条、2条、3条、4条、5条、6条、7条、8条、9条、10条或11条，可以最优选为4条。更进一步优选地，每条所述sgRNA的长度各自独立地为16-22bp，例如可以独立地为16bp、17bp、18bp、19bp、20bp、21bp或22bp。According to the CRISPR-Cas9 system of the present invention, each of the sgRNAs specifically targeting each of the specific gene loci is independently one or more, preferably from 1 to 11, and may be, for example, one, two, or three. Articles, 4, 5, 6, 7, 8, 9, 10 or 11 may be most preferably 4. Still more preferably, the length of each of the sgRNAs is independently 16-22 bp, for example, 16 bp, 17 bp, 18 bp, 19 bp, 20 bp, 21 bp or 22 bp, respectively.
根据本发明的CRISPR-Cas9系统，其中，所述CRISPR-Cas9系统还包括Cas9。优选地，所述Cas9和所述特异性靶向HIV基因组上的特定基因位点的sgRNA可以分别存在于质粒中。According to the CRISPR-Cas9 system of the present invention, the CRISPR-Cas9 system further includes Cas9. Preferably, the Cas9 and the sgRNA specific for a particular gene locus on the HIV genome can be present in a plasmid, respectively.
在本发明的第二方面，提供了制备本发明第一方面所述CRISPR-Cas9系统的方法，该方法包括通过PCR扩增获得sgRNA片段的步骤。In a second aspect of the invention, there is provided a method of making the CRISPR-Cas9 system of the first aspect of the invention, the method comprising the step of obtaining an sgRNA fragment by PCR amplification.
优选地，所述PCR扩增采用的引物选自Cas9-5'LTR-1引物对、Cas9-5'LTR-2引物对、Cas9-5'LTR-3引物对、Cas9-5'LTR-4引物对、Cas9-Pol-1引物对、Cas9-Pol-2引物对、Cas9-Pol-3引物对、Cas9-Pol-4引物对、Cas9-vif-1引物对、Cas9-vif-2引物对、Cas9-vif-3引物对、Cas9-vif-4引物对、Cas9-vpr-1引物对、Cas9-vpr-2引物对、Cas9-vpr-3引物对、Cas9-tat-1引物对、Cas9-tat-2引物对、Cas9-tat-3引物对、Cas9-tat-4引物对、Cas9-rev2-1引物对、 Cas9-rev2-2引物对、Cas9-rev2-3引物对、Cas9-vpu-1引物对、Cas9-vpu-2引物对、Cas9-env-1引物对、Cas9-env-2引物对、Cas9-env-3引物对、Cas9-env-4引物对、Cas9-nef-1引物对、Cas9-nef-2引物对、Cas9-nef-3引物对、Cas9-nef-4引物对、Cas9-3'LTR-1引物对、Cas9-3'LTR-2引物对、Cas9-3'LTR-3引物对、Cas9-Gag-1引物对、Cas9-Gag-2引物对、Cas9-Gag-3引物对和Cas9-Gag-4引物对中的一个或多个。Preferably, the primer used for the PCR amplification is selected from the group consisting of a Cas9-5'LTR-1 primer pair, a Cas9-5'LTR-2 primer pair, a Cas9-5'LTR-3 primer pair, and a Cas9-5'LTR-4. Primer pair, Cas9-Pol-1 primer pair, Cas9-Pol-2 primer pair, Cas9-Pol-3 primer pair, Cas9-Pol-4 primer pair, Cas9-vif-1 primer pair, Cas9-vif-2 primer pair , Cas9-vif-3 Primer Pair, Cas9-vif-4 Primer Pair, Cas9-vpr-1 Primer Pair, Cas9-vpr-2 Primer Pair, Cas9-vpr-3 Primer Pair, Cas9-tat-1 Primer Pair, Cas9 -tat-2 Primer Pair, Cas9-tat-3 Primer Pair, Cas9-tat-4 Primer Pair, Cas9-rev2-1 Primer Pair, Cas9-rev2-2 Primer Pair, Cas9-rev2-3 Primer Pair, Cas9-vpu-1 Primer Pair, Cas9-vpu-2 Primer Pair, Cas9-env-1 Primer Pair, Cas9-env-2 Primer Pair, Cas9- Env-3 Primer Pair, Cas9-env-4 Primer Pair, Cas9-nef-1 Primer Pair, Cas9-nef-2 Primer Pair, Cas9-nef-3 Primer Pair, Cas9-nef-4 Primer Pair, Cas9-3' LTR-1 Primer Pair, Cas9-3'LTR-2 Primer Pair, Cas9-3'LTR-3 Primer Pair, Cas9-Gag-1 Primer Pair, Cas9-Gag-2 Primer Pair, Cas9-Gag-3 Primer Pair and One or more of the Cas9-Gag-4 primer pairs.
更优选地，所述引物对碱基序列按5'-3'方向如下：More preferably, the primer pair base sequence is in the 5'-3' direction as follows:
Cas9-5'LTR-1引物对：Cas9-5'LTR-1 Primer Pair:
gacaagatatccttggtttt，和CAAGGATATCTTGTCcggtg；Gacaagatatccttggtttt, and CAAGGATATCTTGTCcggtg;
Cas9-5'LTR-2引物对：Cas9-5'LTR-2 Primer Pair:
cctatgagcctgcagtttt，和TGCAGGCTCATAGGcggtg；Cctatgagcctgcagtttt, and TGCAGGCTCATAGGcggtg;
Cas9-5'LTR-3引物对：Cas9-5'LTR-3 Primer Pair:
gctttttgcctgtagtttt，和TACAGGCAAAAAGCcggtg；Gctttttgcctgtagtttt, and TACAGGCAAAAAGCcggtg;
Cas9-5'LTR-4引物对：Cas9-5'LTR-4 Primer Pair:
gacccttttagtcagtggtttt，和CACTGACTAAAAGGGTCcggtg；Gacccttttagtcagtggtttt, and CACTGACTAAAAGGGTCcggtg;
Cas9-Pol-1引物对：Cas9-Pol-1 Primer Pair:
aataccacatcccgcagtttt，和TGCGGGATGTGGTATTCcggtg；Aataccacatcccgcagtttt, and TGCGGGATGTGGTATTCcggtg;
Cas9-Pol-2引物对：Cas9-Pol-2 Primer Pair:
ccacagggatggaagtttt，和TTCCATCCCTGTGGcggtg；Ccacagggagtggaagtttt, and TTCCATCCCTGTGGcggtg;
Cas9-Pol-3引物对：Cas9-Pol-3 Primer Pair:
gtcagatttatgcgtttt，和GCATAAATCTGACcggtg；Gtcagatttatgcgtttt, and GCATAAATCTGACcggtg;
Cas9-Pol-4引物对：Cas9-Pol-4 Primer Pair:
cctggattcctgaatgtttt，和ATTCAGGAATCCAGGcggtg；Cctggattcctgaatgtttt, and ATTCAGGAATCCAGGcggtg;
Cas9-vif-1引物对：Cas9-vif-1 Primer Pair:
tcagaagtacacatcccag，和GATGTGTACTTCTGAcggtg；Tcagaagtacacatcccag, and GATGTGTACTTCTGAcggtg;
Cas9-vif-2引物对：Cas9-vif-2 Primer Pair:
acatattggggtctgcatacgtttt，和CAGACCCCAATATGTcggtg；Acatattggggtctgcatacgtttt, and CAGACCCCAATATGTcggtg;
Cas9-vif-3引物对：Cas9-vif-3 Primer Pair:
gccagggagtctccatagaagtttt，和TTCTATGGAGACTCCcggtg；Gccagggagtctccatagaagtttt, and TTCTATGGAGACTCCcggtg;
Cas9-vif-4引物对：Cas9-vif-4 Primer Pair:
gatctctacaatactgtttt，和AGTATTGTAGAGATCcggtg； Gatctctacaatactgtttt, and AGTATTGTAGAGATCcggg;
Cas9-vpr-1引物对：Cas9-vpr-1 Primer Pair:
atggctccatagcttgtttt，和AAGCTATGGAGCCATcggtg；Atggctccatagcttgtttt, and AAGGTATGGAGCCATcggtg;
Cas9-vpr-2引物对：Cas9-vpr-2 Primer Pair:
ggagatacttggacgtttt，和GTCCAAGTATCTCCcggtg；Ggagatacttggacgtttt, and GTCCAAGTATCTCCcggtg;
Cas9-vpr-3引物对：Cas9-vpr-3 Primer Pair:
tgccaacatagcagaatgtttt，和ATTCTGCTATGTTGGCAcggtg；Tgccaacatagcagaatgtttt, and ATTCTGCTATGTTGGCAcggtg;
Cas9-tat-1引物对：Cas9-tat-1 Primer Pair:
agccctggaagcatccgtttt，和GGATGCTTCCAGGGCTcggtg；Agccctggaagcatccgtttt, and GGATGCTTCCAGGGCTcggtg;
Cas9-tat-2引物对：Cas9-tat-2 Primer Pair:
ccaggaagtcagcctgtttt，和AGGCTGACTTCCTGGcggtg；Ccaggaagtcagcctgtttt, and AGGCTGACTTCCTGGcggtg;
Cas9-tat-3引物对：Cas9-tat-3 Primer Pair:
atggcaggaagaaggtttt，和CTTCTTCCTGCCATAcggtg；Atggcaggaagaaggtttt, and CTTCTTCCTGCCATAcggtg;
Cas9-tat-4引物对：Cas9-tat-4 Primer Pair:
cgaaggaatcgaagagtttt，和TCTTCGATTCCTTCGcggtg；Cgaaggaatcgaagagtttt, and TCTTCGATTCCTTCGcggtg;
Cas9-rev2-1引物对：Cas9-rev2-1 Primer Pair:
tcttagcactgttctgtttt，和AGAACAGTGCTAAGAcggtg；Tcttagcactgttctgtttt, and AGAACAGTGCTAAGAcggtg;
Cas9-rev2-2引物对：Cas9-rev2-2 Primer Pair:
acttactcttgattgtagcggtttt，和ACAATCAAGAGTAAGTcggtg；Acttactcttgattgtagcggtttt, and ACAATCAAGAGTAAGTcggtg;
Cas9-rev2-3引物对：Cas9-rev2-3 Primer Pair:
ggtgggaagtcctcatgtttt，和ATATTTGAGGACTTCCCACCcggtg；Ggtgggaagtcctcatgtttt, and ATATTTGAGGACTTCCCACCcggtg;
Cas9-vpu-1引物对：Cas9-vpu-1 Primer Pair:
caataatagcaatagttatagtttt，和TATAACTATTGCTATTATTGcggtg；Caataatagcaatagttatagtttt, and TATAACTATTGCTATTATTGcggtg;
Cas9-vpu-2引物对：Cas9-vpu-2 Primer Pair:
ccatagtattaataaaatatgtttt，和ATATTTTATTAATACTATGGcggtg；Ccatagtattaataaaatatgtttt, and ATATTTTATTAATACTATGGcggtg;
Cas9-env-1引物对：Cas9-env-1 Primer Pair:
catggtagaccagatgcatggtttt，和Catggtagaccagatgcatggtttt, and
CATGCATCTGGTCTACCATGcggtg；CATGCATCTGGTCTACCATGcggtg;
Cas9-env-2引物对：Cas9-env-2 Primer Pair:
tgctctttcaatatcaccacgtttt，和Tgctctttcaatatcaccacgtttt, and
GTGGTGATATTGAAAGAGCAcggtg；GTGGTGATATTGAAAGAGCAcggtg;
Cas9-env-3引物对：Cas9-env-3 Primer Pair:
gtctagcagaagaaggtttt，和CTTCTTCTGCTAGACcggtg； Gtctagcagaagaaggtttt, and CTTCTTCTGCTAGACcggtg;
Cas9-env-4引物对：Cas9-env-4 Primer Pair:
ctgctattaacaagagagtttt，和TCTCTTGTTAATAGCAGcggtg；Ctgctattaacaagagagtttt, and TCTCTTGTTAATAGCAGcggtg;
Cas9-nef-1引物对：Cas9-nef-1 Primer Pair:
aatgggatggcctgctgtaagtttt，和Aatgggatggcctgctgtaagtttt, and
TTACAGCAGGCCATCCCATTcggtg；TTACAGCAGGCCATCCCATTcggtg;
Cas9-nef-2引物对：Cas9-nef-2 Primer Pair:
gagctgagccagcagcagatgtttt，和Gagctgagccagcagcagatgtttt, and
ATCTGCTGCTGGCTCAGCTCcggtg；ATCTGCTGCTGGCTCAGCTCcggtg;
Cas9-nef-3引物对：Cas9-nef-3 Primer Pair:
gggagcagcatctagagaccgtttt，和Gggagcagcatctagagaccgtttt, and
GGTCTCTAGATGCTGCTCCCcggtg；GGTCTCTAGATGCTGCTCCCcggtg;
Cas9-nef-4引物对：Cas9-nef-4 Primer Pair:
tgcctggctagaagcacaaggtttt，和Tgcctggctagaagcacaaggtttt, and
CTTGTGCTTCTAGCCAGGCAcggtg；CTTGTGCTTCTAGCCAGGCAcggtg;
Cas9-3'LTR-1引物对：Cas9-3'LTR-1 Primer Pair:
gatttccactgacctttggagtttt，和Gatttccactgacctttggagtttt, and
TCCAAAGGTCAGTGGAAATCcggtg；TCCAAAGGTCAGTGGAAATCcggtg;
Cas9-3'LTR-2引物对：Cas9-3'LTR-2 Primer Pair:
cggagaaagaagtgttagtggtttt，和Cggagaaagaagtgttagtggtttt, and
CACTAACACTTCTTTCTCCGcggtg；CACTAACACTTCTTTCTCCGcggtg;
Cas9-3'LTR-3引物对：Cas9-3'LTR-3 Primer Pair:
ctttccgctggggactttccgtttt，和Ctttccgctggggactttccgtttt, and
GGAAAGTCCCCAGCGGAAAGcggtg；GGAAAGTCCCCAGCGGAAAGcggtg;
Cas9-Gag-1引物对：Cas9-Gag-1 Primer Pair:
gtcagtattaagtgcgtttt，和GCACTTAATACTGACcggtg；Gtcagtattaagtgcgtttt, and GCACTTAATACTGACcggtg;
Cas9-Gag-2引物对：Cas9-Gag-2 Primer Pair:
cacaggaaaaggcagccgtttt，和GGCTGCCTTTTCCTGTGcggtg；Cacaggaaaaggcagccgtttt, and GGCTGCCTTTTCCTGTGcggtg;
Cas9-Gag-3引物对：Cas9-Gag-3 Primer Pair:
gaacgatttgcagtcaagtttt，和TTGACTGCAAATCGTTCcggtg；Gaacgatttgcagtcaagtttt, and TTGACTGCAAATCGTTCcggtg;
Cas9-Gag-4引物对：Cas9-Gag-4 Primer Pair:
ggaagctttagagaagtttt，和TTCTCTAAAGCTTCCcggtg。Ggaagctttagagaagtttt, and TTCTCTAAAGCTTCCcggtg.
本领域的技术人员可以根据以上提供的引物对确定所扩增的 相应的sgRNA的核苷酸序列，所述sgRNA可以特异性靶向所述HIV基因组上的特定基因位点。Those skilled in the art can determine the amplified according to the primer pairs provided above. The nucleotide sequence of the corresponding sgRNA, which can specifically target a particular gene locus on the HIV genome.
根据本发明的CRISPR-Cas9系统的制备方法，其中，所述PCR扩增的退火温度分别为：According to the preparation method of the CRISPR-Cas9 system of the present invention, the annealing temperatures of the PCR amplification are respectively:
Gag：63℃；Gag: 63 ° C;
Env：63℃；Env: 63 ° C;
Pol：58℃；Pol: 58 ° C;
Tat：51℃；Tat: 51 ° C;
Vif：51℃；Vif: 51 ° C;
Nef：64℃；Nef: 64 ° C;
Vpu：60℃；Vpu: 60 ° C;
Vpr：67℃；Vpr: 67 ° C;
3’LTR：67℃；3'LTR: 67 ° C;
5’LTR：58℃；5'LTR: 58 ° C;
Rev：51℃。Rev: 51 ° C.
优选地，所述PCR扩增的反应条件还可以包括：95℃预变性3min(分钟)，95℃变性30sec(秒)，退火30sec(退火温度可分别采用上述温度)，72℃延伸30sec～1min，30个循环，循环后72℃延伸8min。Preferably, the reaction conditions for the PCR amplification may further include: pre-denaturation at 95 ° C for 3 min (minutes), denaturation at 95 ° C for 30 sec (seconds), annealing for 30 sec (annealing temperature may be respectively used at the above temperature), and extension at 72 ° C for 30 sec to 1 min. 30 cycles, extending at 72 ° C for 8 min after cycling.
PCR扩增的反应体系可以为：无菌ddH2O：37.5μL，10×PCR Buffer(含MgCl2)：5μL，2.5mM dNTP：4μL，相应的上、下游引物各1μL(引物浓度50pmol/μL)，模板DNA(50ng/μL)：1μL，PyrobestTM DNA Polymerase：0.5μL。在温和震荡混匀后，可以按照前文所述的条件进行PCR扩增。The PCR amplification reaction system can be: sterile ddH 2 O: 37.5 μL, 10×PCR Buffer (containing MgCl 2 ): 5 μL, 2.5 mM dNTP: 4 μL, and corresponding upper and downstream primers each 1 μL (primer concentration 50 pmol/μL) ), template DNA (50 ng/μL): 1 μL, PyrobestTM DNA Polymerase: 0.5 μL. After gentle shaking and mixing, PCR amplification can be carried out according to the conditions described above.
更优选地，所述制备方法还可以包括将所扩增的各sgRNA片段分别构建入质粒载体的步骤。More preferably, the preparation method may further comprise the step of separately constructing each of the amplified sgRNA fragments into a plasmid vector.
载体构建的步骤可以包括：用Quigen生产的PCR回收试剂盒按照常规方法进行PCR扩增产物的回收，然后用SalI单酶切PCR片段，SalI和EcoRV双酶切质粒载体p1.0，两者进行连接，转化Top10或DH5α感受态，以SalI和EcoRV酶切筛选出阳性克隆。可以对该阳性克隆进行测序，例如送Invitrogen公司测序。The step of constructing the vector may include: recovering the PCR amplification product according to a conventional method using a PCR recovery kit manufactured by Quigen, and then separately digesting the PCR fragment with SalI, and double-digesting the plasmid vector p1.0 with SalI and EcoRV. Ligation, transformation of Top10 or DH5α competent state, screening positive clones by SalI and EcoRV digestion. The positive clone can be sequenced, for example, sent to Invitrogen for sequencing.
在本发明的第三方面，提供了一种试剂盒，所述试剂盒包括本 发明第一方面所述的CRISPR-Cas9系统或按照本发明第二方面的方法而制备的CRISPR-Cas9系统，或表达所述CRISPR-Cas9系统的表达盒或重组载体。In a third aspect of the invention, a kit is provided, the kit comprising the present The CRISPR-Cas9 system of the first aspect of the invention or the CRISPR-Cas9 system prepared according to the method of the second aspect of the invention, or the expression cassette or recombinant vector expressing the CRISPR-Cas9 system.
在本发明的第四方面，提供了一种药物组合物，所述药物组合物包括药学上可接受的载体，以及本发明第一方面所述的CRISPR-Cas9系统或按照本发明第二方面所述的方法而制备的CRISPR-Cas9系统。本发明第一方面所述的CRISPR-Cas9系统或按照本发明第二方面所述的方法而制备的CRISPR-Cas9系统在所述药物组合物中可以为有效量的或治疗有效量的。In a fourth aspect of the invention, there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier, and the CRISPR-Cas9 system of the first aspect of the invention or according to the second aspect of the invention The CRISPR-Cas9 system prepared by the method described. The CRISPR-Cas9 system of the first aspect of the invention or the CRISPR-Cas9 system prepared according to the method of the second aspect of the invention may be in an effective amount or in a therapeutically effective amount in the pharmaceutical composition.
在本发明的第五方面，提供了一种应用，所述应用为本发明第一方面所述的CRISPR-Cas9系统或按照本发明第二方面所述的方法而制备的CRISPR-Cas9系统或本发明第四方面所述的药物组合物在制备用于预防和/或治疗HIV感染的药物中的应用。In a fifth aspect of the invention, there is provided an application of the CRISPR-Cas9 system according to the first aspect of the invention or the CRISPR-Cas9 system or the method prepared according to the method of the second aspect of the invention Use of the pharmaceutical composition of the fourth aspect of the invention for the manufacture of a medicament for the prevention and/or treatment of HIV infection.
在本发明的第六方面，提供了一种预防和/或治疗HIV感染的方法，所述方法包括向需要的对象给予治疗有效量的如本发明第一方面所述的CRISPR-Cas9系统或按照本发明第二方面所述的方法而制备的CRISPR-Cas9系统或本发明第四方面所述的药物组合物。优选地，所述CRISPR-Cas9系统可以通过高效基因递送系统给药，例如采用基因递送载体。更优选地，所述CRISPR-Cas9系统可以通过纳米载体递送给药。In a sixth aspect of the invention, a method of preventing and/or treating an HIV infection, the method comprising administering to a subject in need thereof a therapeutically effective amount of a CRISPR-Cas9 system according to the first aspect of the invention or according to The CRISPR-Cas9 system prepared by the method of the second aspect of the invention or the pharmaceutical composition of the fourth aspect of the invention. Preferably, the CRISPR-Cas9 system can be administered by a highly efficient gene delivery system, for example using a gene delivery vector. More preferably, the CRISPR-Cas9 system can be administered by nanocarrier delivery.
在本发明的第七方面，提供了一种用于预防和/或治疗HIV感染的CRISPR-Cas9系统，所述CRISPR-Cas9系统为本发明第一方面所述的CRISPR-Cas9系统或按照本发明第二方面所述的方法而制备的CRISPR-Cas9系统。In a seventh aspect of the invention, there is provided a CRISPR-Cas9 system for preventing and/or treating HIV infection, the CRISPR-Cas9 system being the CRISPR-Cas9 system of the first aspect of the invention or according to the invention The CRISPR-Cas9 system prepared by the method described in the second aspect.
与现有技术相比，本发明至少具有但不限于以下有益效果：Compared with the prior art, the present invention has at least but not limited to the following beneficial effects:
本发明的CRISPR-Cas9系统，优化设计针对HIV基因组上11个基因位点(Gag、Env、Pol、Tat、Nef、Vif、Vpr、Vpu、5’LTR、3’LTR、Rev)的sgRNAs，采用高效基因递送系统，将CRISPR-Cas9系统递送进入HIV感染细胞，高效抑制HIV的产生，抑制率高达96％，单从抑制率来讲，达到了和肽类抗HIV药物相当的效果。The CRISPR-Cas9 system of the present invention optimizes the design of sgRNAs targeting 11 gene loci (Gag, Env, Pol, Tat, Nef, Vif, Vpr, Vpu, 5'LTR, 3'LTR, Rev) on the HIV genome. Efficient gene delivery system, the CRISPR-Cas9 system is delivered into HIV-infected cells, and the inhibition of HIV production is highly effective. The inhibition rate is as high as 96%, and the inhibition rate is comparable to that of peptide anti-HIV drugs.
附图的简要说明BRIEF DESCRIPTION OF THE DRAWINGS
图1示出了本发明的CRISPR-Cas9系统的作用示意图。Figure 1 shows the function of the CRISPR-Cas9 system of the present invention.
实施发明的最佳方式The best way to implement the invention
发明人经过广泛而深入的研究，针对HIV基因组上的11个基因位点而设计sgRNAs，继而制备为CRISPR-Cas9系统，可以高效抑制HIV的产生，其抑制率达到了与肽类抗HIV药物相当的效果。在此基础上完成了本发明。After extensive and in-depth research, the inventors designed sgRNAs for 11 loci on the HIV genome, and then prepared the CRISPR-Cas9 system, which can effectively inhibit the production of HIV, and its inhibition rate is equivalent to that of peptide anti-HIV drugs. Effect. The present invention has been completed on this basis.
下面通过具体的实施例进一步说明本发明，但是，应当理解为，这些实施例仅仅是用于更详细具体地说明之用，而不应理解为用于以任何形式限制本发明。The invention is further illustrated by the following examples, which are intended to be in no way intended to
本部分对本发明试验中所使用到的材料以及试验方法进行一般性的描述。虽然为实现本发明目的所使用的许多材料和操作方法是本领域公知的，但是本发明仍然在此作尽可能详细描述。本领域技术人员清楚，在上下文中，如果未特别说明，本发明所用材料和操作方法是本领域公知的。This section provides a general description of the materials used in the tests of the present invention and the test methods. While many of the materials and methods of operation used to accomplish the objectives of the present invention are well known in the art, the present invention is still described in detail herein. It will be apparent to those skilled in the art that, in the context, the materials and methods of operation of the present invention are well known in the art unless otherwise specified.
实施例1～11Examples 1 to 11
实施例1～11用于说明包括特异性靶向HIV基因组上的特定基因位点的sgRNA的CRISPR-Cas9系统及其制备方法，所述特定基因位点分别为Gag、Env、Pol、Tat、Nef、Vif、Vpr、Vpu、5’LTR、3’LTR和Rev(可分别记为实施例1～11)。Examples 1-11 are used to illustrate a CRISPR-Cas9 system comprising sgRNAs that specifically target specific gene loci on the HIV genome, and methods for their preparation, which are Gag, Env, Pol, Tat, Nef, respectively. , Vif, Vpr, Vpu, 5'LTR, 3'LTR and Rev (which can be respectively referred to as Examples 1 to 11).
(1)sgRNA基因的获得(1) Acquisition of sgRNA gene
sgRNAs引物序列直接在invitrogen公司订购获得，相关序列如表1所示。Primer sequences of sgRNAs were obtained directly from invitrogen, and the relevant sequences are shown in Table 1.
表1：sgRNA引物序列Table 1: sgRNA primer sequences
引物序列(5'-3')Primer sequence (5'-3') 引物名称Primer name 序列号serial number
gacaagatatccttggttttGacaagatatccttggtttt Cas9-5'LTR-1Cas9-5'LTR-1 SEQ ID NO:1SEQ ID NO: 1
CAAGGATATCTTGTCcggtgCAAGGATATCTTGTCcggtg Cas9-5'LTR-1Cas9-5'LTR-1 SEQ ID NO:2SEQ ID NO: 2
cctatgagcctgcagttttCctatgagcctgcagtttt Cas9-5'LTR-2Cas9-5'LTR-2 SEQ ID NO:3SEQ ID NO: 3
TGCAGGCTCATAGGcggtgTGCAGGCTCATAGGcggtg Cas9-5'LTR-2Cas9-5'LTR-2 SEQ ID NO:4SEQ ID NO: 4
gctttttgcctgtagttttGctttttgcctgtagtttt Cas9-5'LTR-3Cas9-5'LTR-3 SEQ ID NO:5SEQ ID NO: 5
TACAGGCAAAAAGCcggtgTACAGGCAAAAAGCcggtg Cas9-5'LTR-3Cas9-5'LTR-3 SEQ ID NO:6SEQ ID NO: 6
gacccttttagtcagtggttttGacccttttagtcagtggtttt Cas9-5'LTR-4Cas9-5'LTR-4 SEQ ID NO:7SEQ ID NO:7
CACTGACTAAAAGGGTCcggtgCACTGACTAAAAGGGTCcggtg Cas9-5'LTR-4Cas9-5'LTR-4 SEQ ID NO:8SEQ ID NO:8
aataccacatcccgcagttttAataccacatcccgcagtttt Cas9-Pol-1Cas9-Pol-1 SEQ ID NO:9SEQ ID NO: 9
TGCGGGATGTGGTATTCcggtgTGCGGGATGTGGTATTCcggtg Cas9-Pol-1Cas9-Pol-1 SEQ ID NO:10SEQ ID NO: 10
ccacagggatggaagttttCcacagggagtggaagtttt Cas9-Pol-2Cas9-Pol-2 SEQ ID NO:11SEQ ID NO: 11
TTCCATCCCTGTGGcggtgTTCCATCCCTGTGGcggtg Cas9-Pol-2Cas9-Pol-2 SEQ ID NO:12SEQ ID NO: 12
gtcagatttatgcgttttGtcagatttatgcgtttt Cas9-Pol-3Cas9-Pol-3 SEQ ID NO:13SEQ ID NO: 13
GCATAAATCTGACcggtgGCATAAATCTGACcggtg Cas9-Pol-3Cas9-Pol-3 SEQ ID NO:14SEQ ID NO: 14
cctggattcctgaatgttttCctggattcctgaatgtttt Cas9-Pol-4Cas9-Pol-4 SEQ ID NO:15SEQ ID NO: 15
ATTCAGGAATCCAGGcggtgATTCAGGAATCCAGGcggtg Cas9-Pol-4Cas9-Pol-4 SEQ ID NO:16SEQ ID NO: 16
tcagaagtacacatcccagTcagaagtacacatcccag Cas9-vif-1Cas9-vif-1 SEQ ID NO:17SEQ ID NO: 17
GATGTGTACTTCTGAcggtgGATGTGTACTTCTGAcggtg Cas9-vif-1Cas9-vif-1 SEQ ID NO:18SEQ ID NO:18
acatattggggtctgcatacgttttAcatattggggtctgcatacgtttt Cas9-vif-2Cas9-vif-2 SEQ ID NO:19SEQ ID NO: 19
CAGACCCCAATATGTcggtgCAGACCCCAATATGTcggtg Cas9-vif-2Cas9-vif-2 SEQ ID NO:20SEQ ID NO: 20
gccagggagtctccatagaagttttGccagggagtctccatagaagtttt Cas9-vif-3Cas9-vif-3 SEQ ID NO:21SEQ ID NO: 21
TTCTATGGAGACTCCcggtgTTCTATGGAGACTCCcggtg Cas9-vif-3Cas9-vif-3 SEQ ID NO:22SEQ ID NO: 22
gatctctacaatactgttttGatctctacaatactgtttt Cas9-vif-4Cas9-vif-4 SEQ ID NO:23SEQ ID NO: 23
AGTATTGTAGAGATCcggtgAGTATTGTAGAGATCcggtg Cas9-vif-4Cas9-vif-4 SEQ ID NO:24SEQ ID NO:24
atggctccatagcttgttttAtggctccatagcttgtttt Cas9-vpr-1Cas9-vpr-1 SEQ ID NO:25SEQ ID NO: 25
AAGCTATGGAGCCATcggtgAAGCTATGGAGCCATcggtg Cas9-vpr-1Cas9-vpr-1 SEQ ID NO:26SEQ ID NO:26
ggagatacttggacgttttGgagatacttggacgtttt Cas9-vpr-2Cas9-vpr-2 SEQ ID NO:27SEQ ID NO:27
GTCCAAGTATCTCCcggtgGTCCAAGTATCTCCcggtg Cas9-vpr-2Cas9-vpr-2 SEQ ID NO:28SEQ ID NO:28
tgccaacatagcagaatgttttTgccaacatagcagaatgtttt Cas9-vpr-3Cas9-vpr-3 SEQ ID NO:29SEQ ID NO:29
ATTCTGCTATGTTGGCAcggtgATTCTGCTATGTTGGCAcggtg Cas9-vpr-3Cas9-vpr-3 SEQ ID NO:30SEQ ID NO: 30
agccctggaagcatccgttttAgccctggaagcatccgtttt Cas9-tat-1Cas9-tat-1 SEQ ID NO:31SEQ ID NO: 31
GGATGCTTCCAGGGCTcggtgGGATGCTTCCAGGGCTcggtg Cas9-tat-1Cas9-tat-1 SEQ ID NO:32SEQ ID NO:32
ccaggaagtcagcctgttttCcaggaagtcagcctgtttt Cas9-tat-2Cas9-tat-2 SEQ ID NO:33SEQ ID NO:33
AGGCTGACTTCCTGGcggtgAGGCTGACTTCCTGGcggtg Cas9-tat-2Cas9-tat-2 SEQ ID NO:34SEQ ID NO: 34
atggcaggaagaaggttttAtggcaggaagaaggtttt Cas9-tat-3Cas9-tat-3 SEQ ID NO:35SEQ ID NO: 35
CTTCTTCCTGCCATAcggtgCTTCTTCCTGCCATAcggtg Cas9-tat-3Cas9-tat-3 SEQ ID NO:36SEQ ID NO:36
cgaaggaatcgaagagttttCgaaggaatcgaagagtttt Cas9-tat-4Cas9-tat-4 SEQ ID NO:37SEQ ID NO:37
TCTTCGATTCCTTCGcggtgTCTTCGATTCCTTCGcggtg Cas9-tat-4Cas9-tat-4 SEQ ID NO:38SEQ ID NO:38
tcttagcactgttctgttttTcttagcactgttctgtttt Cas9-rev2-1Cas9-rev2-1 SEQ ID NO:39SEQ ID NO:39
AGAACAGTGCTAAGAcggtgAGAACAGTGCTAAGAcggtg Cas9-rev2-1Cas9-rev2-1 SEQ ID NO:40SEQ ID NO:40
acttactcttgattgtagcggttttActtactcttgattgtagcggtttt Cas9-rev2-2Cas9-rev2-2 SEQ ID NO:41SEQ ID NO:41
ACAATCAAGAGTAAGTcggtgACAATCAAGAGTAAGTcggtg Cas9-rev2-2Cas9-rev2-2 SEQ ID NO:42SEQ ID NO:42
ggtgggaagtcctcatgttttGgtgggaagtcctcatgtttt Cas9-rev2-3Cas9-rev2-3 SEQ ID NO:43SEQ ID NO:43
ATATTTGAGGACTTCCCACCcggtgATATTTGAGGACTTCCCACCcggtg Cas9-rev2-3Cas9-rev2-3 SEQ ID NO:44SEQ ID NO: 44
caataatagcaatagttatagttttCaataatagcaatagttatagtttt Cas9-vpu-1Cas9-vpu-1 SEQ ID NO:45SEQ ID NO:45
TATAACTATTGCTATTATTGcggtgTATAACTATTGCTATTATTGcggtg Cas9-vpu-1Cas9-vpu-1 SEQ ID NO:46SEQ ID NO:46
ccatagtattaataaaatatgttttCcatagtattaataaaatatgtttt Cas9-vpu-2Cas9-vpu-2 SEQ ID NO:47SEQ ID NO:47
ATATTTTATTAATACTATGGcggtgATATTTTATTAATACTATGGcggtg Cas9-vpu-2Cas9-vpu-2 SEQ ID NO:48SEQ ID NO:48
catggtagaccagatgcatggttttCatggtagaccagatgcatggtttt Cas9-env-1Cas9-env-1 SEQ ID NO:49SEQ ID NO:49
CATGCATCTGGTCTACCATGcggtgCATGCATCTGGTCTACCATGcggtg Cas9-env-1Cas9-env-1 SEQ ID NO:50SEQ ID NO: 50
tgctctttcaatatcaccacgttttTgctctttcaatatcaccacgtttt Cas9-env-2Cas9-env-2 SEQ ID NO:51SEQ ID NO: 51
GTGGTGATATTGAAAGAGCAcggtgGTGGTGATATTGAAAGAGCAcggtg Cas9-env-2Cas9-env-2 SEQ ID NO:52SEQ ID NO:52
gtctagcagaagaaggttttGtctagcagaagaaggtttt Cas9-env-3Cas9-env-3 SEQ ID NO:53SEQ ID NO:53
CTTCTTCTGCTAGACcggtgCTTCTTCTGCTAGACcggtg Cas9-env-3Cas9-env-3 SEQ ID NO:54SEQ ID NO: 54
ctgctattaacaagagagttttCtgctattaacaagagagtttt Cas9-env-4Cas9-env-4 SEQ ID NO:55SEQ ID NO: 55
TCTCTTGTTAATAGCAGcggtgTCTCTTGTTAATAGCAGcggtg Cas9-env-4Cas9-env-4 SEQ ID NO:56SEQ ID NO:56
aatgggatggcctgctgtaagttttAatgggatggcctgctgtaagtttt Cas9-nef-1Cas9-nef-1 SEQ ID NO:57SEQ ID NO:57
TTACAGCAGGCCATCCCATTcggtgTTACAGCAGGCCATCCCATTcggtg Cas9-nef-1Cas9-nef-1 SEQ ID NO:58SEQ ID NO: 58
gagctgagccagcagcagatgttttGagctgagccagcagcagatgtttt Cas9-nef-2Cas9-nef-2 SEQ ID NO:59SEQ ID NO:59
ATCTGCTGCTGGCTCAGCTCcggtgATCTGCTGCTGGCTCAGCTCcggtg Cas9-nef-2Cas9-nef-2 SEQ ID NO:60SEQ ID NO: 60
gggagcagcatctagagaccgttttGggagcagcatctagagaccgtttt Cas9-nef-3Cas9-nef-3 SEQ ID NO:61SEQ ID NO: 61
GGTCTCTAGATGCTGCTCCCcggtgGGTCTCTAGATGCTGCTCCCcggtg Cas9-nef-3Cas9-nef-3 SEQ ID NO:62SEQ ID NO: 62
tgcctggctagaagcacaaggttttTgcctggctagaagcacaaggtttt Cas9-nef-4Cas9-nef-4 SEQ ID NO:63SEQ ID NO: 63
CTTGTGCTTCTAGCCAGGCAcggtgCTTGTGCTTCTAGCCAGGCAcggtg Cas9-nef-4Cas9-nef-4 SEQ ID NO:64SEQ ID NO: 64
gatttccactgacctttggagttttGatttccactgacctttggagtttt Cas9-3'LTR-1Cas9-3'LTR-1 SEQ ID NO:65SEQ ID NO: 65
TCCAAAGGTCAGTGGAAATCcggtgTCCAAAGGTCAGTGGAAATCcggtg Cas9-3'LTR-1Cas9-3'LTR-1 SEQ ID NO:66SEQ ID NO:66
cggagaaagaagtgttagtggttttCggagaaagaagtgttagtggtttt Cas9-3'LTR-2Cas9-3'LTR-2 SEQ ID NO:67SEQ ID NO:67
CACTAACACTTCTTTCTCCGcggtgCACTAACACTTCTTTCTCCGcggtg Cas9-3'LTR-2Cas9-3'LTR-2 SEQ ID NO:68SEQ ID NO:68
ctttccgctggggactttccgttttCtttccgctggggactttccgtttt Cas9-3'LTR-3Cas9-3'LTR-3 SEQ ID NO:69SEQ ID NO: 69
GGAAAGTCCCCAGCGGAAAGcggtgGGAAAGTCCCCAGCGGAAAGcggtg Cas9-3'LTR-3Cas9-3'LTR-3 SEQ ID NO:70SEQ ID NO: 70
gtcagtattaagtgcgttttGtcagtattaagtgcgtttt Cas9-Gag-1Cas9-Gag-1 SEQ ID NO:71SEQ ID NO:71
GCACTTAATACTGACcggtgGCACTTAATACTGACcggtg Cas9-Gag-1Cas9-Gag-1 SEQ ID NO:72SEQ ID NO:72
cacaggaaaaggcagccgttttCacaggaaaaggcagccgtttt Cas9-Gag-2Cas9-Gag-2 SEQ ID NO:73SEQ ID NO:73
GGCTGCCTTTTCCTGTGcggtgGGCTGCCTTTTCCTGTGcggtg Cas9-Gag-2Cas9-Gag-2 SEQ ID NO:74SEQ ID NO:74
gaacgatttgcagtcaagttttGaacgatttgcagtcaagtttt Cas9-Gag-3Cas9-Gag-3 SEQ ID NO:75SEQ ID NO:75
TTGACTGCAAATCGTTCcggtgTTGACTGCAAATCGTTCcggtg Cas9-Gag-3Cas9-Gag-3 SEQ ID NO:76SEQ ID NO:76
ggaagctttagagaagttttGgaagctttagagaagtttt Cas9-Gag-4Cas9-Gag-4 SEQ ID NO:77SEQ ID NO:77
TTCTCTAAAGCTTCCcggtgTTCTCTAAAGCTTCCcggtg Cas9-Gag-4Cas9-Gag-4 SEQ ID NO:78SEQ ID NO:78
(2)将sgRNA构建至表达载体(2) Construction of sgRNA into expression vector
a)：sgRNA的PCR扩增a): PCR amplification of sgRNA
反应体系：无菌ddH2O：37.5μL，10×PCR Buffer(含MgCl2)：5μL，2.5mM dNTP：4μL，相应的上、下游引物各1μL(引物浓度50pmol/μL)，模板DNA(50ng/μL)：1μL，PyrobestTM DNA Polymerase：0.5μL。温和震荡混匀后，按表2所示条件进行PCR扩增。Reaction system: Sterile ddH 2 O: 37.5 μL, 10×PCR Buffer (containing MgCl 2 ): 5 μL, 2.5 mM dNTP: 4 μL, corresponding 1 μL of each of the upstream and downstream primers (primer concentration 50 pmol/μL), template DNA (50 ng /μL): 1 μL, PyrobestTM DNA Polymerase: 0.5 μL. After gently shaking and mixing, PCR amplification was carried out under the conditions shown in Table 2.
表2：PCR扩增条件Table 2: PCR amplification conditions
  预变性Pre-denaturation 变性transsexual 退火annealing 延伸extend 循环cycle 延伸extend
GagGag 95℃,3min95 ° C, 3 min 95℃,30sec95°C, 30sec 63℃,30sec63°C, 30sec 72℃,1min72 ° C, 1 min 30个30 72℃,8min72 ° C, 8 min
EnvEnv 95℃,3min95 ° C, 3 min 95℃,30sec95°C, 30sec 63℃,30sec63°C, 30sec 72℃,30sec72°C, 30sec 30个30 72℃,8min72 ° C, 8 min
PolPol 95℃,3min95 ° C, 3 min 95℃,30sec95°C, 30sec 58℃,30sec58°C, 30sec 72℃,30sec72°C, 30sec 30个30 72℃,8min72 ° C, 8 min
TatTat 95℃,3min95 ° C, 3 min 95℃,30sec95°C, 30sec 51℃,30sec51 ° C, 30 sec 72℃,1min72 ° C, 1 min 30个30 72℃,8min72 ° C, 8 min
VifVif 95℃,3min95 ° C, 3 min 95℃,30sec95°C, 30sec 51℃,30sec51 ° C, 30 sec 72℃,30sec72°C, 30sec 30个30 72℃,8min72 ° C, 8 min
NefNef 95℃,3min95 ° C, 3 min 95℃,30sec95°C, 30sec 64℃,30sec64°C, 30sec 72℃,30sec72°C, 30sec 30个30 72℃,8min72 ° C, 8 min
VpuVpu 95℃,3min95 ° C, 3 min 95℃,30sec95°C, 30sec 60℃,30sec60 ° C, 30 sec 72℃,30sec72°C, 30sec 30个30 72℃,8min72 ° C, 8 min
VprVpr 95℃,3min95 ° C, 3 min 95℃,30sec95°C, 30sec 67℃,30sec67°C, 30sec 72℃,30sec72°C, 30sec 30个30 72℃,8min72 ° C, 8 min
3’LTR3’LTR 95℃,3min95 ° C, 3 min 95℃,30sec95°C, 30sec 67℃,30sec67°C, 30sec 72℃,30sec72°C, 30sec 30个30 72℃,8min72 ° C, 8 min
5’LTR5’LTR 95℃,3min95 ° C, 3 min 95℃,30sec95°C, 30sec 58℃,30sec58°C, 30sec 72℃,30sec72°C, 30sec 30个30 72℃,8min72 ° C, 8 min
RevRev 95℃,3min95 ° C, 3 min 95℃,30sec95°C, 30sec 51℃,30sec51 ° C, 30 sec 72℃,1min72 ° C, 1 min 30个30 72℃,8min72 ° C, 8 min
b)：载体构建及鉴定b): vector construction and identification
用Quigen生产的PCR回收试剂盒按照常规方法进行PCR扩增产物的回收，然后用SalI单酶切PCR片段，SalI和EcoRV双酶切质粒载体p1.0，两者进行连接，转化Top10或DH5α感受态，以SalI和EcoRV酶切筛选出阳性克隆并送Invitrogen公司测序。PCR amplification kits were used to recover PCR amplification products according to the conventional method. Then, the PCR fragments were digested with SalI, and the plasmid vector p1.0 was double-digested with SalI and EcoRV. The two were ligated to transform Top10 or DH5α. Positive clones were screened by SalI and EcoRV and sent to Invitrogen for sequencing.
(3)HIV抑制测试(3) HIV inhibition test
选用TZM-BL细胞株，预先用HIV感染6小时，再用纳米载体系统将本发明的CRISPR-Cas9系统递送进入细胞，引导CRISPR-Cas9在细胞内的表达。培养细胞2天后，检测HIV的产量。将单独只感染HIV的细胞组设为阴性对照，既不加HIV也不加CRISPR-Cas9的细胞组设为空白对照；加HIV和已上市的抗HIV药物T20的细胞组设为阳性对照。测试结果如表3所示。The TZM-BL cell line was selected and previously infected with HIV for 6 hours, and the CRISPR-Cas9 system of the present invention was delivered into the cells using a nanocarrier system to direct the expression of CRISPR-Cas9 in the cells. Two days after the cells were cultured, the yield of HIV was examined. The cell group in which only HIV was infected alone was set as a negative control, and the cell group in which neither HIV nor CRISPR-Cas9 was added as a blank control; the cell group to which HIV and the marketed anti-HIV drug T20 were added was set as a positive control. The test results are shown in Table 3.
表3：HIV抑制测试结果Table 3: HIV inhibition test results
CRISPR-Cas9系统CRISPR-Cas9 System 抑制率Inhibition rate
CRISPR-Cas9(Env)CRISPR-Cas9(Env) 0.960.96
CRISPR-Cas9(Pol)CRISPR-Cas9(Pol) 0.960.96
CRISPR-Cas9(Gag)CRISPR-Cas9(Gag) 0.960.96
CRISPR-Cas9(3’LTR)CRISPR-Cas9(3’LTR) 0.920.92
CRISPR-Cas9(5’LTR)CRISPR-Cas9(5’LTR) 0.930.93
CRISPR-Cas9(Vif)CRISPR-Cas9(Vif) 0.960.96
CRISPR-Cas9(Rev)CRISPR-Cas9(Rev) 0.950.95
CRISPR-Cas9(Tat)CRISPR-Cas9(Tat) 0.960.96
CRISPR-Cas9(Nef)CRISPR-Cas9(Nef) 0.900.90
CRISPR-Cas9(Vpr)CRISPR-Cas9(Vpr) 0.880.88
CRISPR-Cas9(Vpu)CRISPR-Cas9(Vpu) 0.730.73
阳性对照Positive control 0.900.90
阴性对照Negative control 00
空白对照Blank control 00
从以上结果可以看出，本发明的CRISPR-Cas9系统对于HIV的抑制率为73％～96％，说明本发明的CRISPR-Cas9系统能抑制HIV在细胞内的产生；阳性对照是已上市的抗HIV药物，其抑制率为90％。单从抑制率来讲，本发明的CRISPR-Cas9系统达到了和市售的抗HIV药物以及肽类抗HIV药物相当的效果。From the above results, it can be seen that the CRISPR-Cas9 system of the present invention has an inhibition rate of HIV of 73% to 96%, indicating that the CRISPR-Cas9 system of the present invention can inhibit the production of HIV in cells; the positive control is a marketed anti-antibody. The HIV drug has an inhibition rate of 90%. In terms of inhibition rate alone, the CRISPR-Cas9 system of the present invention achieves comparable effects to commercially available anti-HIV drugs and peptide anti-HIV drugs.
试剂盒Kit
本发明还提供了一种试剂盒，该试剂盒包括本发明的CRISPR-Cas9系统或按照本发明的方法而制备的CRISPR-Cas9系统，或表达所述CRISPR-Cas9系统的表达盒或重组载体。The present invention also provides a kit comprising the CRISPR-Cas9 system of the present invention or the CRISPR-Cas9 system prepared according to the method of the present invention, or an expression cassette or recombinant vector expressing the CRISPR-Cas9 system.
该试剂盒中还可以包括用于PCR扩增、载体构建等所需的各种试剂，包括但不限于扩增缓冲液、引物、模板DNA、酶等。Various reagents required for PCR amplification, vector construction, and the like, including but not limited to amplification buffers, primers, template DNA, enzymes, and the like, can also be included in the kit.
此外，所述试剂盒中还可包括使用说明书和/或使用/分析软件。In addition, instructions for use and/or use/analysis software may also be included in the kit.
药物组合物Pharmaceutical composition
本发明还提供了一种药物组合物，该药物组合物包括药学上可接受的载体，以及本发明的CRISPR-Cas9系统或按照本发明的方法而制备的CRISPR-Cas9系统。所述CRISPR-Cas9系统在该药物组合物中可以为有效量的或治疗有效量的。The invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier, and a CRISPR-Cas9 system of the invention or a CRISPR-Cas9 system prepared according to the methods of the invention. The CRISPR-Cas9 system can be an effective amount or a therapeutically effective amount in the pharmaceutical composition.
如本文所用，“有效量”是指可对人和/或动物产生功能或活性的且可被人和/或动物所接受的量。As used herein, "effective amount" refers to an amount that is functional or active to a human and/or animal and that is acceptable to humans and/or animals.
如本文所用，“药学上可接受的”的成分是适用于人和/或动物(如哺乳动物和禽类)而无过度不良副反应(如毒性、刺激和变态反应)的，即具有合理的效益/风险比的物质。“药学上可接受的载体”是指用于给药的载体，可以包括各种赋形剂和稀释剂等。As used herein, a "pharmaceutically acceptable" ingredient is suitable for use in humans and/or animals (eg, mammals and birds) without excessive adverse side effects (eg, toxicity, irritation, and allergies), ie, has reasonable benefits / risk ratio substance. "Pharmaceutically acceptable carrier" means a carrier for administration, and may include various excipients, diluents and the like.
本发明的药物组合物可以含有安全有效量的本发明的CRISPR-Cas9系统作为活性成分以及药学上可接受的载体。这类载体可以包括但不限于：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。通常药物制剂应与给药方式相匹配，本发明的药物组合物的剂型可以依需要制备为注射剂、口服制剂(片剂、胶囊、口服液)、透皮剂、稀释剂等。例如用生理盐水或含有葡萄糖和其他辅 料的水溶液通常以常规方法进行制备。该药物组合物更适宜在无菌条件下制造。The pharmaceutical composition of the present invention may contain a safe and effective amount of the CRISPR-Cas9 system of the present invention as an active ingredient together with a pharmaceutically acceptable carrier. Such carriers can include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical preparation should be matched with the administration mode, and the dosage form of the pharmaceutical composition of the present invention can be prepared as an injection, an oral preparation (tablet, capsule, oral liquid), a transdermal agent, a diluent, and the like as needed. For example with saline or with glucose and other supplements The aqueous solution of the material is usually prepared in a conventional manner. The pharmaceutical composition is more suitably manufactured under sterile conditions.
本发明所述的有效量可随给药的模式和待治疗的疾病的严重程度等而变化。优选的有效量的选择可以由本领域普通技术人员根据各种因素来确定(例如通过临床试验)。所述的因素包括但不限于：所述的活性成分的药代动力学参数，例如生物利用率、代谢、半衰期等；患者所要治疗的疾病的严重程度、患者的体重、患者的免疫状况、给药的途径等。通常，当本发明的活性成分每天以约0.00001mg-50mg/kg动物体重(较佳的0.0001mg-10mg/kg动物体重)的剂量给予，能得到令人满意的效果。例如，由治疗状况的迫切要求，可每天给予若干次分开的剂量，或将剂量按比例地减少。The effective amount of the present invention may vary depending on the mode of administration and the severity of the disease to be treated and the like. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., by clinical trials). The factors include, but are not limited to, the pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, The route of medicine, etc. In general, when the active ingredient of the present invention is administered at a dose of about 0.00001 mg to 50 mg/kg of animal body weight per day (preferably 0.0001 mg to 10 mg/kg of animal body weight), a satisfactory effect can be obtained. For example, several separate doses may be administered per day, or the dose may be proportionally reduced, as is critical to the condition of the treatment.
本发明所述的药学上可接受的载体包括但不限于：水、盐水、脂质体、脂质、蛋白、蛋白-抗体缀合物、肽类物质、纤维素、纳米凝胶、或其组合。载体的选择通常应与给药方式相匹配，这是本领域的普通技术人员所熟知的。Pharmaceutically acceptable carriers of the invention include, but are not limited to, water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptide materials, cellulose, nanogels, or combinations thereof . The choice of carrier should generally be matched to the mode of administration, as is well known to those of ordinary skill in the art.
本发明还提供了所述药物组合物的应用，其用于制备预防和/或治疗HIV感染的药物。The invention also provides the use of the pharmaceutical composition for the preparation of a medicament for the prevention and/or treatment of HIV infection.
预防和/或治疗方法Prevention and/or treatment
本发明还提供了一种预防和/或治疗HIV感染的方法。在优选的实施方案中，该方法包括向需要的对象给予治疗有效量的本发明的CRISPR-Cas9系统或按照本发明的方法而制备的CRISPR-Cas9系统，或本发明的药物组合物。The invention also provides a method of preventing and/or treating HIV infection. In a preferred embodiment, the method comprises administering to a subject in need thereof a therapeutically effective amount of a CRISPR-Cas9 system of the invention or a CRISPR-Cas9 system prepared according to the methods of the invention, or a pharmaceutical composition of the invention.
申请人声明，本发明通过上述实施例来说明本发明的工艺方法，但本发明并不局限于上述工艺步骤，即不意味着本发明必须依赖上述工艺步骤才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明所选用原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。 The Applicant declares that the present invention illustrates the process of the present invention by the above-described embodiments, but the present invention is not limited to the above process steps, that is, it does not mean that the present invention must rely on the above process steps to be implemented. It will be apparent to those skilled in the art that any modifications of the present invention, equivalent substitutions of the materials selected for the present invention, and the addition of the auxiliary ingredients, the selection of the specific means, etc., are all within the scope of the present invention.
Figure PCTCN2016076638-appb-000001
Figure PCTCN2016076638-appb-000001
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Claims (10)

  1. 一种CRISPR-Cas9系统，该系统包括特异性靶向HIV基因组上的特定基因位点的sgRNA，所述HIV基因组上的特定基因位点包括Gag、Env、Pol、Tat、Nef、Vif、Vpr、Vpu、5’LTR、3’LTR和Rev中的一个或多个；优选地，所述HIV基因组上的特定基因位点选自Gag、Env、Pol、Tat、Nef、Vif、Vpr、Vpu、5’LTR、3’LTR和Rev中的一个或多个。A CRISPR-Cas9 system comprising sgRNAs that specifically target specific gene loci on the HIV genome, specific genetic loci on the HIV genome including Gag, Env, Pol, Tat, Nef, Vif, Vpr, One or more of Vpu, 5'LTR, 3'LTR and Rev; preferably, the specific genetic locus on the HIV genome is selected from the group consisting of Gag, Env, Pol, Tat, Nef, Vif, Vpr, Vpu, 5 One or more of 'LTR, 3' LTR and Rev.
  2. 根据权利要求1所述的CRISPR-Cas9系统，其特征在于，特异性靶向每个所述特定基因位点的sgRNA各自独立地为1条以上，优选为1～11条，更优选为4条；进一步优选地，每条所述sgRNA的长度各自独立地为16-22bp。The CRISPR-Cas9 system according to claim 1, wherein each of the sgRNAs specifically targeting each of the specific gene loci is independently one or more, preferably from 1 to 11, more preferably four. Further preferably, the length of each of the sgRNAs is independently 16-22 bp.
  3. 根据权利要求1或2所述的CRISPR-Cas9系统，其特征在于，所述CRISPR-Cas9系统还包括Cas9；优选地，所述Cas9和所述特异性靶向HIV基因组上的特定基因位点的sgRNA分别存在于质粒中。The CRISPR-Cas9 system according to claim 1 or 2, wherein the CRISPR-Cas9 system further comprises Cas9; preferably, the Cas9 and the specific targeting of a specific gene locus on the HIV genome sgRNA is present in the plasmid, respectively.
  4. 权利要求1至3中任一项所述的CRISPR-Cas9系统的制备方法，该方法包括通过PCR扩增获得sgRNA片段的步骤；A method of producing a CRISPR-Cas9 system according to any one of claims 1 to 3, which comprises the step of obtaining an sgRNA fragment by PCR amplification;
    优选地，所述PCR扩增采用的引物选自Cas9-5'LTR-1引物对、Cas9-5'LTR-2引物对、Cas9-5'LTR-3引物对、Cas9-5'LTR-4引物对、Cas9-Pol-1引物对、Cas9-Pol-2引物对、Cas9-Pol-3引物对、Cas9-Pol-4引物对、Cas9-vif-1引物对、Cas9-vif-2引物对、Cas9-vif-3引物对、Cas9-vif-4引物对、Cas9-vpr-1引物对、Cas9-vpr-2引物对、Cas9-vpr-3引物对、Cas9-tat-1引物对、Cas9-tat-2引物对、Cas9-tat-3引物对、Cas9-tat-4引物对、Cas9-rev2-1引物对、Cas9-rev2-2引物对、Cas9-rev2-3引物对、Cas9-vpu-1引物对、Cas9-vpu-2引物对、Cas9-env-1引物对、Cas9-env-2引物对、Cas9-env-3引物对、Cas9-env-4引物对、Cas9-nef-1引物对、Cas9-nef-2引物对、Cas9-nef-3引物对、Cas9-nef-4引物对、Cas9-3'LTR-1引物对、Cas9-3'LTR-2引物对、Cas9-3'LTR-3引物对、 Cas9-Gag-1引物对、Cas9-Gag-2引物对、Cas9-Gag-3引物对和Cas9-Gag-4引物对中的一个或多个；Preferably, the primer used for the PCR amplification is selected from the group consisting of a Cas9-5'LTR-1 primer pair, a Cas9-5'LTR-2 primer pair, a Cas9-5'LTR-3 primer pair, and a Cas9-5'LTR-4. Primer pair, Cas9-Pol-1 primer pair, Cas9-Pol-2 primer pair, Cas9-Pol-3 primer pair, Cas9-Pol-4 primer pair, Cas9-vif-1 primer pair, Cas9-vif-2 primer pair , Cas9-vif-3 Primer Pair, Cas9-vif-4 Primer Pair, Cas9-vpr-1 Primer Pair, Cas9-vpr-2 Primer Pair, Cas9-vpr-3 Primer Pair, Cas9-tat-1 Primer Pair, Cas9 -tat-2 Primer Pair, Cas9-tat-3 Primer Pair, Cas9-tat-4 Primer Pair, Cas9-rev2-1 Primer Pair, Cas9-rev2-2 Primer Pair, Cas9-rev2-3 Primer Pair, Cas9-vpu -1 Primer Pair, Cas9-vpu-2 Primer Pair, Cas9-env-1 Primer Pair, Cas9-env-2 Primer Pair, Cas9-env-3 Primer Pair, Cas9-env-4 Primer Pair, Cas9-nef-1 Primer pair, Cas9-nef-2 primer pair, Cas9-nef-3 primer pair, Cas9-nef-4 primer pair, Cas9-3'LTR-1 primer pair, Cas9-3'LTR-2 primer pair, Cas9-3 'LTR-3 primer pair, One or more of a Cas9-Gag-1 primer pair, a Cas9-Gag-2 primer pair, a Cas9-Gag-3 primer pair, and a Cas9-Gag-4 primer pair;
    更优选地，所述引物对碱基序列按5'-3'方向如下：More preferably, the primer pair base sequence is in the 5'-3' direction as follows:
    Cas9-5'LTR-1引物对：Cas9-5'LTR-1 Primer Pair:
    gacaagatatccttggtttt，和CAAGGATATCTTGTCcggtg；Gacaagatatccttggtttt, and CAAGGATATCTTGTCcggtg;
    Cas9-5'LTR-2引物对：Cas9-5'LTR-2 Primer Pair:
    cctatgagcctgcagtttt，和TGCAGGCTCATAGGcggtg；Cctatgagcctgcagtttt, and TGCAGGCTCATAGGcggtg;
    Cas9-5'LTR-3引物对：Cas9-5'LTR-3 Primer Pair:
    gctttttgcctgtagtttt，和TACAGGCAAAAAGCcggtg；Gctttttgcctgtagtttt, and TACAGGCAAAAAGCcggtg;
    Cas9-5'LTR-4引物对：Cas9-5'LTR-4 Primer Pair:
    gacccttttagtcagtggtttt，和CACTGACTAAAAGGGTCcggtg；Gacccttttagtcagtggtttt, and CACTGACTAAAAGGGTCcggtg;
    Cas9-Pol-1引物对：Cas9-Pol-1 Primer Pair:
    aataccacatcccgcagtttt，和TGCGGGATGTGGTATTCcggtg；Aataccacatcccgcagtttt, and TGCGGGATGTGGTATTCcggtg;
    Cas9-Pol-2引物对：Cas9-Pol-2 Primer Pair:
    ccacagggatggaagtttt，和TTCCATCCCTGTGGcggtg；Ccacagggagtggaagtttt, and TTCCATCCCTGTGGcggtg;
    Cas9-Pol-3引物对：Cas9-Pol-3 Primer Pair:
    gtcagatttatgcgtttt，和GCATAAATCTGACcggtg；Gtcagatttatgcgtttt, and GCATAAATCTGACcggtg;
    Cas9-Pol-4引物对：Cas9-Pol-4 Primer Pair:
    cctggattcctgaatgtttt，和ATTCAGGAATCCAGGcggtg；Cctggattcctgaatgtttt, and ATTCAGGAATCCAGGcggtg;
    Cas9-vif-1引物对：Cas9-vif-1 Primer Pair:
    tcagaagtacacatcccag，和GATGTGTACTTCTGAcggtg；Tcagaagtacacatcccag, and GATGTGTACTTCTGAcggtg;
    Cas9-vif-2引物对：Cas9-vif-2 Primer Pair:
    acatattggggtctgcatacgtttt，和CAGACCCCAATATGTcggtg；Acatattggggtctgcatacgtttt, and CAGACCCCAATATGTcggtg;
    Cas9-vif-3引物对：Cas9-vif-3 Primer Pair:
    gccagggagtctccatagaagtttt，和TTCTATGGAGACTCCcggtg；Gccagggagtctccatagaagtttt, and TTCTATGGAGACTCCcggtg;
    Cas9-vif-4引物对：Cas9-vif-4 Primer Pair:
    gatctctacaatactgtttt，和AGTATTGTAGAGATCcggtg；Gatctctacaatactgtttt, and AGTATTGTAGAGATCcggg;
    Cas9-vpr-1引物对：Cas9-vpr-1 Primer Pair:
    atggctccatagcttgtttt，和AAGCTATGGAGCCATcggtg；Atggctccatagcttgtttt, and AAGGTATGGAGCCATcggtg;
    Cas9-vpr-2引物对：Cas9-vpr-2 Primer Pair:
    ggagatacttggacgtttt，和GTCCAAGTATCTCCcggtg；Ggagatacttggacgtttt, and GTCCAAGTATCTCCcggtg;
    Cas9-vpr-3引物对： Cas9-vpr-3 Primer Pair:
    tgccaacatagcagaatgtttt，和ATTCTGCTATGTTGGCAcggtg；Tgccaacatagcagaatgtttt, and ATTCTGCTATGTTGGCAcggtg;
    Cas9-tat-1引物对：Cas9-tat-1 Primer Pair:
    agccctggaagcatccgtttt，和GGATGCTTCCAGGGCTcggtg；Agccctggaagcatccgtttt, and GGATGCTTCCAGGGCTcggtg;
    Cas9-tat-2引物对：Cas9-tat-2 Primer Pair:
    ccaggaagtcagcctgtttt，和AGGCTGACTTCCTGGcggtg；Ccaggaagtcagcctgtttt, and AGGCTGACTTCCTGGcggtg;
    Cas9-tat-3引物对：Cas9-tat-3 Primer Pair:
    atggcaggaagaaggtttt，和CTTCTTCCTGCCATAcggtg；Atggcaggaagaaggtttt, and CTTCTTCCTGCCATAcggtg;
    Cas9-tat-4引物对：Cas9-tat-4 Primer Pair:
    cgaaggaatcgaagagtttt，和TCTTCGATTCCTTCGcggtg；Cgaaggaatcgaagagtttt, and TCTTCGATTCCTTCGcggtg;
    Cas9-rev2-1引物对：Cas9-rev2-1 Primer Pair:
    tcttagcactgttctgtttt，和AGAACAGTGCTAAGAcggtg；Tcttagcactgttctgtttt, and AGAACAGTGCTAAGAcggtg;
    Cas9-rev2-2引物对：Cas9-rev2-2 Primer Pair:
    acttactcttgattgtagcggtttt，和ACAATCAAGAGTAAGTcggtg；Acttactcttgattgtagcggtttt, and ACAATCAAGAGTAAGTcggtg;
    Cas9-rev2-3引物对：Cas9-rev2-3 Primer Pair:
    ggtgggaagtcctcatgtttt，和ATATTTGAGGACTTCCCACCcggtg；Ggtgggaagtcctcatgtttt, and ATATTTGAGGACTTCCCACCcggtg;
    Cas9-vpu-1引物对：Cas9-vpu-1 Primer Pair:
    caataatagcaatagttatagtttt，和TATAACTATTGCTATTATTGcggtg；Caataatagcaatagttatagtttt, and TATAACTATTGCTATTATTGcggtg;
    Cas9-vpu-2引物对：Cas9-vpu-2 Primer Pair:
    ccatagtattaataaaatatgtttt，和ATATTTTATTAATACTATGGcggtg；Ccatagtattaataaaatatgtttt, and ATATTTTATTAATACTATGGcggtg;
    Cas9-env-1引物对：Cas9-env-1 Primer Pair:
    catggtagaccagatgcatggtttt，和CATGCATCTGGTCTACCATGcggtg；Catggtagaccagatgcatggtttt, and CATGCATCTGGTCTACCATGcggtg;
    Cas9-env-2引物对：Cas9-env-2 Primer Pair:
    tgctctttcaatatcaccacgtttt，和GTGGTGATATTGAAAGAGCAcggtg；Tgctctttcaatatcaccacgtttt, and GTGGTGATATTGAAAGAGCAcggtg;
    Cas9-env-3引物对：Cas9-env-3 Primer Pair:
    gtctagcagaagaaggtttt，和CTTCTTCTGCTAGACcggtg；Gtctagcagaagaaggtttt, and CTTCTTCTGCTAGACcggtg;
    Cas9-env-4引物对：Cas9-env-4 Primer Pair:
    ctgctattaacaagagagtttt，和TCTCTTGTTAATAGCAGcggtg；Ctgctattaacaagagagtttt, and TCTCTTGTTAATAGCAGcggtg;
    Cas9-nef-1引物对：Cas9-nef-1 Primer Pair:
    aatgggatggcctgctgtaagtttt，和TTACAGCAGGCCATCCCATTcggtg；Aatgggatggcctgctgtaagtttt, and TTACAGCAGGCCATCCCATTcggtg;
    Cas9-nef-2引物对：Cas9-nef-2 Primer Pair:
    gagctgagccagcagcagatgtttt，和ATCTGCTGCTGGCTCAGCTCcggtg；Gagctgagccagcagcagatgtttt, and ATCTGCTGCTGGCTCAGCTCcggg;
    Cas9-nef-3引物对： Cas9-nef-3 Primer Pair:
    gggagcagcatctagagaccgtttt，和GGTCTCTAGATGCTGCTCCCcggtg；Gggagcagcatctagagaccgtttt, and GGTCTCTAGATGCTGCTCCCcggtg;
    Cas9-nef-4引物对：Cas9-nef-4 Primer Pair:
    tgcctggctagaagcacaaggtttt，和CTTGTGCTTCTAGCCAGGCAcggtg；Tgcctggctagaagcacaaggtttt, and CTTGTGCTTCTAGCCAGGCAcggtg;
    Cas9-3'LTR-1引物对：Cas9-3'LTR-1 Primer Pair:
    gatttccactgacctttggagtttt，和TCCAAAGGTCAGTGGAAATCcggtg；Gatttccactgacctttggagtttt, and TCCAAAGGTCAGTGGAAATCcggtg;
    Cas9-3'LTR-2引物对：Cas9-3'LTR-2 Primer Pair:
    cggagaaagaagtgttagtggtttt，和CACTAACACTTCTTTCTCCGcggtg；Cggagaaagaagtgttagtggtttt, and CACTAACACTTCTTTCTCCGcggtg;
    Cas9-3'LTR-3引物对：Cas9-3'LTR-3 Primer Pair:
    ctttccgctggggactttccgtttt，和GGAAAGTCCCCAGCGGAAAGcggtg；Ctttccgctggggactttccgtttt, and GGAAAGTCCCCAGCGGAAAGcggtg;
    Cas9-Gag-1引物对：Cas9-Gag-1 Primer Pair:
    gtcagtattaagtgcgtttt，和GCACTTAATACTGACcggtg；Gtcagtattaagtgcgtttt, and GCACTTAATACTGACcggtg;
    Cas9-Gag-2引物对：Cas9-Gag-2 Primer Pair:
    cacaggaaaaggcagccgtttt，和GGCTGCCTTTTCCTGTGcggtg；Cacaggaaaaggcagccgtttt, and GGCTGCCTTTTCCTGTGcggtg;
    Cas9-Gag-3引物对：Cas9-Gag-3 Primer Pair:
    gaacgatttgcagtcaagtttt，和TTGACTGCAAATCGTTCcggtg；Gaacgatttgcagtcaagtttt, and TTGACTGCAAATCGTTCcggtg;
    Cas9-Gag-4引物对：Cas9-Gag-4 Primer Pair:
    ggaagctttagagaagtttt，和TTCTCTAAAGCTTCCcggtg。Ggaagctttagagaagtttt, and TTCTCTAAAGCTTCCcggtg.
  5. 根据权利要求4所述的制备方法，其特征在于，所述PCR扩增的退火温度分别为：The preparation method according to claim 4, wherein the annealing temperatures of the PCR amplification are respectively:
    Gag：63℃；Gag: 63 ° C;
    Env：63℃；Env: 63 ° C;
    Pol：58℃；Pol: 58 ° C;
    Tat：51℃；Tat: 51 ° C;
    Vif：51℃；Vif: 51 ° C;
    Nef：64℃；Nef: 64 ° C;
    Vpu：60℃；Vpu: 60 ° C;
    Vpr：67℃；Vpr: 67 ° C;
    3’LTR：67℃；3'LTR: 67 ° C;
    5’LTR：58℃；5'LTR: 58 ° C;
    Rev：51℃；Rev: 51 ° C;
    优选地，所述PCR扩增的反应条件还包括：95℃预变性3min，95 ℃变性30sec，退火30sec，72℃延伸30sec～1min，30个循环，循环后72℃延伸8min；Preferably, the reaction conditions of the PCR amplification further comprise: pre-denaturation at 95 ° C for 3 min, 95 °C denaturation 30 sec, annealing 30 sec, 72 ° C extension 30 sec ~ 1 min, 30 cycles, 72 ° C after the cycle extension 8 min;
    更优选地，所述制备方法还包括将所扩增的各sgRNA片段分别构建入质粒载体的步骤。More preferably, the preparation method further comprises the step of separately constructing each of the amplified sgRNA fragments into a plasmid vector.
  6. 一种试剂盒，所述试剂盒包括权利要求1至3中任一项所述的CRISPR-Cas9系统或按照权利要求4或5所述的方法制备的CRISPR-Cas9系统，或表达所述CRISPR-Cas9系统的表达盒或重组载体。A kit comprising the CRISPR-Cas9 system according to any one of claims 1 to 3 or the CRISPR-Cas9 system prepared according to the method of claim 4 or 5, or expressing the CRISPR- An expression cassette or recombinant vector of the Cas9 system.
  7. 一种药物组合物，所述药物组合物包括药学上可接受的载体，以及权利要求1至3中任一项所述的CRISPR-Cas9系统或按照权利要求4或5所述的方法制备的CRISPR-Cas9系统。A pharmaceutical composition comprising a pharmaceutically acceptable carrier, and a CRISPR-Cas9 system according to any one of claims 1 to 3 or a CRISPR prepared according to the method of claim 4 or 5. - Cas9 system.
  8. 根据权利要求1至3中任一项所述的CRISPR-Cas9系统或按照权利要求4或5所述的方法制备的CRISPR-Cas9系统或权利要求7所述的药物组合物在制备用于预防和/或治疗HIV感染的药物中的应用。The CRISPR-Cas9 system according to any one of claims 1 to 3 or the CRISPR-Cas9 system prepared according to the method of claim 4 or 5 or the pharmaceutical composition according to claim 7 in preparation for prevention and / or the application of drugs for the treatment of HIV infection.
  9. 一种预防和/或治疗HIV感染的方法，所述方法包括向需要的对象给予治疗有效量的如权利要求1至3中任一项所述的CRISPR-Cas9系统或按照权利要求4或5所述的方法制备的CRISPR-Cas9系统或权利要求7所述的药物组合物；优选地，所述CRISPR-Cas9系统通过高效基因递送系统给药；更优选地，所述CRISPR-Cas9系统通过纳米载体递送给药。A method of preventing and/or treating an HIV infection, the method comprising administering to a subject in need thereof a therapeutically effective amount of the CRISPR-Cas9 system according to any one of claims 1 to 3 or according to claim 4 or 5. The CRISPR-Cas9 system prepared by the method or the pharmaceutical composition of claim 7; preferably, the CRISPR-Cas9 system is administered by a highly efficient gene delivery system; more preferably, the CRISPR-Cas9 system is passed through a nanocarrier Delivery is delivered.
  10. 一种用于预防和/或治疗HIV感染的CRISPR-Cas9系统，所述CRISPR-Cas9系统为权利要求1至3中任一项所述的CRISPR-Cas9系统或按照权利要求4或5所述的方法制备的CRISPR-Cas9系统。 A CRISPR-Cas9 system for preventing and/or treating HIV infection, the CRISPR-Cas9 system being the CRISPR-Cas9 system according to any one of claims 1 to 3 or according to claim 4 or 5. Method of preparation of the CRISPR-Cas9 system.
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