An sgRNA for targeted knockout of APP gene and its application

Abstract

The present invention discloses an sgRNA for targeted knockout of APP gene and its application, belonging to the field of animal genetic engineering. By using the CRISPR / Cas9 gene knockout technology, the sgRNA sequence is linked into the PX459 vector, and the PX459-APP targeting vector is transferred into porcine fibroblasts. After resistance screening and PCR sequencing identification, positive cell clones are obtained, and then cloned embryos are obtained through somatic cell nuclear transfer technology; the cloned embryos are transferred into the pig uterus for pregnancy to obtain cloned pigs. The present invention constructs APP gene knockout pigs for the first time, and makes the APP gene not express to obtain ASFV resistance, laying a foundation for the research on breeding against African swine fever.

Description

Technical Field

[0001] The present invention relates to the field of animal genetic engineering, and particularly to an sgRNA for targeted knockout of the APP gene and its application. Background Art

[0002] African swine fever is an acute, febrile, highly contagious disease caused by the African swine fever virus, which has been proven to have a devastating impact on the global pork industry; it usually presents clinical symptoms such as systemic hemorrhage, neurological symptoms, and dyspnea, with a short onset time and a high fatality rate. The African swine fever virus (ASFV) is the only member of the family Asfarviridae and the genus Asfivirus, and is a large double-stranded DNA arbovirus. The main sources of infection of African swine fever are infected pigs and virus-carrying pigs. The virus is contained in infected tissues, blood, secretions, and excretions of pigs, and can attach to objects such as feed and transport vehicles for transmission. In addition to direct contact transmission, ASFV can also be transmitted through ticks.

[0003] Currently, it is considered that the main invasion routes of ASFV are the clathrin-mediated endocytosis pathway (CME) and the macropinocytosis pathway. It has been reported that CD163 is the receptor for ASFV invasion. However, the research by Popescu et al. shows that this receptor is not essential for ASFV infection in some cases. Therefore, the identity of the receptor involved in virus entry into cells is still unknown, which causes great trouble for the development of disease-resistant breeding.

[0004] The viral protein pE248R is located in the viral inner membrane and is involved in the early process of ASFV entry. ASFV enters the cell through the CME or macropinocytosis pathway and needs to be uncoated from the endosome formed by the endocytic pathway and released into the cytoplasm. The uncoating of ASFV involves the loss of the outer layer (outer membrane and protein capsid) of the virus in the late endosome of multivesicular bodies, as well as the late fusion of the inner membrane with the endosome, thereby releasing the naked nucleus into the cytoplasm. Among them, pE248R is closely related to the uncoating of the virus. Rodríguez et al. used pE248R-deficient virus particles for research and found that the infectivity of pE248R-deficient viruses was reduced by at least 100 times. The deletion of the pE248R protein neither affects virus binding nor virus internalization, but does not cause cytopathic effects and can damage early and late gene expression, indicating that this protein is essential for virus infection and early entry. Recent research also points out that this protein is involved in the fusion of the inner membrane with the membrane of multivesicular endosomes and is a key factor for nuclear release.

[0005] The APP gene is often reported as a key gene for the treatment of Alzheimer's disease. However, some studies have also found that the app gene is often associated with the endocytosis of macrophages and is highly expressed in porcine alveolar macrophages. Therefore, the research on the APP gene can provide a theoretical basis for the research related to African swine fever virus (ASFV) infection, and the research on APP gene knockout will lay a foundation for further studying the specific role of the APP receptor in the process of ASFV infection. Currently, there is no report on cloned pigs with APP gene knockout. Summary of the Invention

[0006] The object of the present invention is to provide an sgRNA for targeted knockout of the APP gene and its application, so as to solve the problems existing in the above-mentioned prior art. By using the sgRNA to construct pigs with knockout of the ASFV receptor APP, the APP gene is not expressed in the pigs, thereby obtaining pigs resistant to ASFV, which lays a foundation for the research on anti-African swine fever breeding.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides an sgRNA for targeted knockout of the APP gene, and the nucleotide sequence of the sgRNA is shown as SEQ ID NO.1-2 or SEQ ID NO.3-4.

[0009] The present invention also provides a DNA molecule for encoding the sgRNA.

[0010] The present invention also provides a recombinant vector containing the sgRNA.

[0011] The present invention also provides a transgenic cell containing the recombinant vector.

[0012] The present invention also provides a method for constructing a cell line with knockout of the APP gene, including the step of knocking out the APP gene in the cell line by using the CRISPR / Cas9 system, and the nucleotide sequence of the sgRNA of the CRISPR / Cas9 system is shown as SEQ ID NO.1-2 or SEQ ID NO.3-4.

[0013] Optionally, the cell line includes porcine fibroblasts and porcine alveolar macrophages.

[0014] The present invention also provides a cell line with knockout of the APP gene, which is prepared by the above construction method.

[0015] The present invention also provides the application of the sgRNA in constructing a cell line with knockout of the APP gene.

[0016] The present invention also provides the application of the cell line in any one of the following:

[0017] (1) Application in the preparation of an APP gene function research model;

[0018] (2) Application in the preparation of African swine fever-resistant transgenic pigs;

[0019] (3) Application in African swine fever-resistant breeding.

[0020] The present invention also provides a method for preparing African swine fever-resistant transgenic pigs, comprising the following steps:

[0021] Using the cell line constructed by the described construction method, screening for positive cell clones, and then using the positive cell clones as nuclear transfer donor cells and oocytes as nuclear transfer recipient cells to obtain cloned embryos; transferring the cloned embryos into the uterus of a pig for pregnancy to obtain African swine fever-resistant transgenic pigs after gene knockout.

[0022] The present invention discloses the following technical effects:

[0023] By using the CRISPR / Cas9 technology to knockout the African swine fever receptor APP gene, the present invention constructs a pig fibroblast cell line with APP knockout, and after screening for positive clones, uses them as nuclear transfer donor cells and oocytes as nuclear transfer recipient cells; transferring the cloned embryos into the uterus of a pig for pregnancy to obtain African swine fever-resistant cloned pigs after gene knockout. Through experiments, the APP gene knockout cell line and gene knockout pigs were identified, and it was found that the APP gene was not expressed in the pigs, and the pigs did not die after being infected with African swine fever. This shows that the knockout of APP confers resistance to African swine fever virus, laying a foundation for African swine fever-resistant breeding research. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention, and those of ordinary skill in the art can obtain other drawings without creative efforts based on these drawings.

[0025] Figure 1 It is a schematic diagram of the PX459 vector of the present invention;

[0026] Figure 2 To detect the correlation between the app protein of primary alveolar macrophages infected with African swine fever virus and the African swine fever virus protein pE248R; ACTIN is an internal reference gene;

[0027] Figure 3This is for the identification of the cleavage of the PX459 plasmid on the porcine fibroblast genome by the T7E1 digestion method in Example 2 of the present invention. Among them, the experimental group is the genome of porcine fibroblasts transfected with the px459 plasmid, and the WT group is the genome of wild-type porcine fibroblasts; the maker is the standard DNA molecule.

[0028] Figure 4 This is the Western blot detection result diagram of neonatal piglets in Example 4 of the present invention.

[0029] Figure 5 This is the result of detecting the positive rate of ASFV in Example 4.

[0030] Figure 6 This is to detect the infection efficiency of ASFV by qPCR in Example 5. Detailed implementation manners

[0031] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be regarded as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and implementation manners of the present invention.

[0032] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0033] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can 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 related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0034] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are only exemplary.

[0035] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, that is, they are meant to include but not be limited to.

[0036] During experimental research, the inventor found that the expression of app protein changed significantly in primary alveolar macrophages infected with African swine fever virus. As Figure 2 shown, the APP-HA plasmid with an HA tag and the E248R-FLAG plasmid with a FLAG tag were constructed and co-transfected into 293T cells. Through co-immunoprecipitation experiments, it was found that the app protein interacted with the African swine fever virus protein pE248R. This indicates that the APP gene may be a key gene for African swine fever virus infection. Therefore, further in-depth research on the APP gene was carried out.

[0037] The present invention uses the CRISPR / Cas9 gene knockout technology to construct a CRISPR / Cas9 targeting vector, which is co-transferred into porcine fibroblasts. Positive cell clones are obtained, and the APP gene is not expressed in the positive cell clones. ASFV resistance is obtained under the condition that the African swine fever virus receptor app gene is not expressed.

[0038] Example 1 Construction of a Porcine app Gene Knockout Vector

[0039] S1. Using the CRISPR / Cas9 target design tool (https: / / benchling.com / ), select the CRISPR / Cas9 system knockout target. To maximize frameshift mutations in the gene and demonstrate the phenotype of the silenced gene, select the 20bp sequence of the exon target shared by the gene subtypes as the knockout target, and select the target as close to the 5' end as possible; according to the scores in the self-assessment and prediction results, select the one with the highest score from the candidate target sites.

[0040] The porcine APP gene knockout targets designed in the present invention are as follows:

[0041] Two knockout targets of the porcine APP gene (NCBI: XM_005670302.3) are shown in Table 1. Shanghai Sangon synthesized complementary oligonucleotides according to the sgRNA sequence, and the lowercase letters are restriction enzyme sites.

[0042] Table 1 Oligonucleotides

[0043]

[0044] S2. Construction of the targeting vector

[0045] Using the oligonucleotides in Table 1, the construction process is as follows: anneal the oligonucleotides at 37°C for 30 min, 95°C for 5 min, then 25°C for 10 min, and then immediately place on ice; the PX459 vector (map shown in Figure 1), Mix BbsI endonuclease and T4 ligase, incubate at 37°C for 5 min; then at 25°C for 5 min. After six cycles, the double-stranded sgRNA template is ligated to the PX459 vector. Transform and plate by conventional transformation method. After single colonies grow, pick several for expansion culture and sequencing. Correct sequencing verification indicates that the CRISPR-Cas9 targeting vector of the present invention is successfully constructed.

[0046] Table 2 Reaction system and reaction process

[0047]

[0048] S3. Expansion culture of positive single colonies

[0049] The specific steps are as follows:

[0050] (a) Initial culture: Use an inoculation loop to pick a positive single colony and add it to a sterilized tube containing 10 mL of LB medium. Incubate at 37°C and 250 rpm for 5 h - 28 h;

[0051] (b) Expansion culture: Transfer the overnight culture to a triangular flask containing 100 mL of LB medium at a ratio of 1:500 by volume, and then incubate at 37°C and 250 rpm for 16 h.

[0052] S4. Extraction of endotoxin-free px459 plasmid

[0053] Extract the px459 plasmid according to the method provided in the large-scale endotoxin extraction kit for plasmid regions. The extracted plasmid is used for cell transfection.

[0054] S5. Cell transfection

[0055] Cell transfection is performed using lipo3000 liposome transfection. The specific procedure is as follows:

[0056] (a) Cell pretreatment: Culture the cells in DMEM medium containing 10% fetal bovine serum.

[0057] (b) Mix the plasmid and lipo3000 reagent: Dilute px459 and lipo3000 reagent to appropriate concentrations respectively according to the requirements of the kit instructions. Then, mix equal volumes of the plasmid and lipo3000 reagent and gently shake the tube to mix well.

[0058] (c) Let the mixture stand: Let the mixture stand for 15 - 30 minutes to allow the plasmid and lipo3000 reagent to fully combine to form a complex. Avoid strong shaking and oscillation during this period.

[0059] (d) Transfection of cells: Wash the pre-treated cells twice with cell culture medium to make the cells adhere to the culture dish. Then, add the medium containing the mixture to bring the cells into contact with the mixture of plasmid and lipo3000 reagent, avoiding the generation of bubbles.

[0060] (e) Removal of transfection solution: Wash the cells with the medium containing 10% fetal bovine serum to remove the unadsorbed plasmid and lipo3000 reagent.

[0061] (f) Cultivation of cells: Add the washed cells to fresh medium and continue the cultivation. According to the experimental needs, add the corresponding screening agents and inducers for subsequent screening.

[0062] Example 2 Screening and Identification of Positive Cell Monoclonal

[0063] S1. Screening of positive monoclonal cells

[0064] The PX459 plasmid has puromycin resistance. Therefore, after transfection, screen with puromycin until no cells survive in the blank control. Extract genomic DNA (gDNA) from a part of the cells and continue to culture another part of the cells. Design primers using the NCBI (UCSC, Ensemble, etc. are all acceptable) genomic DNA sequence as a template. The primers are located about 200 - 300 bp upstream and downstream of the sgRNA target position. Using the extracted gDNA as a template, perform PCR amplification to obtain a PCR product of about 400 - 600 bp. Detect the specificity of the PCR product by gel electrophoresis. If the PCR product is a single band, send it for sanger sequencing, and the sequencing primer is the PCR primer. If there are heterozygous bands, re-design primers with strong specificity or increase the annealing temperature, and the latter is effective in some cases.

[0065] S2. Establishment of a permanent cell line

[0066] Gradiently dilute the cells that continue to be cultured after the above puromycin screening to obtain single cells and then continue to culture them. Extract gDNA, perform PCR and sanger sequencing, and select the cells to be edited as required, such as gene knockout or precise editing such as single-base editing, etc. After obtaining the cells, expand the culture and cryopreserve some of them.

[0067] S3. Detection of targeting efficiency

[0068] Extract the cell genome according to the method provided by the genomic extraction kit; using the extracted genome and the genome of wild-type porcine fibroblasts (WT group) as templates, perform PCR with KOD DNA polymerase to amplify a 528-bp fragment. The primers F (shown in SEQ ID NO.5): 5’-GCTGAATGAGTGCAGTAAAATAGTGGTTAC-3’ and R (shown in SEQ ID NO.6): 5’-ACACACTAGATAAATTCATTCTGAGGCAGG-3’ are used. The amplification conditions are 94°C for 2 min; 90°C for 30 sec; 60°C for 30 sec; 65°C for 60 sec; 70°C for 6 min; 45 cycles. Observe the results by 2.0% agarose gel electrophoresis, then recover the PCR product and measure the concentration. Take 400 ng of the recovered product for annealing, and cool the temperature from 95°C to 4°C in a programmed manner. Digest the annealed product with T7E1 enzyme. The reaction system is: 10 μL of the annealed product, 2 μL of NEB buffer2, 0.5 μL of T7E1, and make up to 20 μL with ddH2O. Observe the results by 1% agarose gel electrophoresis after digestion. The results show (see Figure 3 ) that this plasmid can play a role in cleaving the genome on porcine fibroblasts.

[0069] Example 3 Preparation and Identification of ASFV-resistant Cloned Pigs

[0070] S1. Preparation of ASFV-resistant Cloned Pigs

[0071] Using the positive cells that successfully underwent homologous recombination obtained in Example 2 as nuclear transfer donor cells, and in vitro matured prepubertal sow oocytes as nuclear transfer recipient cells, transfer the nuclear transfer donor cells into enucleated oocytes, and construct cloned embryos through electrofusion and activation. Select morphologically excellent cloned embryos and transfer them into the uterus of naturally estrous multiparous sows (6 heads) by surgical method for pregnancy. The steps of surgical embryo transfer are general anesthesia with Zoletil, lying supine and tied on the operating table, make a surgical incision about 8 cm long along the midline of the abdomen, expose the ovaries, fallopian tubes and uterus, insert the embryo transfer tube about 5 cm along the fimbria of the fallopian tube, and transfer the embryos (more than 400 embryos / head) to the junction of the ampulla and isthmus of the fallopian tube. Detect pregnancy by B-mode ultrasound 30 days after embryo transfer.

[0072] S2. Gene Identification of Newborn Piglets

[0073] Collect 1 g - 5 g of tissue from the ear of the newborn pig, immediately freeze it in liquid nitrogen, store it at -80°C, and send it to Sangon Biotech in Shanghai for sequencing identification with sufficient dry ice. The results show that 4 out of 6 piglets are positive piglets.

[0074] S3. Western blot Detection of Newborn Piglets

[0075] Take 1 - 5 g of ear and tail tissues from each cloned piglet, and 1 - 5 g of ear and tail tissues from each ordinary commercial pig as negative controls. Cut the tissues into small pieces, and add 1.5 mL of lysis buffer (add PMSF to the lysis buffer a few minutes before use to make the final concentration of PMSF 10 μM). Homogenize with a glass homogenizer until fully lysed. After complete lysis, centrifuge and take the supernatant to obtain the total protein of each tissue. Measure the concentration using a BCA protein concentration assay kit. Take 20 μg of total protein each, and perform electrophoresis using a 10% SDS - PAGE gel at 100 V for 30 min and then at 120 V for 1.5 h. After electrophoresis, transfer the membrane using a Bio - Rad wet transfer apparatus at 350 mA for 80 min. After membrane transfer, block with 5% non - fat milk for 2 h, then add the anti - APP primary antibody (diluted 1:1000) and incubate overnight. Wash the membrane with TBST 3 times for 10 min each, then incubate with the HRP - labeled goat anti - rabbit secondary antibody (diluted 1:1000) for 2 h, wash the membrane with TBST 3 times for 10 min each, and finally perform BCL color development. The results showed (see Figure 4 ), APP protein was not expressed in the cloned piglets numbered 1, 2, 3, and 5 at birth.

[0076] Example 4 Detection of ASFV resistance of APP gene - knockout pigs

[0077] Perform ASFV resistance detection in an "aerosol - type" infection mode. Raise three ASFV - resistant cloned pigs and three healthy commercial pigs in the same pigsty without biosafety protection for 3 consecutive months, and observe the resistance differences between the two types of pigs under natural infection conditions. Collect blood once a week and detect the positive rate of ASFV using the Mingrida ASFV detection kit (Veterinary Drug Approval No. 010688870). As Figure 5 shown, the detection results showed that ASFV - positive cases were detected in ordinary healthy commercial pigs, while no ASFV infection was detected in the APP gene - knockout cloned pigs.

[0078] Example 5 Detection of ASFV resistance of PAMs cells collected from APP gene - knockout pigs

[0079] After euthanizing the APP gene - knockout pigs, open the chest cavity and completely remove the lung tissue. Collect porcine alveolar macrophages (PAMs) by rinsing with PBS, and culture them in an incubator at 37 °C and 5% CO2. After the cells adhere, perform an ASFV infection experiment with an infection dose of MOI = 1. After 24 hours of infection, wash the cells three times with PBS, extract the mRNA of the cells, and detect the ASFV infection efficiency by qPCR. As Figure 6 shown, the results showed that compared with PAMs of normal pigs, the ASFV infection efficiency of PAMs cells from APP gene - knockout cloned pigs decreased significantly, further indicating that APP gene knockout can significantly improve the resistance of PAMs to ASFV.

[0080] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of a cell line with APP gene knocked out in preparing a research model for APP gene function, characterized in that, The APP gene function research model is an African swine fever-resistant pig model; The method for constructing the cell line with the APP gene knocked out includes the step of knocking out the APP gene in the cell line by using the CRISPR / Cas9 system, and the nucleotide sequences of the sgRNAs of the CRISPR / Cas9 system are as shown in SEQ ID NO.1-2 or SEQ ID NO.3-4.

2. The application according to claim 1, characterized in that, The cell line is porcine fibroblasts.

3. Application of sgRNA for knocking out APP gene in the preparation of a cell model against African swine fever, characterized in that, The nucleotide sequences of the sgRNAs are as shown in SEQ ID NO.1-2 or SEQ ID NO.3-4, and the cells include porcine fibroblasts and porcine alveolar macrophages.

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