WO2023108405A1 - 重组载体及其构建方法和应用 - Google Patents

重组载体及其构建方法和应用 Download PDF

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WO2023108405A1
WO2023108405A1 PCT/CN2021/137799 CN2021137799W WO2023108405A1 WO 2023108405 A1 WO2023108405 A1 WO 2023108405A1 CN 2021137799 W CN2021137799 W CN 2021137799W WO 2023108405 A1 WO2023108405 A1 WO 2023108405A1
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sgrna
plasmid
recombinant vector
backbone
fragment
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PCT/CN2021/137799
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French (fr)
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朱英杰
陈子君
胡靖怡
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中国科学院深圳先进技术研究院
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    • 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
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    • 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
    • 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
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
    • 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
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
    • 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

Definitions

  • the invention relates to the field of biotechnology, in particular to a recombinant vector and its construction method and application.
  • optogenetics is mainly used to activate specific cells, which has the following limitations: 1Invasive. Optogenetics requires embedding a fiber optic ceramic ferrule and is an invasive procedure. 2Limitation of the scope of activities. If wired optogenetic activation is used, it is necessary to link the optical fiber to the ceramic ferrule, which will limit the range of activity of the mouse; if wireless optogenetic activation is used, a magnetic field needs to be arranged with a coil. In order to ensure the strength of the magnetic field, the mouse can move as little as possible. The scope is also limited. 3 Photothermal effect on cell damage.
  • Optogenetic stimulation usually uses blue light, which has a strong photothermal effect, and if it is activated for a long time or multiple times, it may cause damage to the neurons at the tip of the optical fiber. 4Limitations of applicable scenarios. Due to the limitations of the range of motion mentioned in 2 and 3 above and the damage to cells caused by the photothermal effect, this technology is applicable to limited scenarios. When it is necessary to manipulate specific neurons in mice for a long time or in a complex environment, this technology is not suitable. apply again.
  • a method for constructing a recombinant vector comprising the steps of:
  • a sgRNA fragment is inserted into the sgRNA backbone plasmid with chemical genetics activation elements, and the sgRNA fragment is used to knock out vGAT to obtain the recombinant vector.
  • the construction method of the above-mentioned recombinant vector combines CRISPR gene editing technology and chemical genetic activation technology to obtain a recombinant vector that can be used to knock out vGAT of GABAergic neurons, block the release of GABA, and activate the GABA that knocks out vGAT through chemical genetics. It is convenient to activate specific neurons for a long time, so as to achieve the purpose of long-term activation of GABAergic neurons knocked out of vGAT.
  • Chemogenetic activation technology is only through injection or oral administration of corresponding activation drugs, without invasive
  • the operation is non-invasive, improves the adaptability of animals, and no longer needs to give light stimulation through optical fiber connection to LED, reduces the use of experimental equipment during the experiment, improves convenience, and no longer needs to be connected with optical fiber or in a specific Moving in a magnetic field with a wide range, the mouse can move more widely; it avoids the cell damage caused by the photothermal effect, can activate specific neurons for a long time, and has a wider range of applications.
  • the step of constructing the sgRNA backbone plasmid with chemical genetics activation elements comprises:
  • Plasmid recombination of the original sgRNA backbone plasmid with a chemical genetics plasmid carrying the chemical genetics activation element is carried out using a plasmid recombination technique.
  • the step of constructing the sgRNA backbone plasmid with chemical genetics activation elements comprises:
  • the fragment containing the sgRNA backbone and the fragment containing the chemogenetic activation element are connected to obtain the sgRNA backbone plasmid with the chemogenetic activation element.
  • the original sgRNA backbone plasmid includes AAV-U6-sgRNA(backbone)-pCBh-DIO-ChR2-mCherry plasmid;
  • the chemical genetics plasmid includes pAAV-hSyn-DIO-hM3D(Gq)- mCherry plasmid;
  • the steps of carrying out plasmid recombination of the original sgRNA backbone plasmid and the chemical genetics plasmid include:
  • the fragment containing AAV-U6-sgRNA (backbone) and the fragment containing DIO-hM3D(Gq)-mCherry were ligated.
  • the step of inserting the sgRNA fragment into the sgRNA backbone plasmid with chemical genetic activation elements comprises: using plasmid recombination technology to insert the sgRNA fragment into the sgRNA backbone plasmid with chemical genetic activation elements Insert the sgRNA fragment.
  • the recombinant vector is a viral vector
  • the following step is further included: inserting the sgRNA Fragments of recombinant plasmids are packaged into viruses.
  • the sequence of the sgRNA fragment is as shown in SEQ ID No.1.
  • a recombinant vector which is constructed by the method for constructing the above-mentioned recombinant vector.
  • a recombinant vector comprising: a sgRNA backbone plasmid with a chemical genetic activation element, the chemical genetic activation element is used to activate neurons, and an sgRNA fragment is also inserted into the sgRNA backbone plasmid with a chemical genetic activation element , the sgRNA fragment is used to knock out vGAT.
  • the chemogenetic activation element is a chemogenetic excitatory receptor.
  • the sequence of the sgRNA fragment is as shown in SEQ ID No.1.
  • the recombinant vector is a viral vector.
  • the reagent further includes an activator for activating the chemogenetic activation element.
  • a method for activating GABAergic neurons comprising the steps of:
  • the above-mentioned recombinant vector is packaged into a virus and injected into the brain area of an animal model for expression, and then an activator is injected into the animal model, the animal model can express CRISPR nuclease, and the activator is used to activate the chemical genetic activation element.
  • Fig. 1 is AAV-U6-sgRNA(backbone)-pCBh-DIO-ChR2-mCherry plasmid map
  • Fig. 2 is pAAV-hSyn-DIO-hM3D(Gq)-mCherry plasmid map
  • Fig. 3 is the enzyme digestion verification map of AAV-U6-sgRNA(backbone)-pCBh-DIO-hM3D(Gq)-mCherry plasmid;
  • Figure 4 is a sequencing verification map of the AAV-U6-sgRNA(backbone)-pCBh-DIO-hM3D(Gq)-mCherry plasmid;
  • Figure 5 is a sequence verification map of sAAV-U6-sgRNA (vGAT)-pCBh-DIO-hM3D (Gq)-mCherry plasmid;
  • Figure 6a is a schematic diagram of a patch clamp experiment
  • Figure 6b is a statistical diagram of light-induced inhibitory postsynaptic currents dependent on light intensity
  • Figure 6c is a schematic diagram of light-evoked inhibitory postsynaptic currents under different light intensities
  • Fig. 7 is the c-fos immunofluorescence staining figure of embodiment 3.
  • Fig. 8 is a graph comparing the eating behavior of the experimental mice and control mice in Example 4.
  • One embodiment of the present application provides a method for constructing a recombinant vector, which combines CRISPR gene editing technology and chemical genetic activation technology to obtain a recombinant vector that can be used to knock out vGAT of GABAergic neurons, block GABA release, and
  • chemical genetic activation of GABAergic neurons knocked out of vGAT it is convenient to activate specific neurons for a long time to achieve the purpose of long-term activation of GABAergic neurons knocked out of vGAT.
  • the chemical genetic activation technology is only through injection Or take the corresponding activating drugs orally, without invasive surgery, non-invasive, improve the adaptability of animals, and no longer need to give light stimulation through optical fiber connection LED, reduce the use of experimental equipment during the experiment, and improve convenience , and no longer need to connect with optical fiber or move in a specific range of magnetic field, the range of activities of mice is wider; avoiding cell damage caused by photothermal effect, specific neurons can be activated for a long time, and the scope of application is wider .
  • vGAT Vesicular GABA transporter, vesicular ⁇ -aminobutyric acid transporter
  • GAT ⁇ -aminobutyric acid transporter
  • the method for constructing the above-mentioned recombinant vector includes the following steps S110-S120:
  • the step of constructing the sgRNA backbone plasmid with the chemical genetics activation element comprises: performing plasmid recombination of the original sgRNA backbone plasmid and the chemical genetics plasmid with the chemical genetics activation element using plasmid recombination technology.
  • Chemical genetics technology also known as DREADDs (designer receptors exclusively activated by designer drugs, receptors activated only by specific drugs) technology, is a chemical genetics platform modified based on G protein-coupled receptors. Different G protein-coupled receptors are modified so that they can transmit artificially synthesized proteins. The modified receptors can only be activated or inhibited by artificially synthesized special compounds, and activate the corresponding GPCR signaling pathways, thereby triggering different cellular processes. changes in excitability. This technology is widely used to enhance or inhibit the activity of neurons in a cell-specific and non-invasive manner. Although chemical genetics lacks the precise time control ability like optogenetics, since long-term neuronal circuit regulation is most likely to be needed in long-term behavioral research or disease treatment, this technology will be very suitable for this class application.
  • Chemogenetic activation elements are used to activate neurons. Wherein, the chemogenetic activation element is a chemogenetic excitatory receptor. Chemogenetic excitatory receptors include, for example, hM1Dq, hM3Dq, hM5Dq, and the like.
  • steps of S110 include S111-S113:
  • the original sgRNA backbone plasmid is the AAV-U6-sgRNA(backbone)-pCBh-DIO-ChR2-mCherry plasmid. It should be noted that the original sgRNA backbone plasmid is not limited to the AAV-U6-sgRNA(backbone)-pCBh-DIO-ChR2-mCherry plasmid, and can also be other sgRNA backbone plasmids, which can be selected according to needs.
  • the step of digesting the original sgRNA backbone plasmid to obtain a fragment containing the sgRNA backbone includes: digesting the AAV-U6-sgRNA(backbone)-pCBh-DIO-ChR2-mCherry plasmid to obtain a fragment containing AAV-U6- Fragments of sgRNA (backbone).
  • the restriction endonuclease of Asc I and Nhe I-HF restriction endonuclease carries out double digestion.
  • Digestion system 1 ⁇ g of plasmid, 5 ⁇ L of 5x Cutsmart buffer solution, 1 ⁇ L of Asc I endonuclease, 1 ⁇ L of Nhe I-HF endonuclease, and supplement the whole system to 50 ⁇ L with deionized water. Digestion conditions: 37°C, incubate for 3 hours.
  • the chemogenetics plasmid is the pAAV-hSyn-DIO-hM3D(Gq)-mCherry plasmid.
  • the chemical genetics plasmid is not limited to the pAAV-hSyn-DIO-hM3D(Gq)-mCherry plasmid, and can also be other chemical genetics plasmids with chemical genetics activation elements, which can be selected according to needs.
  • the step of digesting the chemical genetics plasmid to obtain a fragment containing the chemical genetics activation element includes: digesting the pAAV-hSyn-DIO-hM3D(Gq)-mCherry plasmid to obtain a fragment containing DIO-hM3D(Gq) ) - fragment of mCherry.
  • the restriction endonuclease of Asc I and Nhe I-HF restriction endonuclease carries out double digestion.
  • Digestion system 1 ⁇ g of plasmid, 5 ⁇ L of 5x Cutsmart buffer solution, 1 ⁇ L of Asc I endonuclease, 1 ⁇ L of Nhe I-HF endonuclease, and supplement the whole system to 50 ⁇ L with deionized water. Digestion conditions: 37°C, incubate for 3 hours.
  • sequence of S111 and S112 is not limited, S111 may be performed first and then S112 may be performed, S112 may be performed first and then S111 may be performed, or S111 and S112 may be performed simultaneously.
  • S113 Ligate the fragment containing the sgRNA backbone with the fragment containing the chemogenetic activation element to obtain the sgRNA backbone plasmid with the chemogenetic activation element.
  • the step of linking the fragment containing the sgRNA backbone and the fragment containing the chemogenetic activation element comprises: linking the fragment comprising AAV-U6-sgRNA (backbone) and the fragment comprising DIO-hM3D(Gq)-mCherry .
  • the following system was used for fragment ligation: 1 ⁇ L of fragments containing AAV-U6-sgRNA (backbone), 1 ⁇ L of fragments containing DIO-hM3D(Gq)-mCherry, 5 ⁇ L of T4 buffer solution, 1 ⁇ L of T4 ligase, Supplement the whole system to 10 ⁇ L with deionized water, and let stand at room temperature for 30 minutes.
  • the step of ligating the fragment containing the sgRNA backbone and the fragment containing the chemogenetic activation element it further includes: a step of amplifying and screening the recombinant backbone plasmid obtained by ligating the above two fragments .
  • the step of amplifying the recombinant backbone plasmid includes: transferring the above ligation system into competent cells for amplifying and culturing. Specifically, 20 ⁇ L of competent cells were added to the above ligation system, allowed to stand on ice for 30 minutes, then heat-shocked at 42°C for 45 seconds, then left to stand on ice for 3 minutes, and then 600 ⁇ L of liquid medium without ampicillin was added, 37 ° C, cultured on a shaker at 220 rpm. After 30-40 minutes, centrifuge at 3500 x g for 4 minutes, suck off the supernatant, and leave about 100 ⁇ L of liquid.
  • Verification methods include enzyme digestion verification and sequencing verification.
  • sequencing verification includes: designing multiple sequencing sites, sending them to a sequencing company for sequencing, and then performing sequence comparison.
  • the primer sequences of the designed sequencing sites are shown in SEQ ID No.2-SEQ ID No.5.
  • sequence shown in SEQ ID No.2 is: 5'-CATAGCGTAAAAGGAGCAACA-3' (ie Primer 1).
  • SEQ ID No.3 The sequence shown in SEQ ID No.3 is: 5'-TCTTTCTTCTGCATTACGGGG-3' (i.e. Primer 2).
  • sequence shown in SEQ ID No.4 is: 5'-GGGAAACGCCTGGTATCTTT-3' (ie Primer 3).
  • sequence shown in SEQ ID No.5 is: 5'-CAGCACAAAAGGAAACTCACC-3' (ie Primer 4).
  • plasmid recombination technology is used to insert sgRNA fragments into sgRNA backbone plasmids with chemical genetic activation elements.
  • sequence of the sgRNA fragment is shown in SEQ ID No.1. Specifically, the sequence shown in SEQ ID No.1 is: 5'-CGGCTTCGTGCATTCACTCG-3'.
  • sequence of the sgRNA fragment is not limited to the sequence shown in SEQ ID No.1, and can also be other sgRNA fragments that knock out vGAT.
  • the recombinant vector is a viral vector
  • the following step is further included: packaging the recombinant plasmid with the sgRNA fragment inserted into Virus.
  • the virus may be, for example, AVV virus (adenovirus). It should be noted that the virus is not limited to the AVV virus, and may be other viruses, which can be selected according to needs.
  • An embodiment of the present application also provides a recombinant vector, which is constructed by the method for constructing a recombinant vector described above. Relevant specific descriptions can be found above, and will not be repeated here.
  • the recombinant vector includes: a sgRNA backbone plasmid with chemical genetic activation elements, the chemical genetic activation elements are used to activate GABAergic neurons, sgRNA fragments are also inserted in the sgRNA backbone plasmid with chemical genetic activation elements, sgRNA Fragments were used to knock out vGAT.
  • the chemogenetic activation element is a chemogenetic excitatory receptor. See the above for detailed description, and will not repeat them here.
  • sequence of the sgRNA fragment is shown in SEQ ID No.1. See the above for detailed description, and will not repeat them here.
  • the recombinant vector is a viral vector. See the above for detailed description, and will not repeat them here.
  • One embodiment of the present application also provides the application of the above-mentioned recombinant vector in the preparation of reagents.
  • the reagent is used to knock down vGAT of GABAergic neurons.
  • the agent is used to activate GABAergic neurons.
  • the reagent is used to knock out vGAT of GABAergic neurons, and to activate the GABAergic neurons knocked out of vGAT.
  • the reagent further includes an activator, and the activator is used to activate the chemogenetic activation element.
  • Activators activate elements through chemical genetics and activate corresponding GPCR signaling pathways, thereby triggering different excitatory changes in cells.
  • the activator is capable of activating vGAT-knockout GABAergic neurons by activating the chemogenetic activation element.
  • the activator is CNO (clozapine nitric oxide, clozapine N-oxide). It should be noted that the activator is not limited to CNO, and may be other activators, and an activator capable of activating the chemogenetic activating element can be selected as required.
  • One embodiment of the present application also provides a method for activating GABAergic neurons, comprising the steps of:
  • the above-mentioned recombinant vector is packaged into a virus and injected into the brain area of the animal model for expression, and then injected into the animal model with an activator, the animal model can express CRISPR nuclease, and the activator is used to activate the chemogenetic activation element.
  • Activators activate elements through chemical genetics and activate corresponding GPCR signaling pathways, thereby triggering different excitatory changes in cells.
  • the activator is capable of activating vGAT-knockout GABAergic neurons by activating the chemogenetic activation element.
  • the activator is CNO (clozapine nitric oxide, clozapine N-oxide). It should be noted that the activator is not limited to CNO, and may be other activators, and an activator capable of activating the chemogenetic activating element can be selected as required.
  • the CRISPR nuclease is Cas9 nuclease. It should be noted that the CRISPR nuclease is not limited to Cas9 nuclease, and other CRISPR nucleases can also be selected according to needs.
  • the animal model is LSL-Cas9 and Nts-ires-Cre double transfected mice. It should be noted that the Cre animal model is not limited to the above-mentioned mice, and other Cre animal models can be selected as needed.
  • the method for activating GABAergic neurons comprises: injecting sAAV-U6-sgRNA(vGAT)-pCBh-DIO-hM3D(Gq )-mCherry virus, three weeks later, the virus was fully expressed. At this moment, the vGAT protein in GABAergic neurons was knocked out, and when CNO (clozapine nitric oxide, clozapine N-oxide) was injected 30 minutes later, the GABAergic neurons knocked out of vGAT protein were activated.
  • the specific brain region may be, for example, the lateral septum (LS). It should be noted that the specific brain region is not limited to the lateral septal nucleus, and may also be a brain region of other neurons, which is only used for illustration and not limitation.
  • CRISPR/Cas9 Clustered Regularly Interspersed Short Palindromic Repeats gene editing technology: CRISPR/Cas9 is an adaptive immune defense formed during the long-term evolution of bacteria and archaea, which can be used to fight against invading viruses and foreign DNA.
  • CRISPR/Cas9 is an adaptive immune defense formed during the long-term evolution of bacteria and archaea, which can be used to fight against invading viruses and foreign DNA.
  • researchers published several articles introducing a new method for gene knockout in cell lines based on CRISPR-Cas9 technology (Wang et.al., Science, 2013; Ophir Shalem et.al.Science, 2013).
  • the CRISPR-Cas9 system consists of two parts, one is the sgRNA (small guide RNA) sequence with a length of about 20 bp, which is used to identify the target genome, and the other is the double-stranded DNA nuclease—Cas9, which exists near the CRISPR site.
  • sgRNA small guide RNA
  • the target site is cut, and finally the broken DNA is repaired through the intracellular non-homologous end joining mechanism and homologous recombination repair mechanism, thereby forming gene knockout and insertion, and finally realizing gene (directed) editing.
  • This technology was quickly applied to the construction of gene knockout mice and rat animal models.
  • Chemical genetics technology also known as DREADDs (designer receptors exclusively activated by designer drugs, receptors activated only by specific drugs) technology, is a chemical genetics platform modified based on G protein-coupled receptors. Different G protein-coupled receptors are modified so that they can transmit artificially synthesized proteins. The modified receptors can only be activated or inhibited by artificially synthesized special compounds, and activate the corresponding GPCR signaling pathways, thereby triggering different cellular processes. changes in excitability. This technology is widely used to enhance or inhibit the activity of neurons in a cell-specific and non-invasive manner.
  • the reagents and instruments used in the examples are all conventional choices in the art unless otherwise specified.
  • the experimental methods without specific conditions indicated in the examples are usually implemented according to conventional conditions, such as the conditions described in literature, books or the method recommended by the kit manufacturer.
  • the reagents used in the examples are all commercially available.
  • Embodiment 1 Construction of sAAV-U6-sgRNA (vGAT)-pCBh-DIO-hM3D (Gq)-mCherry virus
  • the AAV-U6-sgRNA(backbone)-pCBh-DIO-ChR2-mCherry plasmid was obtained from Stanford University, and the pAAV-hSyn-DIO-hM3D(Gq)-mCherry plasmid (Cat. No.: 44361) was purchased from Addgene (a plasmid ordering company).
  • the plasmid map of AAV-U6-sgRNA(backbone)-pCBh-DIO-ChR2-mCherry is shown in Figure 1.
  • the plasmid map of pAAV-hSyn-DIO-hM3D(Gq)-mCherry is shown in FIG. 2 .
  • 1AAV-U6-sgRNA(backbone)-pCBh-DIO-ChR2-mCherry plasmid was digested to obtain a fragment containing AAV-U6-sgRNA(backbone), represented by fragment A.
  • the following system was used for fragment ligation: 1 ⁇ L of A fragment, 1 ⁇ L of B fragment, 5 ⁇ L of T4 buffer solution, 1 ⁇ L of T4 ligase, supplemented the whole system to 10 ⁇ L with deionized water, and stood at room temperature for 30 minutes.
  • Figure 3 is a diagram of restriction enzyme digestion verification of AAV-U6-sgRNA(backbone)-pCBh-DIO-hM3D(Gq)-mCherry plasmid. It can be seen from Figure 3 that 3 single clone colonies were picked and amplified, among which No. 2 and No. 3 were the correct plasmids.
  • Figure 4 is a sequence verification map of the AAV-U6-sgRNA(backbone)-pCBh-DIO-hM3D(Gq)-mCherry plasmid. It can be seen from Figure 4 that the constructed plasmid is correct.
  • the designed sequencing sites are as follows:
  • Primer 1 5'-CATAGCGTAAAAGGAGCAACA-3';
  • Primer 2 5'-TCTTTCTTCTGCATTACGGGG-3';
  • Primer 3 5'-GGGAAACGCCTGGTATCTTT-3';
  • Primer 4 5'-CAGCACAAAAGGAAACTCACC-3'.
  • Insert the sgRNA sequence into the modified backbone plasmid Using plasmid recombination construction technology, insert the synthesized sgRNA sequence into the backbone plasmid (ie AAV-U6-sgRNA(backbone)-pCBh-DIO -hM3D(Gq)-mCherry plasmid), the sAAV-U6-sgRNA(vGAT)-pCBh-DIO-hM3D(Gq)-mCherry plasmid was obtained.
  • the confirmed sAAV-U6-sgRNA(vGAT)-pCBh-DIO-hM3D(Gq)-mCherry plasmid was packaged into the corresponding AAV virus to obtain sAAV-U6-sgRNA(vGAT)-pCBh-DIO-hM3D(Gq)- mCherry virus.
  • Example 2 Using patch clamp whole-cell recordings to prove that vGAT is knocked out in specific cells and GABA is no longer released
  • the sAAV-U6-sgRNA(vGAT)-pCBh-DIO-hM3D(Gq)-mCherry virus and AAV-hsyn-DIO-ChR2-EYFP virus were injected on LSL-Cas9 (#024857) and Nts-ires-Cre (#017525 ) lateral septum (Lateral septum, LS) of double-transformed mice (commercially available mice, all imported from Jackson lab). Three weeks later, virus expression was sufficient, and both viruses were co-expressed in Nts-positive neurons in the LS brain region.
  • Animals in the control group ie ctrl: co-express green fluorescent protein EYFP and optogenetic virus ChR2 in Nts-positive neurons in the LS brain area; animals in the experimental group (ie vGAT sgRNA): co-express sgRNA (vGAT) in Nts-positive neurons in the LS brain area Sequence and optogenetic virus ChR2.
  • Animals in the control group ie vGAT sgRNA
  • vGAT co-express sgRNA
  • Using a vibrating microtome in artificial cerebrospinal fluid obtain LS brain slices with a thickness of 300 ⁇ m. Light-evoked postsynaptic currents were recorded by clamping a non-fluorescent cell in the LS brain region using the patch-clamp technique. The measurement results are shown in Figure 6 in detail.
  • Figure 6a is a schematic diagram of the patch clamp experiment.
  • Fig. 6b is a statistical diagram of the light intensity-dependent light-evoked inhibitory postsynaptic current.
  • Figure 6c is a schematic diagram of light-evoked inhibitory postsynaptic currents under different light intensities.
  • Example 3 Immunofluorescence staining of c-fos (early instant gene, which indicates that cells are activated) to prove that specific cells are activated by chemogenetics
  • mice Inject sAAV-U6-sgRNA(vGAT)-pCBh-DIO-hM3D(Gq)-mCherry virus into LSL-Cas9 (#024857) and Nts-ires-Cre (#017525) double transfected mice (commercially available mice , were introduced from Jackson lab) lateral septum (Lateral septum, LS). After three weeks, the virus was fully expressed. After intraperitoneal injection of 2 mg/kg of CNO (clozapine nitric oxide, clozapine N-oxide), 30 minutes later, the mice were perfused and brains were taken, followed by c-fos immunofluorescent staining.
  • CNO clozapine nitric oxide, clozapine N-oxide
  • Fig. 7 is the c-fos immunofluorescent staining image of Example 3.
  • the sAAV-U6-sgRNA(vGAT)-pCBh-DIO-hM3D(Gq)-mCherry virus and the control virus sAAV-U6-sgRNA(LacZ)-pCBh-DIO-hM3D(Gq)-mCherry were injected on LSL-Cas9 (# 024857) and Nts-ires-Cre (#017525) double-transformed mice (commercially available mice, both imported from Jackson lab) lateral septum (LS). After three weeks, the virus was fully expressed.
  • Fig. 8 is a graph comparing the eating behavior of the experimental mice and control mice in Example 4.
  • mice compared with control mice (ie injected with sAAV-U6-sgRNA(LacZ)-pCBh-DIO-hM3D(Gq)-mCherry virus
  • experimental mice ie injected with sAAV-U6- sgRNA(vGAT)-pCBh-DIO-hM3D(Gq)-mCherry virus
  • significantly increased the intake of high-sugar food indicating that the vGAT protein in Nts-positive neurons in the LS brain region has an important function in feeding behavior and also
  • the behavioral differences show that the method of the present application has indeed knocked out the vGAT protein in the Nts-positive neurons in the LS brain region.
  • knockdown of vGAT in GABAergic neurons in specific brain regions increases intake of high-sugar foods.
  • the method of this application combines CRISPR gene editing technology and chemical genetic activation technology, can knock out vGAT of GABAergic neurons, block GABA release, and activate GABAergic neurons knocked out of vGAT through chemical genetics, which is convenient Long-term activation of specific neurons to achieve the purpose of long-term activation of GABA-ergic neurons knocked out of vGAT.
  • Chemogenetic activation technology is only through injection or oral administration of corresponding activation drugs, without invasive surgery, and is non-invasive.

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Abstract

本申请涉及一种重组载体及其构建方法和应用。该重组载体的构建方法包括如下步骤：构建带有化学遗传学激活元件的sgRNA骨架质粒；向带有化学遗传学激活元件的sgRNA骨架质粒中插入sgRNA片段，sgRNA片段用于敲除vGAT，得到重组载体。上述重组载体的构建方法将CRISPR基因编辑技术和化学遗传激活技术结合，得到重组载体能够用于敲除GABA能神经元的vGAT，并通过能化学遗传学激活敲除vGAT的GABA能神经元，达到长时程激活敲除vGAT的GABA能神经元的目的。

Description

重组载体及其构建方法和应用 技术领域
本发明涉及生物技术领域，特别是涉及一种重组载体及其构建方法和应用。
背景技术
目前，主要采用光遗传学对特定的细胞进行激活，具有以下局限性：①有创性。光遗传学需要埋置光纤陶瓷插芯，是一个有创性手术。②活动范围的局限性。若采用有线光遗传激活，需要将光纤与陶瓷插芯链接，这会限制小鼠的活动范围；若采用无线光遗传激活，需要用线圈布置一个磁场，为了保证磁场的强度，小鼠能活动的范围也是有限的。③光热效应对细胞的损伤。光遗传学刺激通常采用蓝光，蓝光的光热效应较强，长时间或者多次激活，可能对光纤尖端的神经元造成损伤。④适用场景的局限性。正由于前面②和③提到的活动范围的局限性和光热效应对细胞的损伤，该技术适用的场景有限，需要长时间或者在复杂环境中对小鼠的特定神经元进行操纵时，该技术不再适用。
发明内容
基于此，有必提供一种重组载体及其构建方法和应用。
一种重组载体的构建方法，包括如下步骤：
构建带有化学遗传学激活元件的sgRNA骨架质粒；及
向所述带有化学遗传学激活元件的sgRNA骨架质粒中插入sgRNA片段，所述sgRNA片段用于敲除vGAT，得到所述重组载体。
上述重组载体的构建方法将CRISPR基因编辑技术和化学遗传激活技术结合，得到重组载体能够用于敲除GABA能神经元的vGAT，阻断GABA释放，并通过能化学遗传学激活敲除vGAT的GABA能神经元，便于对特定神经元进行长时间激活，达到长时程激活敲除vGAT的GABA能神经元的目的，化学遗传学激活技术是仅通过注射或口服相应的激活药物，无需进行有创手术， 具有无创性，提高了动物的适应性，且不再需要通过光纤连接LED给予光刺激，减少了实验过程中的实验装置使用，提高了便利性，也不再需要与光纤连接或者在特定范围的磁场中运动，小鼠的活动范围更加广泛；避免了光热效应带来的细胞损伤，可以长时间对特定神经元进行激活处理，适用范围更广泛。
在其中一个实施例中，构建所述带有化学遗传学激活元件的sgRNA骨架质粒的步骤包括：
采用质粒重组技术将原始sgRNA骨架质粒与化学遗传学质粒进行质粒重组，所述化学遗传学质粒带有所述化学遗传学激活元件。
在其中一个实施例中，构建所述带有化学遗传学激活元件的sgRNA骨架质粒的步骤包括：
将所述原始sgRNA骨架质粒进行酶切，得到含有sgRNA骨架的片段；
将所述化学遗传学质粒进行酶切，得到含有所述化学遗传学激活元件的片段；及
将所述含有sgRNA骨架的片段和所述含有所述化学遗传学激活元件的片段连接，得到所述带有化学遗传学激活元件的sgRNA骨架质粒。
在其中一个实施例中，所述原始sgRNA骨架质粒包括AAV-U6-sgRNA(backbone)-pCBh-DIO-ChR2-mCherry质粒；所述化学遗传学质粒包括pAAV-hSyn-DIO-hM3D(Gq)-mCherry质粒；
所述将原始sgRNA骨架质粒与化学遗传学质粒进行质粒重组的步骤包括：
将AAV-U6-sgRNA(backbone)-pCBh-DIO-ChR2-mCherry质粒进行酶切，得到包含AAV-U6-sgRNA(backbone)的片段；
将pAAV-hSyn-DIO-hM3D(Gq)-mCherry质粒进行酶切，得到包含DIO-hM3D(Gq)-mCherry的片段；及
将包含AAV-U6-sgRNA(backbone)的片段和包含DIO-hM3D(Gq)-mCherry的片段连接。
在其中一个实施例中，所述向所述带有化学遗传学激活元件的sgRNA骨架质粒中插入sgRNA片段的步骤包括：采用质粒重组技术向所述带有化学遗传学激活元件的sgRNA骨架质粒中插入所述sgRNA片段。
在其中一个实施例中，所述重组载体为病毒载体，所述向所述带有化学遗 传学激活元件的sgRNA骨架质粒中插入sgRNA片段的步骤之后，还包括如下步骤：将插入有所述sgRNA片段的重组质粒包装成病毒。
在其中一个实施例中，所述sgRNA片段的序列如SEQ ID No.1所示。
一种重组载体，由上述重组载体的构建方法构建得到。
一种重组载体，包括：带有化学遗传学激活元件的sgRNA骨架质粒，所述化学遗传学激活元件用于激活神经元，所述带有化学遗传学激活元件的sgRNA骨架质粒中还插入sgRNA片段，所述sgRNA片段用于敲除vGAT。
在其中一个实施例中，所述化学遗传学激活元件为化学遗传学兴奋性受体。
在其中一个实施例中，所述sgRNA片段的序列如SEQ ID No.1所示。
在其中一个实施例中，所述重组载体为病毒载体。
上述重组载体在制备试剂中的应用，所述试剂用于敲除GABA能神经元的vGAT。
上述重组载体在制备试剂中的应用，所述试剂用于激活GABA能神经元；
上述重组载体在制备试剂中的应用，所述试剂用于敲除GABA能神经元的vGAT，并且激活所述GABA能神经元。
在其中一个实施例中，所述试剂还包括激活剂，所述激活剂用于激活所述化学遗传学激活元件。
一种激活GABA能神经元的方法，包括如下步骤：
将上述重组载体包装成病毒后注入动物模型的脑区中表达，然后向所述动物模型注入激活剂，所述动物模型能够表达CRISPR核酸酶，所述激活剂用于激活所述化学遗传学激活元件。
附图说明
图1为AAV-U6-sgRNA(backbone)-pCBh-DIO-ChR2-mCherry质粒图谱；
图2为pAAV-hSyn-DIO-hM3D(Gq)-mCherry质粒图谱；
图3为AAV-U6-sgRNA(backbone)-pCBh-DIO-hM3D(Gq)-mCherry质粒的酶切验证图；
图4为AAV-U6-sgRNA(backbone)-pCBh-DIO-hM3D(Gq)-mCherry质粒的测序验证图；
图5为sAAV-U6-sgRNA(vGAT)-pCBh-DIO-hM3D(Gq)-mCherry质粒的测序验证图；
图6a为膜片钳实验示意图；
图6b为光强依赖的光诱发的抑制性突触后电流的统计图；
图6c为不同光强下光诱发的抑制性突触后电流的示意图；
图7为实施例3的c-fos免疫荧光染色图；
图8为实施例4的实验小鼠和对照小鼠的摄食行为学比对图。
具体实施方式
为使本发明的上述目的、特征和优点能够更加明显易懂，下面结合具体实施例及附图对本发明的具体实施方式做详细的说明。在下面的描述中阐述了很多具体细节以便于充分理解本发明。但是本发明能够以很多不同于在此描述的其它方式来实施，本领域技术人员可以在不违背本发明内涵的情况下做类似改进，因此本发明不受下面公开的具体实施的限制。
本申请一实施方式提供一种重组载体的构建方法，该构建方法将CRISPR基因编辑技术和化学遗传激活技术结合，得到重组载体能够用于敲除GABA能神经元的vGAT，阻断GABA释放，并通过能化学遗传学激活敲除vGAT的GABA能神经元，便于对特定神经元进行长时间激活，达到长时程激活敲除vGAT的GABA能神经元的目的，化学遗传学激活技术是仅通过注射或口服相应的激活药物，无需进行有创手术，具有无创性，提高了动物的适应性，且不再需要通过光纤连接LED给予光刺激，减少了实验过程中的实验装置使用，提高了便利性，也不再需要与光纤连接或者在特定范围的磁场中运动，小鼠的活动范围更加广泛；避免了光热效应带来的细胞损伤，可以长时间对特定神经元进行激活处理，适用范围更广泛。
vGAT(Vesicular GABA transporter，囊泡γ-氨基丁酸转运体)是脑内主要的抑制性神经递质，通过γ-氨基丁酸转运体(GAT)重摄取是胞外GA-BA消除的重要机制。
具体地，上述重组载体的构建方法包括如下步骤S110-S120：
S110、构建带有化学遗传学激活元件的sgRNA骨架质粒。
其中，构建带有化学遗传学激活元件的sgRNA骨架质粒的步骤包括：采用质粒重组技术将原始sgRNA骨架质粒与化学遗传学质粒进行质粒重组，化学遗传学质粒带有所述化学遗传学激活元件。
化学遗传学技术，又被称为DREADDs(designer receptors exclusively activated by designer drugs，只由特定药物激活的受体)技术，是一种基于G蛋白偶联受体所改造的化学遗传学平台，通过将不同的G蛋白偶联受体进行改造，让它能传递人工合成的蛋白质，修改后的受体只能由人工合成的特殊化合物来激活或者抑制，并激活相应的GPCR信号通路，从而引发细胞不同的兴奋性变化。该项技术被广泛用于以细胞特异性、无创地增强或抑制神经元的活动。虽然化学遗传学缺乏像光遗传学那样精准的时间控制能力，但是由于在进行长时程行为研究或者疾病治疗时，最有可能需要的是长期神经元环路调节，该项技术会非常适合这类应用。
化学遗传学激活元件用于激活神经元。其中，化学遗传学激活元件为化学遗传学兴奋性受体。化学遗传学兴奋性受体例如包括hM1Dq、hM3Dq、hM5Dq等。
具体地，S110的步骤包括S111-S113：
S111、将原始sgRNA骨架质粒进行酶切，得到含有sgRNA骨架的片段。
在一个具体示例中，原始sgRNA骨架质粒为AAV-U6-sgRNA(backbone)-pCBh-DIO-ChR2-mCherry质粒。需要说明的是，原始sgRNA骨架质粒不限于为AAV-U6-sgRNA(backbone)-pCBh-DIO-ChR2-mCherry质粒，也可以为其他sgRNA骨架质粒，可以根据需要进行选择。
具体地，将原始sgRNA骨架质粒进行酶切，得到含有sgRNA骨架的片段的步骤包括：将AAV-U6-sgRNA(backbone)-pCBh-DIO-ChR2-mCherry质粒进 行酶切，得到包含AAV-U6-sgRNA(backbone)的片段。其中，酶切为Asc I和Nhe I-HF限制性内切酶酶进行双酶切。酶切的体系：1μg的质粒、5μL的5x Cutsmart缓冲溶液、1μL的Asc I内切酶、1μL的Nhe I-HF内切酶，用去离子水将整个体系补充至50μL。酶切条件：37℃，孵育3小时。
S112、将化学遗传学质粒进行酶切，得到含有化学遗传学激活元件的片段。
在一个具体示例中，化学遗传学质粒为pAAV-hSyn-DIO-hM3D(Gq)-mCherry质粒。需要说明的是，化学遗传学质粒不限于为pAAV-hSyn-DIO-hM3D(Gq)-mCherry质粒，也可以为其他带有化学遗传学激活元件的化学遗传学质粒，可以根据需要进行选择。
具体地，将化学遗传学质粒进行酶切，得到含有化学遗传学激活元件的片段的步骤包括：将pAAV-hSyn-DIO-hM3D(Gq)-mCherry质粒进行酶切，得到包含DIO-hM3D(Gq)-mCherry的片段。其中，酶切为Asc I和Nhe I-HF限制性内切酶酶进行双酶切。酶切的体系：1μg的质粒、5μL的5x Cutsmart缓冲溶液、1μL的Asc I内切酶、1μL的Nhe I-HF内切酶，用去离子水将整个体系补充至50μL。酶切条件：37℃，孵育3小时。
需要说明的是，S111和S112的顺序不限，可以先进行S111再进行S112，也可以先进行S112再进行S111，还可以同时进行S111和S112。
S113、将含有sgRNA骨架的片段和所述含有化学遗传学激活元件的片段连接，得到带有化学遗传学激活元件的sgRNA骨架质粒。
具体地，将含有sgRNA骨架的片段和所述含有化学遗传学激活元件的片段连接的步骤包括：将包含AAV-U6-sgRNA(backbone)的片段和包含DIO-hM3D(Gq)-mCherry的片段连接。其中，采用下述体系进行片段连接：1μL的包含AAV-U6-sgRNA(backbone)的片段、1μL的包含DIO-hM3D(Gq)-mCherry的片段、5μL的T4缓冲溶液、1μL的T4连接酶，用去离子水将整个体系补充至10μL，室温静置30分钟。
在其中一个实施例中，将含有sgRNA骨架的片段和所述含有化学遗传学激活元件的片段连接的步骤之后，还包括：将上述两个片段连接得到的重组骨架质粒进行扩增和筛选的步骤。
其中，重组骨架质粒进行扩增的步骤包括：将上述连接体系转入感受态细 胞中进行扩增培养。具体地，将上述连接体系中加入20μL感受态细胞，冰上静置30分钟，然后42℃热激45秒，接着冰上静置3分钟，随后加入600μL没有加入氨苄抗生素的液体培养基，37℃，220rpm转速下摇床培养。30～40分钟后，用3500x g转速离心4分钟，吸去上清，留约100μL液体，在无菌环境下，利用涂菌棒均匀涂布在含有氨苄抗生素的平板上，37℃培养。约18小时后，从平板上挑取单克隆，进行液体培养基培养。约18小时后，菌液变得浑浊，利用试剂盒进行质粒提取，具体步骤根据不同公司的试剂盒说明书确定。
其中，重组骨架质粒扩增完成后进行验证。验证方法包括酶切验证和测序验证。具体地，测序验证包括：设计多个测序位点，送测序公司进行测序，随后进行序列比对。在一个具体示例中，设计的测序位点的引物序列如SEQ ID No.2-SEQ ID No.5所示。
具体地，如SEQ ID No.2所示的序列为：5'-CATAGCGTAAAAGGAGCAACA-3'(即Primer 1)。
如SEQ ID No.3所示的序列为：5'-TCTTCTTCTGCATTACGGGG-3'(即Primer 2)。
如SEQ ID No.4所示的序列为：5'-GGGAAACGCCTGGTATCTTT-3'(即Primer 3)。
如SEQ ID No.5所示的序列为：5'-CAGCACAAAAGGAAACTCACC-3'(即Primer 4)。
S120、向带有化学遗传学激活元件的sgRNA骨架质粒中插入sgRNA片段，sgRNA片段用于敲除vGAT，得到重组载体。
其中，采用质粒重组技术向带有化学遗传学激活元件的sgRNA骨架质粒中插入sgRNA片段。
其中，sgRNA片段的序列如SEQ ID No.1所示。具体地，如SEQ ID No.1所示的序列为：5'-CGGCTTCGTGCATTCACTCG-3'。sgRNA片段的序列不限于为如SEQ ID No.1所示的序列，也可以为其他敲除vGAT的sgRNA片段。
在其中一个实施例中，重组载体为病毒载体，向所述带有化学遗传学激活元件的sgRNA骨架质粒中插入sgRNA片段的步骤之后，还包括如下步骤：将 插入有sgRNA片段的重组质粒包装成病毒。其中，病毒例如可以为AVV病毒(腺病毒)。需要说明的是，病毒不限于为AVV病毒，也可以为其他病毒，可以根据需要进行选择。
本申请一实施方式还提供一种重组载体，由上述重组载体的构建方法构建得到。相关具体描述详见上文，此处不再赘述。
具体地，重组载体包括：带有化学遗传学激活元件的sgRNA骨架质粒，化学遗传学激活元件用于激活GABA能神经元，带有化学遗传学激活元件的sgRNA骨架质粒中还插入sgRNA片段，sgRNA片段用于敲除vGAT。
其中，化学遗传学激活元件为化学遗传学兴奋性受体。具体描述详见上文，此处不再赘述。
其中，sgRNA片段的序列如SEQ ID No.1所示。具体描述详见上文，此处不再赘述。
其中，重组载体为病毒载体。具体描述详见上文，此处不再赘述。
本申请一实施方式还提供上述重组载体在制备试剂中的应用。
其中，试剂用于敲除GABA能神经元的vGAT。
其中，试剂用于激活GABA能神经元。
其中，试剂用于敲除GABA能神经元的vGAT，并且激活敲除vGAT的GABA能神经元。
在其中一个实施例中，试剂还包括激活剂，激活剂用于激活化学遗传学激活元件。激活剂通过化学遗传学激活元件，并激活相应的GPCR信号通路，从而引发细胞不同的兴奋性变化。在本申请中，激活剂通过激活化学遗传学激活元件，能够激活敲除vGAT的GABA能神经元。
在一个具体示例中，激活剂为CNO(氯氮平一氧化氮，clozapine N-oxide)。需要说明的是，激活剂不限为CNO，也可以为其他激活剂，可以根据需要进行选择能够激活化学遗传学激活元件的激活剂。
本申请一实施方式还提供一种激活GABA能神经元的方法，包括如下步骤：
将上述重组载体包装成病毒后注入动物模型的脑区中表达，然后向动物模型注入激活剂，动物模型能够表达CRISPR核酸酶，激活剂用于激活化学遗传 学激活元件。
激活剂通过化学遗传学激活元件，并激活相应的GPCR信号通路，从而引发细胞不同的兴奋性变化。在本申请中，激活剂通过激活化学遗传学激活元件，能够激活敲除vGAT的GABA能神经元。
在一个具体示例中，激活剂为CNO(氯氮平一氧化氮，clozapine N-oxide)。需要说明的是，激活剂不限为CNO，也可以为其他激活剂，可以根据需要进行选择能够激活化学遗传学激活元件的激活剂。
其中，CRISPR核酸酶为Cas9核酸酶。需要说明的是，CRISPR核酸酶不限于为Cas9核酸酶，也可以根据需要选择其他的CRISPR核酸酶。
其中，动物模型为LSL-Cas9和Nts-ires-Cre双转小鼠。需要说明的是，Cre动物模型不限于上述指出的小鼠，可以根据需要选择其他Cre动物模型。
在一个具体示例中，激活GABA能神经元的方法包括：在LSL-Cas9和Nts-ires-Cre双转小鼠的特定脑区注射sAAV-U6-sgRNA(vGAT)-pCBh-DIO-hM3D(Gq)-mCherry病毒，三周后，病毒表达充分。此刻，GABA能神经元中的vGAT蛋白被敲除，当注射CNO(氯氮平一氧化氮，clozapine N-oxide)30分钟后，敲除了vGAT蛋白的GABA能神经元被激活。其中，特定脑区例如可以为外侧隔核(Lateral septum,LS)。需要说明的是，特定脑区不限于为外侧隔核，也可以为其他神经元的脑区，此处仅用于举例说明，不做限制。
CRISPR/Cas9(Clustered Regularly Interspersed Short Palindromic Repeats)基因编辑技术：CRISPR/Cas9是细菌和古细菌在长期演化过程中形成的一种适应性免疫防御，可用来对抗入侵的病毒及外源DNA。2013年，研究者们发表多篇文章介绍了基于CRISPR-Cas9技术在细胞系中进行基因敲除的新方法(Wang et.al.,Science,2013；Ophir Shalem et.al.Science,2013)。CRISPR-Cas9系统由两部分组成，一部分是用来识别靶基因组的，长度为20bp左右的sgRNA(small guide RNA)序列，另外一部分是存在于CRISPR位点附近的双链DNA核酸酶—Cas9，能在sgRNA的引导下对靶位点进行切割，最终通过细胞内的非同源性末端连接机制和同源重组修复机制对形成断裂的DNA进行修复，从而形成基因的敲除和插入，最终实现基因的(定向)编辑。该技术迅速被运用 到基因敲除小鼠和大鼠动物模型的构建之中。化学遗传学技术，又被称为DREADDs(designer receptors exclusively activated by designer drugs，只由特定药物激活的受体)技术，是一种基于G蛋白偶联受体所改造的化学遗传学平台，通过将不同的G蛋白偶联受体进行改造，让它能传递人工合成的蛋白质，修改后的受体只能由人工合成的特殊化合物来激活或者抑制，并激活相应的GPCR信号通路，从而引发细胞不同的兴奋性变化。该项技术被广泛用于以细胞特异性、无创地增强或抑制神经元的活动。虽然化学遗传学缺乏像光遗传学那样精准的时间控制能力，但是由于在进行长时程行为研究或者疾病治疗时，最有可能需要的是长期神经元环路调节，该项技术会非常适合这类应用。本申请通过将上述两种技术同时应用于AAV(腺相关病毒)技术中，实现在特定细胞中进行蛋白敲除或者神经元兴奋性操纵，例如用于长时程激活敲除vGAT蛋白的GABA能神经元。
以下为具体实施例部分。
实施例中采用试剂和仪器如非特别说明，均为本领域常规选择。实施例中未注明具体条件的实验方法，通常按照常规条件，例如文献、书本中所述的条件或者试剂盒生产厂家推荐的方法实现。实施例中所使用的试剂均为市售。
实施例1：sAAV-U6-sgRNA(vGAT)-pCBh-DIO-hM3D(Gq)-mCherry病毒的构建
(1)原始骨架质粒的准备：
从斯坦福大学获得AAV-U6-sgRNA(backbone)-pCBh-DIO-ChR2-mCherry质粒，从Addgene(质粒订购公司)购入pAAV-hSyn-DIO-hM3D(Gq)-mCherry质粒(货号：44361)。AAV-U6-sgRNA(backbone)-pCBh-DIO-ChR2-mCherry质粒图谱如图1所示。pAAV-hSyn-DIO-hM3D(Gq)-mCherry质粒图谱如图2所示。
(2)改造原始骨架质粒，得到带有化学遗传学激活元件的骨架质粒。利用质粒重组构建技术，构建新的骨架质粒：AAV-U6-sgRNA(backbone)-hSyn-DIO-hM3D(Gq)-mCherry质粒。
具体过程如下：
①AAV-U6-sgRNA(backbone)-pCBh-DIO-ChR2-mCherry质粒酶切，得到包 含AAV-U6-sgRNA(backbone)的片段，用片段A表示。
采用下述体系进行Asc I和Nhe I-HF限制性内切酶酶切：1μg的质粒、5μL的5x Cutsmart缓冲溶液、1μL的Asc I内切酶、1μL的Nhe I-HF内切酶，用去离子水将整个体系补充至50μL，37℃下孵育3小时。酶切完成后，进行琼脂糖凝胶电泳。配置0.7％浓度的琼脂糖凝胶，在3V/cm的电场强度下，电泳40分钟。紫外扫描仪下观察，找到大小为5500bp左右的条带，为目的条带，即片段A。用干净的刀片进行切割，保存在离心管中。
②pAAV-hSyn-DIO-hM3D(Gq)-mCherry质粒酶切，得到包含DIO-hM3D(Gq)-mCherry的片段，用片段B表示。
采用下述体系进行Asc I和Nhe I-HF限制性内切酶酶切：1μg的质粒、5μL的5x Cutsmart缓冲溶液、1μL的Asc I内切酶、1μL的Nhe I-HF内切酶，用去离子水将整个体系补充至50μL，37℃下孵育3小时。酶切完成后，进行琼脂糖凝胶电泳。配置0.7％浓度的琼脂糖凝胶，在3V/cm的电场强度下，电泳40分钟。紫外扫描仪下观察，找到大小为2500bp左右的条带，为目的条带，即片段B。用干净的刀片进行切割，保存在离心管中。
③利用胶回收试剂盒(市售试剂盒)，纯化在①和②步骤中切割下来包含片段A和片段B的凝胶。具体的过程详见试剂盒的说明书。
④将片段A和片段B连接，得到AAV-U6-sgRNA(backbone)-pCBh-DIO-hM3D(Gq)-mCherry骨架质粒。
采用下述体系进行片段连接：1μL的A片段、1μL的B片段、5μL的T4缓冲溶液、1μL的T4连接酶，用去离子水将整个体系补充至10μL，室温静置30分钟。
⑤AAV-U6-sgRNA(backbone)-pCBh-DIO-hM3D(Gq)-mCherry重组骨架质粒的扩增和筛选。
将上述连接体系中加入20μL DH5α感受态细胞，冰上静置30分钟，然后42℃热激45秒，接着冰上静置3分钟，随后加入600μL没有加入氨苄抗生素的肉膏蛋白胨液体培养基，37℃，220rpm转速下摇床培养。30～40分钟后，用3500x g转速离心4分钟，吸去上清，留约100μL液体，在无菌环境下，利用涂菌棒均匀涂布在含有氨苄抗生素的平板上，37℃培养。约18小 时后，从平板上挑取单克隆，进行液体培养基培养。约18小时后，菌液变得浑浊，利用市售质粒提取试剂盒进行质粒提取，具体步骤详见试剂盒说明书。
新的骨架质粒扩增完成后，进行验证：
方法一：酶切验证
利用Asc I和Nhe I-HF双限制性内切酶进行酶切验证，若被切成2500bp和5500bp两个片段，则说明质粒构建正确。检测结果如图3所示。图3为AAV-U6-sgRNA(backbone)-pCBh-DIO-hM3D(Gq)-mCherry质粒的酶切验证图。从图3可以看出，对挑取3个单克隆菌落进行扩增，其中，2和3号为正确质粒。
方法二：测序验证
设计多个测序位点，送测序公司进行测序，随后进行序列比对，结果如图4所示。图4为AAV-U6-sgRNA(backbone)-pCBh-DIO-hM3D(Gq)-mCherry质粒的测序验证图。从图4可以看出，构建的质粒正确无误。
其中，设计的测序位点如下：
Primer 1：5'-CATAGCGTAAAAGGAGCAACA-3'；
Primer 2：5'-TCTTCTTCTGCATTACGGGG-3'；
Primer 3：5'-GGGAAACGCCTGGTATCTTT-3'；
Primer 4：5'-CAGCACAAAAGGAAACTCACC-3'。
由上述结果可知，成功构建了改造骨架质粒：AAV-U6-sgRNA(backbone)-pCBh-DIO-hM3D(Gq)-mCherry质粒。
(3)准备sgRAN序列：设计高效敲除vGAT的sgRNA序列：5'-CGGCTTCGTGCATTCACTCG-3'，可直接通过公司进行序列合成。
(4)将sgRNA序列插入改造后的骨架质粒中：利用质粒重组构建技术，将合成好的sgRAN序列插入加入化学遗传学激活元件的骨架质粒(即AAV-U6-sgRNA(backbone)-pCBh-DIO-hM3D(Gq)-mCherry质粒)中，获得sAAV-U6-sgRNA(vGAT)-pCBh-DIO-hM3D(Gq)-mCherry质粒。
(5)验证sAAV-U6-sgRNA(vGAT)-pCBh-DIO-hM3D(Gq)-mCherry质粒的序列正确性：对构建好的质粒进行测序验证，在验证骨架质粒正确的基础上，验证质粒中是否成功插入sgRNA(vGAT)序列。测序结果如图5所示。图5为 sAAV-U6-sgRNA(vGAT)-pCBh-DIO-hM3D(Gq)-mCherry质粒的测序验证图。从图5可以看出，已经成功插入相应的序列，sAAV-U6-sgRNA(vGAT)-pCBh-DIO-hM3D(Gq)-mCherry质粒构建成功。
(6)包装病毒：
将确认无误的sAAV-U6-sgRNA(vGAT)-pCBh-DIO-hM3D(Gq)-mCherry质粒包装成相应的AAV病毒，得到sAAV-U6-sgRNA(vGAT)-pCBh-DIO-hM3D(Gq)-mCherry病毒。
实施例2：利用膜片钳全细胞记录，证明在特定细胞中敲除了vGAT，不再释放GABA
将sAAV-U6-sgRNA(vGAT)-pCBh-DIO-hM3D(Gq)-mCherry病毒和AAV-hsyn-DIO-ChR2-EYFP病毒注射在LSL-Cas9(#024857)和Nts-ires-Cre(#017525)双转小鼠(为市售小鼠，均从Jackson lab引进)的外侧隔核(Lateral septum,LS)。三周后，病毒表达充分，在LS脑区的Nts阳性神经元中共同表达两种病毒。对照组动物(即ctrl)：在LS脑区Nts阳性神经元中共表达绿色荧光蛋白EYFP和光遗传病毒ChR2；实验组动物(即vGAT sgRNA)：在LS脑区Nts阳性神经元中共表达sgRNA(vGAT)序列和光遗传病毒ChR2。在人工脑脊液中利用震动切片机，得到厚度为300μm的LS脑区脑片。利用膜片钳技术，在LS脑区钳制一个非荧光细胞，记录光诱发的突触后电流。测定结果详见图6。图6a为膜片钳实验示意图。图6b为光强依赖的光诱发的抑制性突触后电流的统计图。图6c为不同光强下光诱发的抑制性突触后电流的示意图。
从图6可以看出，对于对照组动物，随着刺激光强的增加，光诱发的突触后电流随之增加；对于实验组动物，光诱发的突触后电流显著减少。
实施例3：利用c-fos(早期即时基因，表征细胞被激活)免疫荧光染色，证明特定细胞被化学遗传学激活
将sAAV-U6-sgRNA(vGAT)-pCBh-DIO-hM3D(Gq)-mCherry病毒注射在LSL-Cas9(#024857)和Nts-ires-Cre(#017525)双转小鼠(为市售小鼠，均从 Jackson lab引进)的外侧隔核(Lateral septum,LS)。三周后，病毒表达充分。通过腹腔注射2mg/kg的CNO(氯氮平一氧化氮，clozapine N-oxide)，30分钟后，对小鼠进行灌流取脑，随后进行c-fos免疫荧光染色。同时，通过腹腔注射等量的生理盐水作为对照，3分钟后，对小鼠进行灌流取脑，随后进行c-fos免疫荧光染色。测定结果详见图7。图7为实施例3的c-fos免疫荧光染色图。
从图7可以看出，与注射生理盐水(saline)的小鼠相比，注射CNO的小鼠的LS脑区的Nts阳性神经元(被红色荧光标记)被大比例激活。
实施例4：摄食行为学检测
将sAAV-U6-sgRNA(vGAT)-pCBh-DIO-hM3D(Gq)-mCherry病毒和对照病毒sAAV-U6-sgRNA(LacZ)-pCBh-DIO-hM3D(Gq)-mCherry注射在LSL-Cas9(#024857)和Nts-ires-Cre(#017525)双转小鼠(为市售小鼠，均从Jackson lab引进)的外侧隔核(Lateral septum,LS)。三周后，病毒表达充分。通过腹腔注射2mg/kg的CNO(氯氮平一氧化氮，clozapine N-oxide)，并通过腹腔注射等量的生理盐水作为对照。注射30分钟后，对小鼠进行摄食行为学检测。测定结果详见图8。图8为实施例4的实验小鼠和对照小鼠的摄食行为学比对图。
从图8可以看出，与对照小鼠(即注射有sAAV-U6-sgRNA(LacZ)-pCBh-DIO-hM3D(Gq)-mCherry病毒)相比，实验小鼠(即注射有sAAV-U6-sgRNA(vGAT)-pCBh-DIO-hM3D(Gq)-mCherry病毒)对高糖食物的摄入显著增加，说明LS脑区中Nts阳性神经元中的vGAT蛋白在摄食行为中具有中要功能，也从行为学的差异表明利用本申请的方法确实敲除了LS脑区中Nts阳性神经元中的vGAT蛋白。综上，在特定脑区的GABA能神经元中敲除vGAT后，增加了对高糖食物的摄入。
综上，本申请的方法将CRISPR基因编辑技术和化学遗传激活技术结合，能够敲除GABA能神经元的vGAT，阻断GABA释放，并通过化学遗传学激活敲除vGAT的GABA能神经元，便于对特定神经元进行长时间激活，达到长时程激活敲除vGAT的GABA能神经元的目的，化学遗传学激活技术是仅通过注射或口服相应的激活药物，无需进行有创手术，具有无创性，提高了动 物的适应性，且不再需要通过光纤连接LED给予光刺激，减少了实验过程中的实验装置使用，提高了便利性，也不再需要与光纤连接或者在特定范围的磁场中运动，小鼠的活动范围更加广泛；避免了光热效应带来的细胞损伤，可以长时间对特定神经元进行激活处理，适用范围更广泛。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (13)

  1. 一种重组载体的构建方法，其特征在于，包括如下步骤：
    构建带有化学遗传学激活元件的sgRNA骨架质粒，所述化学遗传学激活元件用于激活神经元；及
    向所述带有化学遗传学激活元件的sgRNA骨架质粒中插入sgRNA片段，所述sgRNA片段用于敲除vGAT，得到所述重组载体。
  2. 根据权利要求1所述的重组载体的构建方法，其特征在于，构建所述带有化学遗传学激活元件的sgRNA骨架质粒的步骤包括：
    采用质粒重组技术将原始sgRNA骨架质粒与化学遗传学质粒进行质粒重组，所述化学遗传学质粒带有所述化学遗传学激活元件。
  3. 根据权利要求2所述的重组载体的构建方法，其特征在于，构建所述带有化学遗传学激活元件的sgRNA骨架质粒的步骤包括：
    将所述原始sgRNA骨架质粒进行酶切，得到含有sgRNA骨架的片段；
    将所述化学遗传学质粒进行酶切，得到含有所述化学遗传学激活元件的片段；及
    将所述含有sgRNA骨架的片段和所述含有所述化学遗传学激活元件的片段连接，得到所述带有化学遗传学激活元件的sgRNA骨架质粒。
  4. 根据权利要求2所述的重组载体的构建方法，其特征在于，所述原始sgRNA骨架质粒包括AAV-U6-sgRNA(backbone)-pCBh-DIO-ChR2-mCherry质粒；所述化学遗传学质粒包括pAAV-hSyn-DIO-hM3D(Gq)-mCherry质粒；
    所述将原始sgRNA骨架质粒与化学遗传学质粒进行质粒重组的步骤包括：
    将AAV-U6-sgRNA(backbone)-pCBh-DIO-ChR2-mCherry质粒进行酶切，得 到包含AAV-U6-sgRNA(backbone)的片段；
    将pAAV-hSyn-DIO-hM3D(Gq)-mCherry质粒进行酶切，得到包含DIO-hM3D(Gq)-mCherry的片段；及
    将包含AAV-U6-sgRNA(backbone)的片段和包含DIO-hM3D(Gq)-mCherry的片段连接。
  5. 根据权利要求1所述的重组载体的构建方法，其特征在于，所述向所述带有化学遗传学激活元件的sgRNA骨架质粒中插入sgRNA片段的步骤包括：采用质粒重组技术向所述带有化学遗传学激活元件的sgRNA骨架质粒中插入所述sgRNA片段。
  6. 根据权利要求1所述的重组载体的构建方法，其特征在于，所述重组载体为病毒载体，所述向所述带有化学遗传学激活元件的sgRNA骨架质粒中插入sgRNA片段的步骤之后，还包括如下步骤：将插入有所述sgRNA片段的重组质粒包装成病毒。
  7. 根据权利要求1～6任一项所述的重组载体的构建方法，其特征在于，所述sgRNA片段的序列如SEQ ID No.1所示。
  8. 一种重组载体，其特征在于，由权利要求1-7任一项所述的重组载体的构建方法构建得到。
  9. 一种重组载体，其特征在于，包括：带有化学遗传学激活元件的sgRNA骨架质粒，所述化学遗传学激活元件用于激活神经元，所述带有化学遗传学激活元件的sgRNA骨架质粒中还插入sgRNA片段，所述sgRNA片段用于敲除vGAT。
  10. 根据权利要求9所述的重组载体，其特征在于，所述化学遗传学激活元件为化学遗传学兴奋性受体；
    及/或，所述sgRNA片段的序列如SEQ ID No.1所示；
    及/或，所述重组载体为病毒载体。
  11. 权利要求8-10任一项所述的重组载体在制备试剂中的应用，其特征在于，所述试剂用于敲除GABA能神经元的vGAT；
    或者，所述试剂用于激活GABA能神经元；
    或者，所述试剂用于敲除GABA能神经元的vGAT，并且激活所述GABA能神经元。
  12. 根据权利要求11所述的应用，其特征在于，所述试剂还包括激活剂，所述激活剂用于激活所述化学遗传学激活元件。
  13. 一种激活GABA能神经元的方法，其特征在于，包括如下步骤：
    将权利要求8-10任一项所述的重组载体包装成病毒后注入动物模型的脑区中表达，然后向所述动物模型注入激活剂，所述动物模型能够表达CRISPR核酸酶，所述激活剂用于激活所述化学遗传学激活元件。
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