DC-SIGN humanized gene editing mouse and application thereof

Abstract

The invention discloses a DC-SIGN humanized gene editing mouse and an application of the DC-SIGN humanized gene editing mouse. The invention specifically discloses a method for constructing a novel coronavirus susceptible animal model, which comprises the following steps: expressing human or humanized CD209 protein in a non-human animal, and reducing or deleting the expression of endogenous CD209 protein in the non-human animal. According to the invention, a CRISPR / Cas9 gene editing technology is utilized to directly replace a coding region of a human CD209 gene genome into a coding region of a non-human animal CD209 gene genome, so that whole-gene humanization of CD209 is realized, and physiological characteristics of CD209 and immune response of CD209 during infection of new coronavirus are reproduced. The animal model can stably express the human CD209 gene and protein, the model is convenient to prepare, the flow time is short, the vacancy of the whole-gene humanized CD209 animal model in the current market is filled, and the subsequent research and utilization are greatly facilitated.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering, and specifically relates to a DC-SIGN humanized gene-edited mouse and uses thereof. Background Art

[0002] The novel coronavirus (COVID-19) is a respiratory infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It is a global outbreak, posing a serious threat to human health. Patients present with symptoms such as cough, fever, and fatigue. Severe cases may lead to respiratory distress, multiple organ failure, and even death. Currently, the development of vaccines and antiviral drugs for the novel coronavirus, as well as related basic research, requires further development.

[0003] Animal models are crucial for elucidating viral pathogenesis and host immune responses and are fundamental for evaluating vaccine efficacy and drug effects. Therefore, developing animal models susceptible to the novel coronavirus is a crucial step in drug and vaccine research and development. Although non-human primates share a high degree of similarity to humans, they are unsuitable for widespread application due to their high cost, long breeding cycles, and limited availability. Mouse models, however, offer advantages such as a clear genetic background, low cost, and a short breeding cycle, and are widely used to study the pathogenesis of human coronaviruses. Human angiotensin-converting enzyme II (ACE2) is widely considered to be the functional receptor for coronaviruses and mediates the entry of the novel coronavirus into host cells. However, ACE2 expression patterns show peak expression in the intestine, with low expression in the lungs, suggesting the possible involvement of other receptors in regulating viral infection. Dendritic cell-specific intercellular adhesion molecule-3 (DC-SIGN / CD209), a C-type lectin primarily expressed on dendritic cells, plays a crucial role in both innate and adaptive immunity. Studies have found that CD209 is widely expressed in the lungs and throughout the body, and can also enhance the replication of SARS-CoV-2, suggesting it serves as an alternative receptor independent of ACE2. However, due to the low sequence similarity between mouse and human CD209, mice cannot be directly used to simulate novel coronavirus infection. Currently, the only available CD11c promoter-humanized CD209 mouse model also suffers from random insertions and long cycle times, making it unable to quickly and intuitively reflect viral infection. Therefore, a humanized CD209 mouse is needed that closely mimics the characteristics of in vivo infection and is susceptible to the virus. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide an animal model that is convenient, rapid, and highly simulates the characteristics of in vivo infection and is susceptible to the new coronavirus. The technical problem to be solved is not limited to the technical subject matter described above, and those skilled in the art will clearly understand other technical subjects not mentioned herein through the following description.

[0005] To solve the above technical problems, the present invention first provides a method for constructing a novel coronavirus susceptible animal model, which comprises expressing human or humanized CD209 protein in a non-human animal, while reducing or eliminating the expression of endogenous CD209 protein in the non-human animal.

[0006] The non-human animal described herein may be a mouse.

[0007] The human or humanized CD209 protein described herein may be any of the following:

[0008] C1) the humanized CD209 protein comprises all or part of a human CD209 protein;

[0009] C2) the humanized CD209 protein comprises all or part of the signal peptide, extracellular region, transmembrane region and / or cytoplasmic region of the human CD209 protein;

[0010] C3) the humanized CD209 protein comprises all or part of the protein encoded by the human CD209 gene 201 transcript (GenBank sequence number: NM_001144894.2);

[0011] C4) The humanized CD209 protein comprises all or part of exons 1 to 5 of the human CD209 gene 201 transcript (GenBank sequence number: NM_001144894.2).

[0012] C5) The amino acid sequence of the human CD209 protein (human CD209 protein) is shown in SEQ ID NO: 8.

[0013] The non-human animal endogenous CD209 protein described herein may be a mouse endogenous CD209 protein, and the amino acid sequence of the mouse endogenous CD209 protein may be as shown in SEQ ID NO:7.

[0014] Furthermore, the method includes replacing the coding region of the non-human animal CD209 gene genome with the coding region of the human CD209 gene genome, wherein the coding region of the genome includes all or part of the exons.

[0015] Furthermore, the method includes replacing a nucleic acid molecule encoding an endogenous mouse CD209 protein (SEQ ID NO: 7) with a nucleic acid molecule encoding a human CD209 protein (SEQ ID NO: 8).

[0016] Furthermore, the humanization described herein may be full humanization.

[0017] The coding region of the non-human animal CD209 gene genome described herein may be the coding region of the mouse CD209 gene genome, which may include exons 1 to 7 of the mouse CD209 gene, as specifically shown in SEQ ID NO: 1.

[0018] The coding region of the human CD209 gene genome described herein may include exons 1 to 5 of the human CD209 gene.

[0019] In the above method, the nucleotide sequence of the coding region of the human CD209 gene genome may be as shown in SEQ ID NO: 2.

[0020] Furthermore, the method includes replacing the coding region of the mouse CD209 gene genome with the nucleotide sequence of SEQ ID NO: 1 with the coding region of the human CD209 gene genome with the nucleotide sequence of SEQ ID NO: 2.

[0021] In the above method, the replacement can be performed using the CRISPR / Cas9 gene editing system, which includes sgRNA, and the target sequence of the sgRNA can be SEQ ID NO: 5 (sgRNA4) and SEQ ID NO: 6 (sgRNA9).

[0022] The sgRNAs (i.e., sgRNA4 and sgRNA9) are designed near the recombination sites of exon 1 (Exon 1) and exon 7 (Exon 7) of the mouse CD209 gene, respectively, for gene editing of the mouse CD209 gene.

[0023] The gene editing described herein may include gene knockout, gene knockin, gene mutation, gene fragment replacement or gene modification, etc.

[0024] In the above method, the CRISPR / Cas9 gene editing system may further include a targeting vector, which may contain a nucleic acid molecule encoding a human or humanized CD209 protein.

[0025] The nucleic acid molecule encoding the human or humanized CD209 protein described herein may be any of the following:

[0026] D1) a DNA molecule encoding all or part of the human CD209 protein;

[0027] D2) a DNA molecule encoding all or part of the signal peptide, extracellular region, transmembrane region and / or cytoplasmic region of the human CD209 protein;

[0028] D3) all or part of exons 1 to 5 of the human CD209 gene;

[0029] D4) A DNA molecule having a nucleotide sequence of SEQ ID NO: 2.

[0030] In the above method, the targeting vector may further contain an upstream homology arm and a downstream homology arm. The nucleotide sequence of the upstream homology arm may be as shown in SEQ ID NO: 3, and the nucleotide sequence of the downstream homology arm may be as shown in SEQ ID NO: 4.

[0031] Furthermore, the CRISPR / Cas9 gene editing system may also include a Cas protein (such as a Cas9 protein).

[0032] Furthermore, the method may include injecting the targeting vector, the sgRNA and Cas9 mRNA into the fertilized egg cell of the non-human animal, and transplanting it into the fallopian tube of a surrogate animal. After genotyping of the obtained non-human animal or its offspring, the non-human animal carrying the human CD209 gene is the novel coronavirus susceptible animal model.

[0033] The present invention also provides a novel coronavirus-susceptible animal model constructed using any of the methods described herein, or the use of the novel coronavirus-susceptible animal model in preparing a CD209 humanized animal model infected with the novel coronavirus, studying the pathogenic mechanism of the novel coronavirus, and developing vaccines or drugs.

[0034] The present invention also provides a biomaterial or any of the following uses of the biomaterial:

[0035] A1) Application in mouse CD209 gene editing;

[0036] A2) Use in the preparation of CD209 gene humanized mouse models or novel coronavirus susceptible animal models;

[0037] The biological material may be any of the following:

[0038] B1) sgRNA described herein;

[0039] B2) the targeting vector described herein;

[0040] B3) any of the CRISPR / Cas9 gene editing systems described herein;

[0041] B4) A recombinant cell containing the sgRNA described in B1), the targeting vector described in B2, or the CRISPR / Cas9 gene editing system described in B3).

[0042] The method for constructing a novel coronavirus susceptible animal model described herein may include the following steps:

[0043] E1) preparing sgRNA, wherein the target sequence of the sgRNA may be SEQ ID NO: 5 (sgRNA4) and SEQ ID NO: 6 (sgRNA9);

[0044] E2) constructing a targeting vector containing the coding region (SEQ ID NO: 2), upstream homology arm (SEQ ID NO: 3), and downstream homology arm (SEQ ID NO: 4) of the human CD209 gene genome;

[0045] E3) Microinjection: Microinjecting sgRNA4 and sgRNA9 obtained in step E1), Cas9 mRNA, and the targeting vector obtained in step E2) into mouse fertilized eggs to obtain embryos;

[0046] E4) Embryo transfer: The embryos obtained in step E3) are briefly cultured in culture medium and then transferred into the oviduct of the mother mouse until the offspring mice are born;

[0047] E5) Genotype identification: The genotype of the offspring mice obtained in step E4) is identified. The offspring mice carrying the humanized CD209 gene and their offspring are the CD209 gene humanized mouse models.

[0048] The genome of the CD209 gene humanized mouse model described herein may contain a gene for a human or humanized CD209 protein (ie, a human or humanized CD209 gene).

[0049] The humanized CD209 gene may comprise a portion of the human CD209 gene.

[0050] The humanized CD209 gene can be designed with reference to the 201 transcript (GenBank sequence number: NM_001144894.2).

[0051] The humanized CD209 gene may comprise all or part of exons 1 to 7 of the human CD209 gene 201 transcript (GenBank sequence number: NM_001144894.2).

[0052] The present invention also provides a method for constructing a CD209 humanized animal model infected with the novel coronavirus, which may include infecting the novel coronavirus-susceptible animal model described herein with the novel coronavirus.

[0053] Furthermore, the infection can be carried out by nasal drops.

[0054] Furthermore, the novel coronavirus may be the Omicron BA.2 strain.

[0055] Furthermore, the inoculation amount of the Omicron BA.2 strain can be 3×10 3 TCID50 / mouse.

[0056] The animal may be a mouse.

[0057] The present invention also provides a CD209 humanized animal model infected with the novel coronavirus obtained according to the method for constructing a CD209 humanized animal model infected with the novel coronavirus.

[0058] The present invention also provides the application of the CD209 humanized animal model infected with the novel coronavirus in the study of the pathogenic mechanism of the novel coronavirus and in the development of vaccines or drugs.

[0059] DC-SIGN and CD209 mentioned herein have the same meaning and can be used interchangeably.

[0060] At present, the new coronavirus is still mutating, which continues to threaten people's health and safety. However, basic research on the development of effective drugs, vaccines, and antibodies for the new coronavirus still needs to be strengthened. Research on the key mechanisms of the new coronavirus infecting the body will provide new ideas for the effective prevention and control of the virus. DC-SIGN / CD209, as one of the C-type lectin molecules, has been reported to be able to promote infection of the body as an alternative receptor for the new coronavirus. The humanized CD209 mice prepared by the present invention can reproduce the physiological characteristics of CD209 and the immune response of CD209 during new coronavirus infection in mice. This will play an important reference role in researchers' understanding of the characteristics and pathogenesis of new coronavirus infection, and provide new ideas for the preparation of vaccines, antibodies and the development of drugs.

[0061] This study utilizes CRISPR / Cas9 gene editing technology to directly replace the coding region of the human CD209 gene genome with the coding region of the mouse CD209 gene genome, achieving full gene humanization of CD209 and successfully expressing human DC-SIGN / CD209 in mice. This mouse model addresses the issues of random gene insertion, inaccurate human gene expression, and complex model preparation processes that plague existing CD11c promoter-humanized DC-SIGN mouse models. It stably expresses the human CD209 gene and protein, and is easy to prepare with a short preparation time. This model fills the current market gap for fully humanized CD209 animal models and greatly facilitates subsequent research and utilization. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 Targeting strategy for CD209 humanized mice.

[0063] Figure 2 This is the sgRNA activity detection result.

[0064] Figure 3 For the enzyme digestion identification and targeting map of the targeting vector.

[0065] Figure 4 This is the identification result of F0 generation mice.

[0066] Figure 5 This is the identification result of F1 generation mice.

[0067] Figure 6 This is the identification result of F2 generation mice.

[0068] Figure 7 The following are the results of PCR and sequencing identification of the offspring mice tail.

[0069] Figure 8 Figure 2 is a graph of mouse weight and lifespan.

[0070] Figure 9 Count the peripheral blood cells of mice.

[0071] Figure 10 This is the pathological condition of the spleen, lung, kidney and liver of mice.

[0072] Figure 11 The viral loads in the lungs and nasal turbinates of mice were measured on the third day after infection.

[0073] Figure 12 The lung tissue pathology and scoring of mice on the third day after infection. DETAILED DESCRIPTION

[0074] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0075] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0076] BamHI, HindIII, XhoI, BstZ171, EcoRI and SalI enzymes were purchased from NEB with catalog numbers: R3136M, R3104M, R0146M, R3594L, R3101M, respectively.

[0077] C57BL / 6 mice were obtained from the National Rodent Laboratory Animal Center of the China Food and Drug Administration.

[0078] The UCA kit was obtained from Biocytogen, catalog number: BCG-DX-001.

[0079] MEGAshortscript TM Kit (Ambion in vitro transcription kit) was purchased from Thermo Fisher, catalog number: AM1354.

[0080] Cas9 mRNA is from SIGMA, product number: CAS9MRNA-1EA.

[0081] Example 1. Preparation of CD209 gene humanized mice

[0082] 1. Target shooting strategy

[0083] Mouse CD209 gene background: NCBI gene ID: 170786, Primary source: MGI: 2157942, Uniprot ID: Q91ZX1, located on chromosome 8, NC_000074.7, positions 3793397-3798984, based on transcript NM_133238.5 and its encoded protein NP_573501.1 (SEQ ID NO: 7); Human CD209 gene background: NCBI gene ID: 30835, Primary source: HGNC: 1641, Uniprot ID: Q9NNX6, located on chromosome 19, NC_000019.10, positions 7739993-7747534, based on transcript NM_001144894.2 and its encoded protein NP_001138366.1 (SEQ ID NO: 8).

[0084] To analyze the gene structure of the CD209 gene, the EGE system developed based on CRISPR / Cas9 can be used to replace the coding region of the mouse CD209 gene genome (see SEQ ID NO: 1 in the sequence listing) with the coding region of the human CD209 gene genome (see SEQ ID NO: 2 in the sequence listing) to achieve the purpose of initiating the expression of the human sequence under the mouse promoter. The specific strategy diagram is shown in the figure below. Figure 1 shown.

[0085] 2. Cas9 / sgRNA Design and Activity Detection

[0086] sgRNAs were designed near the Exon1 and Exon7 recombination sites of the mouse CD209 gene. Based on the design principles of sgRNA, 8 sgRNAs were designed in the 5' target site and 3' target site regions respectively. The activity of sgRNA was confirmed using the UCA kit. Based on the sgRNA specificity and cutting position, sgRNA4 and sgRNA 9 were comprehensively selected for the next step of the experiment. The test results are as follows Figure 2 shown.

[0087] The target sequence of sgRNA4 is: 5'-TCAGTCATATATAGGGTAGTCGG-3' (SEQ ID NO: 5), and the target sequence of sgRNA9 is: 5'-GAGTTGGCCATCACTTGCTAGGG-3' (SEQ ID NO: 6).

[0088] 3. RNA Preparation of Cas9 / sgRNA

[0089] The sgRNA4 and sgRNA9 target DNAs were in vitro transcribed according to the Ambion in vitro transcription kit method to prepare RNA for microinjection with Cas9 mRNA.

[0090] 4. Construction of Targeting Vector

[0091] Targeting vector construction can be performed using conventional methods, such as enzyme digestion, ligation, direct synthesis, etc. According to the targeting plan, a targeting vector is constructed, and enzyme digestion and sequencing are performed to confirm that the targeting vector is correctly constructed. The targeting vector is then prepared for microinjection.

[0092] The vector LentiCRISPERv2 vector plasmid (Addgene, catalog number 52961) was digested with HindIII, XhoI, BstZ171, EcoRI and SalII. Figure 3 As shown in FIG, the CRISPR / Cas9 targeting vector was constructed by connecting the upstream homology arm (SEQ ID NO: 3), the coding region of the human CD209 gene genome (SEQ ID NO: 2), and the downstream homology arm (SEQ ID NO: 4) to construct a CRISPR / Cas9 vector plasmid for the target sequence. The CRISPR / Cas9 targeting vector that cuts the target sequence was constructed, and the enzyme digestion identification and targeting map were shown in FIG. Figure 3 shown.

[0093] 5. Microinjection and Embryo Transfer

[0094] Fertilized C57BL / 6 mouse eggs were collected and pronuclearly injected with sgRNAs (sgRNA4 and sgRNA9), Cas9 mRNA, and a targeting vector. Microinjection of zygotes was performed according to the method described in the Mouse Embryo Experiment Manual (3rd Edition) (Andras Nagy, Chemical Industry Press, 2006). The injected zygotes were briefly cultured in culture medium and then transplanted into the oviducts of surrogate mice. F0 mice were then awaited for birth.

[0095] 6. Genotype identification

[0096] 1. After birth, F0 generation mice were numbered and their toes were taken for identification. Two pairs of primers were designed based on the sequence information to screen positive mice. The sequences of the two pairs of primers are detailed in Table 1. Based on PCR amplification and product sequencing, E3N21-0003, E3N21-0011, and E3N21-0022 were identified as positive F0 generation mice. The identification results are detailed in Figure 4 .

[0097] Table 1. F0 / F1 generation primer names and specific sequences

[0098]

[0099] 2. The positive mice obtained in step 1 were mated with wild-type mice to obtain F1 generation mice with stable genotypes. The tail genotypes were identified (the primers were the same as those used to identify F0 generation mice). A total of 13 positive mice were obtained from the initial screening. The tail DNA of these 13 mice was tested by Southern blot. The results are shown in Figure 5 Finally, a positive mouse was obtained, numbered 1E3N21-0009. This mouse showed correct recombination and no random insertion.

[0100] 3. The F1 generation positive mice numbered 1E3N21-0009 obtained in step 2 were mated with wild-type mice to obtain F2 generation mice with stable genotypes. Two pairs of primers were designed in the wild-type and human sequences respectively to screen positive F2 generation mice. The sequences of the two pairs of primers are detailed in Table 2. 13 positive F2 generation mice were successfully screened, and the PCR identification results are detailed in Table 2. Figure 6 The prepared CD209 gene humanized mice were named C57-hCD209 mice.

[0101] Table 2. F2 generation primer names and specific sequences

[0102]

[0103] Example 2: Identification of CD209 gene humanized mice

[0104] 1. Genotype and Sequencing Identification

[0105] To verify whether the genotype of the offspring mice was mutated, the ten-day-old mice from the same litter were subjected to tail PCR and sequencing. The results are shown in Figure 7 According to the designed primers, the sequence amplified from homozygous mice was successfully replaced with the human sequence in the CD209 promoter region, which was 100% consistent with the target sequence by sequencing verification.

[0106] 2. Weight and lifespan assessment

[0107] The body weight of wild-type and CD209 humanized mice was recorded weekly from the third week after birth until the fifteenth week; the lifespan of the mice was recorded until the fortieth week after birth. Figure 8 ), the human gene did not affect the weight and lifespan of mice.

[0108] 3. Peripheral Blood and Organ Pathology Assessment

[0109] The numbers of white blood cells, platelets, lymphocytes, neutrophils, and monocytes in the peripheral blood of wild-type and CD209 humanized mice were counted to analyze whether the knock-in of the humanized gene had an effect on the physiological condition of the mice. Figure 9 As shown in Figure 2, the physiological conditions indicated by the peripheral blood of humanized mice were normal, with no significant differences from those of wild-type mice. The effects of humanized genes on tissue pathology were detected in four common tissues: spleen, lung, kidney, and liver. The results are shown in Figure 2. Figure 10 There were no obvious lesions in these four tissues in both groups of mice, and the structures were intact without inflammatory infiltration or necrosis.

[0110] Example 3: Evaluation of novel coronavirus in CD209 gene humanized mice

[0111] 1. Viral load testing

[0112] 1. Experimental animals: 8-week-old C57-WT (wild-type C57BL / 6 mice) and C57-hCD209 mice, randomized in sex.

[0113] 2. Grouping: The experimental animals were divided into two groups (6 animals in each group) according to genotype and challenged with nasal drops according to the protocol shown in Table 3. Omicron.BA.2 was provided by the Military Veterinary Research Institute of the Academy of Military Medical Sciences.

[0114] 3. Results: On the third day after infection, the viral loads in the lungs and nasal turbinates of the two groups of mice were detected. Figure 11 As shown in Figure 2, compared with the control group, the experimental group showed a more significant increase in viral load in the lungs and nasal turbinates, with P < 0.001. This result demonstrates that humanized mice are more susceptible than wild-type mice, and that the viral load in the nasal turbinates is higher than in the lung tissue, consistent with the characteristics of novel coronavirus infection.

[0115] Table 3. Mouse challenge plan

[0116]

[0117] 2. Lung Pathology Testing

[0118] 1. Experimental animals: 8-week-old C57-WT and C57-hCD209 mice, randomized in sex.

[0119] 2. Grouping: The experimental animals were divided into two groups (6 animals in each group) according to genotype and challenged with nasal drops according to the protocol shown in Table 3. Omicron.BA.2 was provided by the Military Veterinary Research Institute of the Academy of Military Medical Sciences.

[0120] 3. Results: On the third day after infection, the lung tissues of mice were collected for pathological sections and HE staining to observe cell necrosis, apoptosis, and changes in tissue structure. Figure 12 ), both groups of mice developed varying degrees of pneumonia. The dotted line to the left shows lung tissue from uninfected mice, while the dotted line to the right shows lung tissue from both groups of mice after infection. Arrows point to degenerated and necrotic cells in both groups of mice, while triangles indicate areas of alveolar wall fracture and fusion, as well as enlarged alveolar cavities. Mirror images show extensive inflammatory cell infiltration. Pathological scoring for inflammatory cell infiltration, alveolar wall thickening, hemorrhage, and alveolar expansion revealed more severe lesions in humanized mice, but the difference was not significant.

[0121] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. A method for constructing a susceptible animal model of novel coronavirus, characterized in that, The method includes expressing a human or humanized CD209 protein in a non-human animal while reducing or eliminating the expression of the endogenous CD209 protein in the non-human animal.

2. The method according to claim 1, wherein The method includes replacing the coding region of the genomic CD209 gene of the non-human animal with the coding region of the genomic human CD209 gene, and the coding region of the genome includes all or part of the exon region.

3. The method according to claim 1 or 2, characterized in that, The nucleotide sequence of the coding region of the genomic human CD209 gene is SEQ ID NO:

2.

4. The method according to claim 2 or 3, characterized in that, The replacement is carried out using the CRISPR / Cas9 gene editing system, and the CRISPR / Cas9 gene editing system includes sgRNA, and the target sequences of the sgRNA are SEQ ID NO:5 and SEQ ID NO:

6.

5. The method according to claim 4, characterized in that, The CRISPR / Cas9 gene editing system further includes a targeting vector, and the targeting vector contains a nucleic acid molecule encoding a human or humanized CD209 protein.

6. The method according to claim 5, wherein The targeting vector further contains an upstream homology arm and a downstream homology arm, the nucleotide sequence of the upstream homology arm is as shown in SEQ ID NO:3, and the nucleotide sequence of the downstream homology arm is as shown in SEQ ID NO:

4.

7. The method according to any one of claims 1 to 6, characterized in that The method includes injecting the targeting vector described in claim 5 or 6, the sgRNA described in claim 4, and Cas9 mRNA into the fertilized egg cells of the non-human animal and transplanting them into the fallopian tubes of a surrogate animal. After the non-human animal or its offspring is genotyped, the non-human animal carrying the human CD209 gene is the susceptible animal model for the novel coronavirus.

8. A susceptible animal model for the novel coronavirus constructed by any of the methods described in claims 1-7, or the application of the susceptible animal model for the novel coronavirus in the preparation of a CD209 humanized animal model infected with the novel coronavirus, the study of the pathogenic mechanism of the novel coronavirus, and the research and development of vaccines or drugs.

9. A biomaterial or any of the following applications of the biomaterial: A1) Application in the gene editing of mouse CD209; A2) Application in the preparation of a CD209 gene humanized mouse model or a susceptible animal model for the novel coronavirus; The biomaterial is any of the following: B1) The sgRNA described in claim 4; B2) The targeting vector described in claim 6; B3) The CRISPR / Cas9 gene editing system described in any of claims 4-6; B4) A recombinant cell containing the sgRNA described in B1), the targeting vector described in B2), or the CRISPR / Cas9 gene editing system described in B3).

10. A method for constructing a CD209 humanized animal model infected with novel coronavirus, characterized in that, The construction method includes infecting the susceptible animal model for the novel coronavirus described in claim 8 with the novel coronavirus.

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