Construction method of primary dilated cardiomyopathy genetic engineering animal model

Abstract

The invention provides a construction method of a primary dilated cardiomyopathy genetic engineering animal model, which comprises the following steps: knocking out the 117th A basic group of the ninth exon of an animal NEXN gene to cause the change of an NEXN gene reading frame and cause the generation of truncated NEXN protein, thereby constructing the primary dilated cardiomyopathy genetic engineering animal model. Based on NEXN gene mutation, the DCM mouse construction method is simple and easy to implement, and the model character is remarkable.

Description

Technical Field

[0001] The present invention relates to the technical field of constructing medical experimental models, and particularly to a method for constructing a DCM gene engineering mouse animal model. Background Art

[0002] DCM is a type of cardiomyopathy characterized by the enlargement of the left ventricle or both ventricles of the heart and accompanied by systolic dysfunction. It has many causes, and infections, immune responses, genetics, poisons, and metabolic disorders can all cause DCM. The symptoms of dilated cardiomyopathy are diverse, mainly including heart failure, arrhythmia, sudden death, etc. In the early stage, there may be no symptoms or mild symptoms, while in the late stage of the disease, there will be obvious heart failure symptoms. Patients will experience dyspnea during activities, paroxysmal nocturnal dyspnea, decreased exercise tolerance, etc., and may also include decreased appetite, abdominal distension, and lower limb edema. DCM causes great damage and has a high fatality rate, which is an important factor endangering human health.

[0003] The cause of DCM is complex, and there is currently a lack of effective treatment methods in clinical practice. Therefore, simulating the pathological changes and disease development of human dilated cardiomyopathy through animal models is very important for studying the pathogenesis of dilated cardiomyopathy and drug screening. Through biological methods, human DCM models can be replicated in experimental animals such as mice, rats, zebrafish, and beagle dogs [1-3] , but the breeding cost of large animals is relatively high and the surgical operation is difficult [4] , so currently the research on DCM models mainly focuses on small rodents. Doxorubicin (DOX) can induce mice and rats to produce pathological manifestations similar to those of human DCM, and it is the most commonly used animal model for DCM research, but the model induced by DOX cannot simulate the characteristics of primary DCM [5] . DCM caused by gene mutations is a common form of primary cardiomyopathy. The DCM animal models constructed based on gene mutations can more comprehensively reflect the essence of human cardiomyopathy. The previous primary DCM animal models were mostly constructed based on gene mutations such as MYH7, SCN5A, TTN, and LMNA [6] , and the forms of gene mutations not only include base deletions and insertions, but also point mutations. Primary DCM caused by point mutations is also an important way to construct animal models [1,7] . NEXN is a key gene related to the occurrence of DCM discovered in 2009 [8] , although there is currently a gene engineering mouse model caused by NEXN point mutation [9] , but for NEXN, the gene polymorphism determines that there are other point mutations that cause DCM.

[0004] References

[0005] 1. Yang L, Sun J, Chen Z, Liu L, Sun Y, Lin J, Hu X, Zhao M, Ma Y, Lu D, Li Y, Guo Y, Dong E. The LMNA p.R541C mutation causes dilated cardiomyopathy in human and mice. Int J Cardiol 2022, 363:149 - 158.

[0006] 2. Chen Q, Zeng Y, Yang X, Wu Y, Zhang S, Huang S, Zhong Y, Chen M. Resveratrol ameliorates myocardial fibrosis by regulating Sirt1 / Smad3 deacetylation pathway in rat model with dilated cardiomyopathy. BMC Cardiovasc Disord 2022, 22(1):17.

[0007] 3. Hofeichner J, Gahr BM, Huber M, Boos A, Rottbauer W, Just S. CRISPR / Cas9-mediated nexilin deficiency interferes with cardiac contractile function in zebrafish in vivo. Sci Rep 2023, 13(1):22679.

[0008] 4. Zhang D, Zhu JY, Zhang M, Li B. Establishment and evaluation of animal models of dilated cardiomyopathy. Laboratory Animal Science 2023, 40(4):55 - 61.

[0009] 5. Yu W, Deng D, Li Y, Ding K, Qian Q, Shi H, Luo Q, Cai J, Liu J. Cardiomyocyte-specific Tbk1 deletion aggravated chronic doxorubicin cardiotoxicity via inhibition of mitophagy. Free Radic Biol Med 2024, 222:244 - 258.

[0010] 6. Jin Jiamin, Gong Qian, Zhuang Lenan. Research progress on animal models and treatments of dilated cardiomyopathy. Journal of Zhejiang University (Agriculture and Life Sciences), 2024, 50(1): 1-11.

[0011] 7. Cai ZJ, Lee YK, Lau YM, Ho JC, Lai WH, Wong NL, Huang D, Hai JJ, Ng KM, Tse HF, Siu CW. Expression of Lmna-R225X nonsense mutation results in dilated cardiomyopathy and conduction disorders (DCM-CD) in mice: Impact of exercise training. Int J Cardiol 2020, 298: 85-92.

[0012] 8. Hassel D, Dahme T, Erdmann J, Meder B, Huge A, Stoll M, Just S, Hess A, Ehlermann P, Weichenhan D, Grimmler M, Liptau H, Hetzer R, Regitz-Zagrosek V, Fischer C, Nürnberg P, Schunkert H, Katus HA, Rottbauer W. Nexilin mutations destabilize cardiac Z-disks and lead to dilated cardiomyopathy. Nat Med 2009, 15(11): 1281-1288.

[0013] 9. Liu C, Spinozzi S, Feng W, Chen ZE, Zhang L, Zhu S, Wu T, Fang X, Ouyang K, Evans SM, Chen J. Homozygous G650del nexilin variant causes cardiomyopathy in mice. JCI Insight 2020, 5(16). Summary of the Invention

[0014] In view of the defects or deficiencies of the prior art, the present invention provides a method for constructing a genetic engineering animal model of primary dilated cardiomyopathy.

[0015] To this end, the construction method provided by the present invention includes knocking out the 117th A base of exon 9 of the animal NEXN gene, causing a change in the reading frame of the NEXN gene and resulting in the production of truncated NEXN protein, thereby constructing a genetic engineering animal model of primary dilated cardiomyopathy.

[0016] An alternative is that the animal is a mouse.

[0017] An alternative is to specifically knock out the 117th A base of exon 9 of the mouse NEXN gene using the CRISPR / Cas9 technology. Further, the method includes the following steps:

[0018] (1) Design a corresponding sgRNA sequence near the 117th site of exon 9 of the mouse NEXN gene;

[0019] (2) According to the principle of homologous recombination, design and synthesize an ssODN sequence for specific knockout of the 117th A base; the ssODN sequence is shown as SEQ ID NO.3;

[0020] (3) Microinject a mixture of Cas9 mRNA, sgRNA and ssODN into mouse fertilized eggs, and the obtained offspring mice after transplantation are continuously mated until homozygous animals are obtained.

[0021] An alternative is that the sgRNA sequence is shown as SEQ ID NO.2; the ssODN sequence is shown as SEQ ID NO.3.

[0022] An alternative is that step (1) includes synthesizing oligos according to the sgRNA sequence, and obtaining sgRNA by in vitro transcription for microinjection.

[0023] An alternative is that in step (3), a mixture of Cas9 mRNA, sgRNA and ssODN is microinjected into mouse fertilized eggs. After transplantation of the fertilized eggs into recipient mice, F0 generation mice with the 117th A base of exon 9 knocked out are obtained. Positive F0 generation mice are mated with wild-type mice to obtain F1 generation mice, and after F1 generation mice are intercrossed, homozygous mice with the 117th A base of exon 9 knocked out are obtained.

[0024] The present invention also provides an evaluation method for the animal model obtained by the above method. The method includes: identifying the animal model with the 117th A base of exon 9 deleted by PCR and Sanger sequencing;

[0025] Detect the changes in the heart volume, heart mass, body weight, left ventricular cavity, heart function, left ventricular end-diastolic diameter, end-systolic diameter, left ventricular anterior wall thickness at end-systole, and left ventricular posterior wall thickness at end-diastole of postnatal animals; among them, an animal model with an enlarged heart volume, increased heart mass, increased body weight, enlarged left ventricular cavity, decreased heart function, increased left ventricular end-diastolic diameter, increased end-systolic diameter, decreased left ventricular anterior wall thickness at end-systole, and increased left ventricular posterior wall thickness at end-diastole is a primary dilated cardiomyopathy animal model.

[0026] The present invention solves the problems that there are few breeds and strains of primary DCM animal models at present, the stability and repeatability are poor, and the pathological manifestations and pathogenesis of human primary DCM cannot be comprehensively reflected. This scheme has been experimentally verified and can spontaneously develop symptoms similar to dilated cardiomyopathy after the birth of mice, and can be used for the screening of DCM treatment methods and disease-related research.

[0027] The primary DCM gene engineering mouse model of the present invention is based on the 117th base point mutation in exon 9 of NEXN, has good heredity, high stability and repeatability, and significant model traits, and can be used for the research on the molecular mechanism of the occurrence and development of primary DCM and drug screening. This model can be used to study the molecular mechanism of the occurrence and development of primary DCM, prepare and screen drugs for the treatment of DCM, etc. Brief Description of the Drawings

[0028] Figure 1 It is a schematic diagram of gene editing and sgRNA sequence in the embodiment of the present invention.

[0029] Figure 2 It is the in vitro transcription result of sgRNA in the embodiment of the present invention.

[0030] Figure 3 It is the PCR detection result of the model mice in the embodiment of the present invention; among them, 1-14 are the genotype PCR detections of the F0 generation mice at birth; wt is the wild type; bl is the blank control; M is the marker.

[0031] Figure 4 It is the genotype detection of the model mice in the embodiment of the present invention; among them, A is the analysis of the mutant genotype by Sanger sequencing; B is the amino acid sequence of the mutant NEXN protein.

[0032] Figure 5 It is the appearance inspection of the model mice in the embodiment of the present invention.

[0033] Figure 6 It is the gross anatomy of the heart and the heart / body weight ratio of the model mice in the embodiment of the present invention;; among them, A is the gross anatomy of the mouse heart; B is the mouse heart / body weight ratio.

[0034] Figure 7Detection of Nexilin protein expression level in the model mice of the embodiments of the present invention.

[0035] Figure 8 HE and Masson staining of the model mice in the embodiments of the present invention.

[0036] Figure 9 Echocardiogram detection of the model mice in the embodiments of the present invention. Detailed implementation manners

[0037] Unless otherwise specified, scientific and technical terms and related abbreviations in this article are understood according to the knowledge of those of ordinary skill in the relevant fields.

[0038] The technical solutions of the present invention will be described in detail and completely below in conjunction with specific embodiments. The described embodiments are only a part of the present invention, rather than all embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts fall within the scope of protection of the present invention.

[0039] Unless otherwise specified, test conditions and operations not mentioned in the embodiments of the present invention are carried out according to conventional methods in the art or conditions recommended by the manufacturer.

[0040] Example 1: Design and synthesis of sgRNA sequences

[0041] (1) Design of sgRNA sequences

[0042] According to the characteristics of the NEXN gene, a mouse animal model of dilated cardiomyopathy was constructed by site-directed deletion of A at the 117th nt of exon 9. Exon 9 consists of 198 bp nucleotides, and the sequence is shown in SEQ ID NO.1. The above exon 9 sequence of the NEXN gene was uploaded to the CRISPOR and CHOPCHOP websites to analyze potential sgRNA sequences that can be used for gene knockout; according to the analysis results, two 20 bp sequences with relatively high scores on both websites and a GC content of 40%-70% were selected as candidate sgRNAs and synthesized ( Figure 1 ), and the sgRNA sequences are shown in SEQ ID NO.2.

[0043] (2) Synthesis of sgRNA

[0044] When synthesizing, a 17 bp T7 promoter sequence 5'-TAATACGACTCACTATA-3' was introduced at the 5' end of the sgRNA, a 20 bp RNA scaffold sequence was introduced at the 3' end, and according to the codon preference, 3 Gs were added before the sgRNA to improve the transcription efficiency. This 60 bp sequence (5'- TAATACGACTCACTATAgggagaagaaagacgacgaacagGTTTTAGAGCTAGAAATAGC-3’, where the underlined part is the T7 promoter sequence, the lowercase letters are the sgRNA sequence, and the uppercase letters are the scaffold sequence) was synthesized by Beijing Tsingke Biotechnology Co., Ltd. and used as the upstream primer (sgRNA-F) for sgRNA PCR amplification. In addition, according to the characteristics of the pX330 plasmid, a 20-bp sequence of 5’-AGCACCGACTCGGTGCCACT-3’ was designed and synthesized as the downstream primer (sgRNA-R) for PCR amplification.

[0045] Example 2: In vitro transcription of sgRNA

[0046] (1) PCR amplification and purification of the sgRNA sequence

[0047] Using the pX330 plasmid as a template, PCR amplification was performed with sgRNA-F and sgRNA-R as the upstream and downstream primers. The PCR reaction system is shown in Table 1 below:

[0048] Table 1

[0049] Reagent Volume (μl) pX330 1 2×Taq MIX 20 sgRNA-F 2 sgRNA-R 2 <![CDATA[ddH2O]]> 25

[0050] The PCR reaction conditions are shown in Table 2 below:

[0051] Table 2

[0052]

[0053] The PCR product was purified using the Tiangen DNA purification kit. After equilibrating the adsorption column, the PCR product was added to the adsorption column, and the elution buffer was added twice for elution. The purified DNA was collected from the adsorption column and quantified.

[0054] (2) In vitro transcription of sgRNA

[0055] 1) Prepare the reaction system according to Table 3 below

[0056] Table 3

[0057] PCR Amplification Product 800 ng 5X TranscriptAid Reaction Buffer 4 μl ATP / CTP / GTP / UTP Mix 8 μl TranscriptAid Enzyme Mix 2 μl <![CDATA[DEPC H2O]]> Up to 20 μl

[0058] For in vitro transcription of sgRNA, the TranscriptAid T7 High Yield Transcription Kit (Thermo Scientific TM Company) was used and operated according to the instructions. The specific steps are as follows:

[0059] 2) In vitro transcription

[0060] After mixing the above components, incubate at 37 °C for 2 h, and collect the product for the next purification step.

[0061] 3) Purification of RNA products

[0062] The in vitro transcription product of sgRNA was purified using the MEGAclear TM kit (ThermoFisher Scientific TM Company) according to the instructions.

[0063] Add 20 μl of the transcription product to 100 μl of Elution Solution, 350 μl of Binding Solution, and 250 μl of ethanol, mix well, slowly transfer it into the elution column, centrifuge at 12,000 rpm for 30 s, discard the centrifugate, wash twice with 500 μl of Wash Solution, and elute with 20 μl of Wash Solution preheated at 95 °C. Collect the eluate, and after quantification with NanoDrop, it is the purified sgRNA. The size of the sgRNA was identified by 1% agarose gel electrophoresis to be approximately 120 bp, which is consistent with the expectation ( Figure 2 ). Quantitative analysis showed that sgRNA OD260 / OD280 > 1.8, OD260 / OD230 > 2.3, the content was 1.2 μg / μl, and it was stored at -80 °C for later use.

[0064] Example 3: Design and synthesis of ssODN

[0065] According to the characteristic of specifically knocking out the 117th A base in exon 9 of NEXN, a 120-bp sequence was designed for gene homologous recombination. This sequence consists of 60-bp 5'-left and 3'-right homologous arms respectively, and the homologous arm sequences are exactly the same as the wild type, with the A base deleted in the middle. The sequence is shown in SEQ ID NO.3, and after the sequence design was completed, it was synthesized into ssODN by Beijing Tsingke Biotechnology Co., Ltd.

[0066] Example 4: Microinjection and transplantation of fertilized eggs

[0067] (1) Obtaining of C57BL / 6J mouse fertilized eggs

[0068] Female and male mice inoculated with 5 U of PMSG and HCG were caged together. The mated mice were sacrificed the next day, and mouse fertilized egg cells were collected from the ampulla of the fallopian tube under a stereomicroscope.

[0069] (2) Microinjection

[0070] 100 ng of Cas9 mRNA (ThermoFisher Scientific TMThe 50 ng sgRNA obtained in Example 2 and the synthesized 50 ng ssODN were mixed in proportion to prepare a Cas9 / sgRNA / ssODN mixture. The Cas9 / sgRNA / ssODN mixture was injected into the cytoplasm of fertilized C57BL / 6J mouse oocytes using an Eppendorf NK2 microinjector. The injected fertilized oocytes were cultured at 37 °C in 5% CO2 for 24 h until the 2-cell stage.

[0071] (3) Transfer of fertilized oocytes

[0072] The 2-cell embryos were transferred to recipient ICR mice. The offspring mice were F0 generation animals for genotype identification after birth.

[0073] Example 5: Genotype identification of F0 mice

[0074] (1) Extraction of genomic DNA from F0 mice

[0075] The genomic DNA of F0 generation mice was extracted using a Blood / Tissue / Cell Genomic DNA Extraction Kit (Tiangen Biochemical Technology Co., Ltd.). The specific method is as follows:

[0076] 1) Cut the tail tissue of F0 generation mice at 7 days after birth. Add 200 μl of GA solution and 20 μl of Proteinase K to the sample, and incubate overnight at 56 °C until the tissue is lysed and digested;

[0077] 2) Add 200 μl of GB solution to the lysate, invert and mix well. Place at 70 °C until the solution becomes clear; add 200 μl of absolute ethanol, shake and mix well; pour the mixture into an adsorption column, centrifuge at 12000 rpm for 30 s, and discard the waste liquid;

[0078] 3) Add 500 μl of GD to the adsorption column, centrifuge at 12000 rpm for 30 s, and discard the waste liquid; add 600 μl of PW, centrifuge at 12000 rpm for 30 s and elute continuously twice;

[0079] 4) Drop 50 μl of TE elution buffer onto the adsorption membrane of the adsorption column, let it stand for 5 min, centrifuge at 12000 rpm for 30 s, and collect the genomic DNA for identification.

[0080] (2) Genomic identification of F0 mice

[0081] The corresponding fragment of the NEXN gene was amplified using 2×Taq MIX (Takara Co., Ltd.) and subjected to sequencing analysis. The specific method is as follows:

[0082] The primers required for PCR were synthesized by Beijing Tsingke Biotechnology Co., Ltd. The primers used are as follows:

[0083] Forward primer: 5’-CCTCCATATCACAGATACCCCAG-3’; Reverse primer: 5’-CCTTGCTACACTCTACCAGGTC-3’.

[0084] The PCR reaction system is shown in Table 4:

[0085] Table 4

[0086] Reagent Volume (μl) Genomic DNA 2 2×Taq MIX 20 Forward Primer 2 Reverse Primer 2 <![CDATA[ddH2O]]> 24

[0087] The PCR reaction conditions are shown in Table 5:

[0088] Table 5

[0089]

[0090] After the PCR products were identified by 1% agarose gel electrophoresis ( Figure 3 ), Beijing Tsingke Biotechnology Co., Ltd. sequenced and analyzed the genotypes by Sanger sequencing method. The sequencing results showed that the 117th A base of exon 9 of the NEXN gene in F0 mice was deleted (Δ1) ( Figure 4 -A); the wild-type NEXN gene encodes a Nexilin protein of 672 amino acids, and the deletion of the A base leads to a frameshift change, generating a TAG stop codon at 1186 - 1188 bp, resulting in premature termination of translation and only a truncated Nexilin protein of 396 amino acids (truncated NEXN protein) can be translated ( Figure 4 -B).

[0091] Example 6: Establishment of DCM homozygous mice

[0092] The Δ1 F0 mice were mated with wild-type mice to obtain F1 heterozygous mice, and the F1 heterozygous mice were intercrossed to obtain F2 mice. The F2 mice of different genders were continuously intercrossed until DCM homozygous mice with the Δ1 mutation were obtained.

[0093] Example 7: Identification and evaluation of DCM homozygous mice

[0094] In this example, the appearance and gross anatomy of the heart of the DCM homozygous mice constructed by the method of Example 6 were observed; the expression level of NEXN protein was detected by Western blot; the pathological changes of the mouse heart tissue were detected by HE and Masson staining; and the cardiac function changes of the mice were detected by echocardiography.

[0095] (1) General status observation and gross anatomy

[0096] After conventional feeding, the wild mice survived well, but the Δ1 mutant mice were thin and had sparse hair ( Figure 5 ). After dissection, it was found that the heart volume was significantly increased (Figure 6 A), the heart weight ratio increased ( Figure 6 B).

[0097] (2) The expression level of NEXN protein in the heart tissue of model mice

[0098] Fresh heart tissue of mice was excised, ground, and lysed with an appropriate amount of RIPA lysis buffer. After protein quantification, SDS-PAGE electrophoresis was performed using a 10% separating gel. The separated gel was transferred to a PVDF membrane at a voltage of 100 V for 1.5 h. The PVDF membrane was blocked, and a 1:500 anti-NEXN antibody (Wuhan Sanying Biotechnology Co., Ltd.) was added and incubated overnight at 4 °C. Then, the secondary antibody was added, and the membrane was washed with TBST and developed with ECL. The results showed that compared with wild-type mice, the expression of NEXN in the heart tissue of DCM homozygous mice decreased significantly ( Figure 7 ).

[0099] (3) Pathological changes in the heart tissue of model mice

[0100] Fresh hearts of mice were excised, rinsed with PBS, and fixed with 4% paraformaldehyde for 48 h. After paraffin embedding and sectioning, the sections were deparaffinized and stained with HE and Masson according to the conventional method. The HE- and Masson-stained sections were scanned with a 3DHISTECHs and analyzed. HE staining showed that the left ventricular cavity of DCM homozygous mice was significantly enlarged, the volume increased, and the ventricular wall became thinner, which was consistent with the pathological manifestations of dilated cardiomyopathy. However, Masson staining showed that the characteristics of myocardial cell fibrosis were not obvious ( Figure 8 ).

[0101] (4) Evaluation of cardiac function in model mice

[0102] Mice at 6 days after birth were placed on their backs, and conductive gel was applied to their chests. A small animal photoacoustic ultrasound imaging system (Fujifilm VisualSonics Perform echocardiography on the hearts of mice using LAZR-X. Based on the obtained M-mode echocardiogram images, calculate the following indices: left ventricular anterior wall thickness in diastole (LVAWd), left ventricular anterior wall thickness in systole (LVAWs), left ventricular posterior wall thickness in diastole (LVPWd), left ventricular posterior wall thickness in systole (LVPWs), left ventricle internal diameter in diastole (LVIDd), left ventricle internal diameter in systole (LVIDs), ejection fraction (EF), and fractional shortening (FS). The results showed that the cardiac function of DCM homozygous mice was severely impaired, with decreased LVAWd and LVAWs, increased LVIDd, LVIDs, and LVPWd, and significantly decreased LVEF and LVFS, while LVPWs remained unchanged ( Figure 9 ).

[0103] The above results, in combination with the accompanying drawings, have described in detail the embodiments of the present disclosure. The methods and means established in the present invention can be used to establish a stable and efficient genetically engineered animal model of primary DCM in mice. This model can simulate the development process and body changes of human dilated cardiomyopathy and can be used for research such as screening of therapeutic drugs for primary DCM and the mechanism of disease development. However, the present disclosure is not limited to the specific details in the above embodiments. Within the technical scope of the present disclosure, various modifications and optimizations can be made to the technical solutions of the present disclosure, and these simple optimizations all fall within the protection scope of the present disclosure.

[0104] In addition, it should be noted that, in the case of no conflict, the various specific technical features described in the above specific embodiments can be combined in any suitable manner. To avoid unnecessary repetition, the present disclosure will not separately describe various possible combination methods.

[0105] Furthermore, any combination can be made between various different embodiments of the present disclosure, as long as it does not violate the idea of the present disclosure, and it should also be regarded as the content disclosed by the present disclosure.

[0106] Sequence Listing of the Specification

[0107]

Claims

1. A method for constructing a genetically engineered animal model of primary dilated cardiomyopathy, characterized in that: The method includes knocking out the 117th A base of exon 9 of the animal NEXN gene, causing a change in the NEXN gene reading frame, resulting in the production of truncated NEXN protein, thereby constructing a genetically engineered animal model of primary dilated cardiomyopathy.

2. The method for constructing a genetically engineered animal model of primary dilated cardiomyopathy according to claim 1, characterized in that: The animal is a mouse.

3. The method for constructing a genetically engineered animal model of primary dilated cardiomyopathy according to claim 1, characterized in that: CRISPR / Cas9 technology was used to specifically knock out the 117th A base in exon 9 of the mouse NEXN gene.

4. The method for constructing a genetically engineered animal model of primary dilated cardiomyopathy according to claim 3, characterized in that: The following steps are involved: (1) Designing the corresponding sgRNA sequence near site 117 of exon 9 of the mouse NEXN gene; (2) According to the principle of homologous recombination, a ssODN sequence was designed and synthesized for specific knockout of the 117th A base; the ssODN sequence is shown in SEQ ID NO.3; (3) The mixture of Cas9 mRNA, sgRNA, and ssODN is microinjected into mouse fertilized eggs, and the offspring mice obtained after transplantation are continuously mated until homozygous animals are obtained.

5. The method for constructing a genetically engineered animal model of primary dilated cardiomyopathy according to claim 4, characterized in that: The sgRNA sequence is shown in SEQ ID NO.2; the ssODN sequence is shown in SEQ ID NO.

3.

6. The method for constructing a genetically engineered animal model of primary dilated cardiomyopathy according to claim 4, characterized in that: Step (1) includes synthesizing oligos according to the sgRNA sequence, and obtaining sgRNA by in vitro transcription for microinjection.

7. The method for constructing a genetically engineered animal model of primary dilated cardiomyopathy according to claim 4, characterized in that: In step (3), the mixture of Cas9 mRNA, sgRNA and ssODN is microinjected into mouse fertilized eggs. After the fertilized eggs are transplanted into recipient mice, F0 generation mice with A base knockout at position 117 of exon 9 are obtained. Positive F0 generation mice are mated with wild-type mice to obtain F1 generation mice. After the F1 generation is intercrossed, homozygous mice with A base knockout at position 117 of exon 9 are obtained.

8. A method for evaluating an animal model obtained by the construction method according to any one of claims 1 to 7, characterized in that: PCR and Sanger sequencing are used to identify an animal model with a deletion of the A base at position 117 of exon 9; To detect the changes of heart volume, heart mass, body weight, left ventricular cavity, heart function, left ventricular end-diastolic diameter, end-systolic diameter, left ventricular end-systolic anterior wall thickness and left ventricular end-diastolic posterior wall thickness of postnatal animals; in, An animal model with increased heart volume, increased heart mass, increased body weight, enlarged left ventricular cavity, decreased heart function, increased left ventricular end-diastolic diameter, increased end-systolic diameter, decreased left ventricular end-systolic anterior wall thickness and increased left ventricular end-diastolic posterior wall thickness is an animal model of primary dilated cardiomyopathy.

9. Use of the animal model constructed by the method according to any one of claims 1 to 7 for drug research on primary dilated cardiomyopathy.

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