GRNA and application thereof in construction of Ireb2-p.D826V point mutation mouse model

Abstract

The invention relates to the technical field of biological medicines, in particular to gRNA (guide Ribonucleic Acid) and application thereof in constructing an Ireb2-p.D826V point mutation mouse model. The invention provides gRNA of a specific targeting Ireb2 gene mutation pathogenic site, a mouse model carrying IREB2-p.D826V mutation is constructed by using the gRNA, and the phenotype of the nervous system of the mouse model is deeply identified to find that the mutation has great influence on the nervous system of the mouse. The mouse model is expected to open up a new path for research on pathogenesis of neurodegenerative diseases such as Alzheimer's disease, and a novel animal model with great value is provided for screening potential drug targets.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a gRNA and an application thereof in constructing an Ireb2-p.D826V point mutation mouse model. Background Art

[0002] IREB2 (Ireb2), or Iron Responsive Element Binding protein 2, is a class of iron regulatory proteins that occupy a core position in the cellular iron metabolism regulatory network. IREB2 plays an irreplaceable and key role in maintaining the delicate balance of intracellular iron homeostasis. It can accurately recognize and specifically bind to a specific mRNA sequence - Iron Response Element (IRE), and then finely regulate the expression of a series of genes closely related to iron metabolism, including the ferritin heavy chain (FTH) gene that plays an important role in the iron storage process. When the intracellular iron content is insufficient, IREB2 is activated, and in an active state, it quickly binds to the IRE sequence of FTH mRNA. This binding is like putting a layer of "protective clothing" on FTH mRNA, effectively preventing nucleases from degrading it, allowing FTH to be continuously synthesized, thereby promoting the cell's storage of iron. Conversely, when the intracellular iron reserves are sufficient, free iron ions will bind tightly to IREB2, and this binding event will trigger a significant change in the spatial conformation of IREB2. After the conformational change, IREB2 can no longer stably bind to the IRE sequence of FTH mRNA and thus dissociates from it. FTH mRNA without the protection of IREB2 becomes fragile and is easily attacked and degraded by nucleases, and its translation efficiency is greatly reduced, and the cell's iron storage activity is correspondingly weakened.

[0003] In recent years, researchers have found that mutations in the IREB2 gene are closely related to an extremely rare inherited neurological disease, NEURODEGENERATION, EARLY-ONSET, WITH CHOREOATHETOID MOVEMENTS AND MICROCYTIC ANEMIA (NDCAMA). This disease usually begins to show typical symptoms of neurodegenerative diseases in childhood, accompanied by clinical manifestations such as choreoathetosis and microcytic anemia.

[0004] Currently, the process of constructing transgenic neurodegenerative disease animal models is cumbersome and complex, and the disease cycle is long. However, constructing a neurodegenerative disease mouse model and conducting in-depth identification of its nervous system phenotype is expected to open up a new path for studying the pathogenesis of neurodegenerative diseases such as Alzheimer's disease, and also provide a valuable new animal model for screening potential drug targets. Summary of the invention

[0005] The purpose of the present invention is to provide a gRNA and its application in constructing an Ireb2-p.D826V point mutation mouse model to solve the problems existing in the above-mentioned prior art.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a gRNA that specifically targets the Ireb2 gene mutation pathogenic site, the gRNA comprising gRNA-1 and gRNA-2; the nucleotide sequence of the gRNA-1 is shown in SEQ ID NO.3; the nucleotide sequence of the gRNA-2 is shown in SEQ ID NO.4; the Ireb2 gene mutation pathogenic site is NM_022655.3: exon20: c.2477A>T: p.D826V.

[0008] The present invention provides the use of the above-mentioned gRNA in preparing a product that specifically targets the pathogenic site of Ireb2 gene mutation.

[0009] Preferably, the product comprises a reagent, a kit or a chip.

[0010] The present invention provides a product that specifically targets the pathogenic site of Ireb2 gene mutation, and the product includes the above-mentioned gRNA.

[0011] The present invention provides the use of the above-mentioned gRNA or the above-mentioned product in constructing an Ireb2-p.D826V point mutation mouse model.

[0012] The present invention provides a method for constructing an Ireb2-p.D826V point mutation mouse model based on CRISPR-Cas9 technology, comprising the following steps:

[0013] The mixture containing the gRNA, template DNA and Cas9 protein was injected into mouse fertilized eggs to obtain mutant F 0 The nucleotide sequence of the template DNA is shown in SEQ ID NO.2;

[0014] The mutant F 0 After self-fertilization of the first generation heterozygous mice, the Ireb2-p.D826V point mutation mouse model was obtained.

[0015] Preferably, the concentration of gRNA in the mixture is 2 pmol / μL, the concentration of template DNA is 15 ng / μL, and the concentration of Cas9 protein is 30 ng / μL.

[0016] Preferably, the mass ratio of gRNA-1 to gRNA-2 in the gRNA is 1:1.

[0017] Preferably, the injection dosage is 1-2PL.

[0018] The present invention provides the use of an Ireb2-p.D826V point mutation mouse model constructed by the above method in the preparation and / or screening of drugs for treating neurodegenerative diseases.

[0019] Further preferably, the neurodegenerative disease includes Alzheimer's disease and / or early-onset neurodegeneration with choreoathetosis and microcytic anemia.

[0020] The present invention discloses the following technical effects:

[0021] The present invention provides a gRNA that specifically targets the pathogenic site of the Ireb2 gene mutation. The gRNA is used to construct a mouse model carrying the IREB2-p.D826V mutation, and its nervous system phenotype is deeply identified. It is found that the mutation has a great impact on the nervous system of the mouse. The mouse model is expected to open up a new path for the study of the pathogenesis of neurodegenerative diseases such as Alzheimer's disease, early-onset neurodegenerative lesions with choreoathetosis and microcytic anemia, and also provide a valuable new animal model for screening potential drug targets. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 Comparison of the amino acid conservation of human and mouse Ireb2 proteins; NP_004127.2 is the reference amino acid sequence of human Ireb2 protein; NP_073146.2 is the reference amino acid sequence of mouse Ireb2 protein; the box is aspartic acid (Asp, abbreviated as D) at position 826;

[0024] Figure 2 The strategy for constructing Ireb2-D826V homozygous mutant mice;

[0025] Figure 3 This is the result of genome identification of Ireb2-D826V homozygous mutant mice;

[0026] Figure 4 The behavioral test results of Ireb2-D826V homozygous mutant mice; A and B are the results of movement distance and average speed in the mine field experiment of wild-type mice and Ireb2-D826V homozygous mutant mice, respectively; C and D are the results of the number of times reached and the residence time in the Y maze experiment of wild-type mice and Ireb2-D826V homozygous mutant mice, respectively; E is the results of the water maze experiment of wild-type mice and Ireb2-D826V homozygous mutant mice, the horizontal axis represents the number of days, and the vertical axis represents the time required to get on the stage; the values ​​represent means±SEM, **P<0.01, ***P<0.001, and the difference analysis methods include T test (Student's t-test) and two-way analysis of variance (Two-wayANOVA);

[0027] Figure 5 The morphological test results of the hippocampus of D826V homozygous mutant mice; A is the immunofluorescence staining of Iba-1 in the hippocampus of wild-type mice and D826V homozygous mutant mice; B is the quantitative analysis of the fluorescence intensity of Iba-1 in the hippocampus of wild-type mice and D826V homozygous mutant mice; C is the Golgi staining of the hippocampus of wild-type mice and D826V homozygous mutant mice; D is the quantification of dendritic spines in the hippocampus of wild-type mice and D826V homozygous mutant mice; the values ​​represent means±SEM, **P<0.01, and the difference analysis method is T test (Student's t-test);

[0028] Figure 6 The results of hippocampal synaptic plasticity detection in D826V homozygous mutant mice; A is the detection of evoked field excitatory postsynaptic potential (fEPSP) in brain slices of wild-type mice and D826V homozygous mutant mice; B is the difference analysis of fEPSP; C is the ratio of the field potential amplitude induced by the second stimulus to the field potential amplitude induced by the first stimulus in mouse brain slices; the values ​​represent means±SEM, **P<0.01, ***P<0.001, and the difference analysis methods include Student's t-test and one-way ANOVA;

[0029] Figure 7The results of Ireb2 protein detection in the hippocampus of D826V homozygous mutant mice; A is the protein expression detection of Ireb2 and Fth in the hippocampus of wild-type mice and D826V homozygous mutant mice; B and C are quantitative analysis of Ireb2 and Fth protein expression; values ​​represent means±SEM, *P<0.05, **P<0.01, difference analysis method: T test (Student's t-test). DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] Our team previously discovered the p.D826V mutation on the IREB2 gene through whole exome sequencing technology combined with functional validation experiments. This mutation causes the IREB2 protein to undergo ubiquitination degradation, thereby disrupting the normal expression of key iron-regulating genes such as FTH, and ultimately breaking the originally stable iron homeostasis in the cell. However, it is not yet clear whether this mutation is the direct cause of the phenotype of early-onset neurodegenerative diseases.

[0036] Example 1 Method for constructing an Ireb2-p.D826V point mutation mouse model

[0037] 1. Mutation type: missense mutation.

[0038] 2. Mutation source: Based on the new pathogenic gene mutation site that causes early-onset Alzheimer's neurodegenerative genetic disease identified by the research team using whole exome sequencing (PMID: 39587636).

[0039] 3. Mutation location:

[0040] The position information of the mutation is shown in Table 1.

[0041] Table 1 Comparison of missense mutations in human and mouse Ireb2 genes

[0042] Species Gene Genomic location Transcript Mutation information Exon location Amino acid changes people IREB2 Chr15:78786488 NM_004136.4 c.2477A>T Exon 20 Aspartic acid / Valine Mouse IREB2 Chr9:54906805 NM_022655.3 c.2477A>T Exon 20 Aspartic acid / Valine

[0043] Note: The human reference genome is hg19 and the mouse reference genome is mm10.

[0044] 4. Comparison of amino acid sequences of human and mouse Ireb2 proteins:

[0045] Comparison of amino acid conservation between human and mouse Ireb2 proteins Figure 1 As shown. Figure 1 It can be seen that this mutation site is highly conserved between humans and mice.

[0046] 5. Construction method:

[0047] 5.1. Construction of mouse Ireb2 genome sequence containing mutation site

[0048] Using BAC clone RP23-382D20 as a template, high-fidelity Taq DNA polymerase was used to construct template DNA for homologous recombination, namely the mouse Ireb2 genomic sequence containing the mutation site.

[0049] The nucleotide sequence of RP23-382D20 is shown in SEQ ID NO.1, specifically:

[0050]

[0051] Among them, the italic part is the homology arm, the bold part is the mouse Ireb2 genomic DNA, the bold italic underlined part is exon 19, the bold italic wavy line part is exon 20, and the bold italic double wavy line part is the mutation region (GAT mutates to GTT);

[0052] The mouse Ireb2 genome sequence containing the mutation site is shown in SEQ ID NO.2, specifically:

[0053]

[0054]

[0055] Among them, the italic underlined part is exon 19, the wavy part is exon 20, and the double wavy part is the mutation region (GAT mutates to GTT);

[0056] 5.2. Construction of mutant F 0 Heterozygous mice

[0057] The gRNA targeting the mouse Ireb2 gene (SEQ ID NO.3 and SEQ ID NO.4, whose specific sequences are shown in Table 2), the template DNA for homologous recombination (SEQ ID NO.2) and the Cas9 protein (purchased from: NEB, catalog number: M0646M) were co-injected into mouse fertilized eggs, that is, a mixture containing the gRNA targeting the mouse Ireb2 gene, the template DNA for homologous recombination and the Cas9 protein was microinjected into the mouse fertilized eggs, and the injection volume was 1-2PL to produce mutant F 0 Heterozygous mice ( Figure 2 ), wherein the concentrations of gRNA, template DNA for homologous recombination and CRISPR protein in the mixture were 2 pmol / μL, 15 ng / μL and 30 ng / μL, respectively, and the mass ratio of gRNA-1 and gRNA-2 in the gRNA was 1:1.

[0058] Table 2 gRNA sequences used for mouse model construction

[0059]

[0060] Note: “[]” indicates the part where the target sequence is identical to the gRNA sequence.

[0061] 5.2. Construction of mutant F 1 Heterozygous mice

[0062] 5.2.1 Mutant F 0 Self-fertilization of heterozygous mice produces F 1 The first generation of mice was identified by extracting the mouse tail genome, PCR and Sanger sequencing.

[0063] 5.2.2 Mouse genome extraction method

[0064] The mouse genome was extracted using the SteadyPure Universal Genomic DNA Extraction Kit (Cat. No. AG21009). The specific steps are as follows:

[0065] a. Add 180 μL of Buffer LS-2, 20 μL of Proteinase K, and 10 μL of RNase A (10 mg / mL) to the tissue.

[0066] b. Incubate in a 56°C water bath until the tissue is completely lysed (overnight lysis).

[0067] c. Centrifuge at 12,000 rpm for 2 minutes.

[0068] d. Add 200 μL Buffer BS-2 and 200 μL 100% ethanol to the lysate and mix thoroughly by pipetting.

[0069] e. Transfer the above solution to the Universal DNA Mini Column, let it stand at room temperature for 1 minute, centrifuge at 12,000 rpm at room temperature for 1 minute, and discard the filtrate.

[0070] f. Add 500 μL of Buffer WA to the Mini Column, centrifuge at 12,000 rpm for 1 minute at room temperature, and discard the filtrate.

[0071] g. Add 750 μL of Buffer WB to the Mini Column, centrifuge at 12,000 rpm for 1 minute at room temperature, and discard the filtrate.

[0072] h. Repeat step "g" once.

[0073] i. Place the Mini Column on a new 2 mL Collection Tube and centrifuge at 12,000 rpm for 2 minutes at room temperature.

[0074] j. Place the Mini Column in a new 1.5 mL centrifuge tube, add 50 μL of Eliution Buffer to the center of the Mini Column membrane, let stand at room temperature for 5 minutes, and then centrifuge at 12,000 rpm for 2 minutes at room temperature to elute the DNA.

[0075] 5.2.3 PCR

[0076] The PCR reaction system is shown in Table 3, and the PCR reaction conditions are shown in Table 4.

[0077] Table 3 PCR reaction system

[0078] Components volume Rat tail genomic DNA 1.5μL Upstream primer (10 μM) 1μL Downstream primer (10 μM) 1μL 2× Taq enzyme (Novozyme, catalog number #P222) 12.5μL Sterile water 9μL Total volume 25μL

[0079] Note: The nucleotide sequence of the upstream primer is shown in SEQ ID NO.7; the nucleotide sequence of the downstream primer is shown in SEQ ID NO.8.

[0080] Table 4 PCR reaction conditions

[0081]

[0082] 5.2.4 Sanger sequencing identification

[0083] Table 5 Primer sequences for identification of point mutation mouse genotypes

[0084] Primer sequences SEQ ID NO. Stripe size Upstream primer 5'-CAAGAGGCACATTTGCAAACATCA-3' 7 545bp Downstream primer 5'-CTTCACTGCGTCCATACAGACTTC-3' 8 545bp

[0085] 5.5.5. Interpretation of genotype identification results

[0086] like Figure 3 As shown in Table 5, the point mutation mice were identified by PCR of mouse genome and then by Sanger sequencing. There are three main identification results, namely wild type G A T(Asp), heterozygous G A / T T(Asp / Val) and homozygous G T T(Val), named as WT, D826V + / - and D826V - / - .

[0087] Next, adult homozygous male mice D826V - / -(Ireb2-D826V homozygous mutant mice, D826V homozygous mutant homozygous mice or D826V - / - Mice) for follow-up studies (8-12 weeks).

[0088] In this example, a total of 270 eggs were injected, 37 eggs were born, 4 of which were positive, with a birth rate of 13.7% and a positive rate of 10.8%.

[0089] Example 2 Ireb2-D826V homozygous mutation (D826V - / - ) Mouse models for neurodegenerative diseases

[0090] 1. D826V mutation causes behavioral abnormalities in mice

[0091] The effects of the D826V mutation on the physiological and psychological activities of mice, such as cognition, memory, learning and movement, were studied through a variety of behavioral experiments, including the Open Field Test (OFT), Morris Water Maze (MWM) and Y-Maze. - / - The mice's motor ability was significantly reduced, including the distance and average speed ( Figure 4 AB in the Y-maze test and the water maze test results suggest that their spatial learning, cognitive and memory abilities are significantly reduced ( Figure 4 CE in ).

[0092] 2. D826V mutation causes morphological abnormalities in the mouse hippocampus

[0093] Iba-1 (Ionized calcium binding adapter molecule 1) is a calcium-binding protein specifically expressed in microglia in the central nervous system and has been widely used as a microglia marker. Microglia are intrinsic immune effector cells in the central nervous system and play an extremely important role in the physiological processes of the central nervous system. Their activation state is closely related to the severity of the damaged part in the brain. The activation degree of microglia in different regions of the hippocampus (including CA1, CA3 and DG regions) was detected by Iba-1 immunofluorescence, and it was found that D826V - / - The number and fluorescence intensity of Iba-1 positive cells in the hippocampus were significantly higher than those in wild-type mice, suggesting that D826V - / - The activation of microglia in mice suggests the presence of central nervous system inflammation ( Figure 5 A and B in ).

[0094] Dendritic spines are the parts of the dendrites of neurons where synapses are formed, and are closely related to learning and memory. Golgi staining uses the silver-loving properties of neurons to well display the morphology of neurons and dendritic spines. - / - The number of dendritic spines in the hippocampus of mice was significantly reduced, indicating that the synaptic transmission function of nerve cells was reduced ( Figure 5 C and D in ).

[0095] 3. D826V mutation causes impaired synaptic plasticity in the mouse hippocampus

[0096] Learning and memory, from the perspective of neuroscience, a memory is a connection network formed by a large group of neurons with various functional types on a macro level, and a change in the efficacy of a single synapse on a micro level. The application of patch clamp in brain science is mainly the study of brain slice patch clamp. Brain slice patch clamp can detect and record the activity of ion channels between cells in isolated brain slices, that is, electrophysiological signals in a brain environment close to the real physiological state. By analyzing these recorded signals, the discharge of brain cells can be observed relatively quickly and intuitively. It is often used to study the interaction between single cells or different regions in the nervous system, such as synaptic transmission, neuronal excitability and rhythmicity, and is also widely used in synaptic plasticity, learning and memory, central nervous system diseases and anti-aging research. The inventor induced field excitatory postsynaptic potential (fEPSP) by electrical stimulation, using 0.1ms square wave for stimulation, gradually increasing the stimulation power until the induced fEPSP no longer increases, and after determining the maximum power, adjusting the stimulation power to 50% of the maximum power to induce fEPSP basal value (baseline fEPSP). Observe whether the induced fEPSP waveform is stable. After stabilization, baseline fEPSP is recorded for 20min. After completing the baseline fEPSP recording, 2 series of 100 pulses at 100Hz with an interval of 20s are given through the stimulating electrode. High-frequency stimulation (HFS) to induce LTP, and continuous recording for 60min. The fEPSP amplitude changes are statistically measured to measure the whole-cell mode recording of long-term potentiation (Long-Term Potentiation, LTP). At the same time, combined with the changes in the paired-pulse facilitation (PPF) effect after LTP (PPF2 / PPF1), synaptic plasticity, learning and memory, and other cellular synaptic mechanisms were studied. - / - The LTP amplitude in the hippocampus decreased significantly, while the PPF increased significantly, indicating that its synaptic plasticity was impaired ( Figure 6 ).

[0097] 4. The D826V mutation causes Ireb2 protein degradation and abnormal iron metabolism gene expression in the mouse hippocampus

[0098] Ireb2 (iron responsive element binding protein 2) is a key gene for regulating intracellular iron homeostasis. It affects the concentration of intracellular iron ions by regulating the expression of downstream iron metabolism-related genes such as ferritin heavy chain (Fth). The loss of Ireb2 gene expression can lead to abnormal neuronal function and cognitive impairment in mice. - / - ) The samples of the hippocampus of the mouse brain were collected, protein was extracted and western blot was performed (the antibodies used in the detection process are shown in Table 6). It was found that after the D826V mutation, the D826V - / - The expression of Ireb2 protein in the mouse hippocampus was significantly reduced, while the expression of the downstream regulatory gene Fth was significantly increased, suggesting that the D826V mutation caused the degradation of Ireb2 protein and affected the expression of genes related to iron metabolism ( Figure 7 ).

[0099] Table 6 Specific information of antibodies

[0100] Antibody factory Part Number concentration Ireb2 Proteintech 29976-1-AP 1：1000 F Affinity DF6278 1：1000 Actb (internal reference) Affinity AF7018 1：3000

[0101] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A gRNA specifically targeting the pathogenic site of Ireb2 gene mutation, characterized in that: The gRNA includes gRNA-1 and gRNA-2; the nucleotide sequence of the gRNA-1 is shown in SEQ ID NO.3; the nucleotide sequence of the gRNA-2 is shown in SEQ ID NO.4; the Ireb2 gene mutation pathogenic site is NM_022655.3: exon20: c.2477A>T: p.D826V.

2. Use of the gRNA according to claim 1 in preparing a product that specifically targets the pathogenic site of Ireb2 gene mutation.

3. The product according to claim 2, characterized in that The product includes a reagent, a kit or a chip.

4. A product specifically targeting the pathogenic site of Ireb2 gene mutation, characterized in that: The product includes the gRNA of claim 1.

5. Use of the gRNA described in claim 1 or the product described in claim 4 in constructing an Ireb2-p.D826V point mutation mouse model.

6. A method for constructing an Ireb2-p.D826V point mutation mouse model based on CRISPR-Cas9 technology, characterized in that: The following steps are involved: Injecting a mixture containing the gRNA, template DNA and Cas9 protein according to claim 1 into mouse fertilized eggs to obtain mutant F0 generation heterozygous mice; the nucleotide sequence of the template DNA is shown in SEQ ID NO.2; The Ireb2-p.D826V point mutation mouse model was obtained after self-fertilization of the mutant F0 generation heterozygous mice.

7. The method according to claim 6, characterized in that The concentration of gRNA in the mixture is 2 pmol / μL, the concentration of template DNA is 15 ng / μL, and the concentration of Cas9 protein is 30 ng / μL.

8. The method according to claim 6, characterized in that The mass ratio of gRNA-1 to gRNA-2 in the gRNA is 1:

1.

9. The method according to claim 6, characterized in that The injection dosage is 1-2PL.

10. Use of the Ireb2-p.D826V point mutation mouse model constructed by the method according to any one of claims 6 to 9 in the preparation and / or screening of drugs for treating neurodegenerative diseases.

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