SgRNA for targeted knockout of APP gene and application of sgRNA

Abstract

The invention discloses sgRNA for targeted knockout of an APP gene and application of the sgRNA, and belongs to the field of animal genetic engineering. A CRISPR / Cas9 gene knockout technology is utilized, an sgRNA sequence is linked to a PX459 vector, a PX459-APP targeting vector is transferred into porcine fibroblasts, positive cell clone is obtained after resistance screening and PCR sequencing identification, and a cloned embryo is obtained through a somatic cell nuclear transfer technology; transferring the cloned embryo into a pig uterus for pregnancy to obtain a cloned pig. The APP gene knockout pig is constructed for the first time, the APP gene is not expressed, ASFV resistance is obtained, and a foundation is laid for African swine fever resistance breeding research.

Description

Technical Field

[0001] The present invention relates to the field of animal genetic engineering, and in particular to a sgRNA for targeted knockout of an APP gene and an application thereof. 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 manifests itself as clinical symptoms such as systemic bleeding, neurological symptoms and dyspnea, with a short onset and high mortality rate. African swine fever virus (ASFV) is the only member of the Asfarviridae family and the Asfivirus genus, and is a large double-stranded DNA arbovirus. The main source of infection of African swine fever is infected pigs and carriers of the virus. The virus is contained in infected tissues, blood, secretions and excrement of pigs. The virus can be attached to objects such as feed and transport vehicles for transmission. In addition to direct contact transmission, ASFV can also be transmitted by ticks.

[0003] It is currently believed that the main invasion pathways of ASFV are clathrin-mediated endocytosis (CME) and macropinocytosis. It has been reported that CD163 is a receptor for ASFV invasion. However, studies by Popescu et al. have shown that this receptor is not essential for ASFV infection in some cases. Therefore, the identity of the receptor involved in the entry of the virus into the cell is still unknown, which has caused great trouble in the development of disease-resistant breeding.

[0004] The viral protein pE248R is located in the inner membrane of the virus 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. ASFV uncoating involves the loss of the outer layer of the virus body (outer membrane and protein capsid) in the late multivesicular endosome, and 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 defective virus particles for research and found that the infectivity of the pE248R defective virus was reduced by at least 100 times, while the absence of pE248R protein did not affect the binding or internalization of the virus, but did not cause cytopathic effects and could impair early and late gene expression, indicating that the protein is essential for viral infection and early entry. Recent studies have also pointed out that the protein is involved in the fusion of the inner membrane with the multivesicular endosomal membrane and is a key factor in nuclear release.

[0005] The APP gene is often reported as a key gene for the treatment of Alzheimer's disease, but some studies have found that the APP gene is often associated with the endocytosis of macrophages and is highly expressed in porcine alveolar macrophages. Therefore, the study of the APP gene can provide a theoretical basis for the study of African swine fever virus infection, and the study of APP gene knockout will lay the foundation for further research on the specific role of APP receptors in the ASFV infection process. At present, there have been no reports of cloned pigs with APP gene knockout. Summary of the invention

[0006] The purpose of the present invention is to provide a sgRNA for targeted knockout of the APP gene and its application to solve the problems existing in the above-mentioned prior art. The African swine fever virus receptor APP knockout pig is constructed using sgRNA, and the APP gene is not expressed in the pig body, thereby obtaining African swine fever virus-resistant pigs, which lays the foundation for breeding research on resistance to African swine fever.

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

[0008] The present invention provides a sgRNA for targeted knockout of the APP gene, wherein the nucleotide sequence of the sgRNA is shown in 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 invention also provides a transgenic cell containing the recombinant vector.

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

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

[0014] The present invention also provides a cell line for knocking out the APP gene, which is prepared by the construction method.

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

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

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

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

[0019] (3) Application in breeding for resistance to African swine fever.

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

[0021] The cell line constructed by the construction method is used to screen positive cell clones, and then cloned embryos are obtained by using the positive cell clones as nuclear transplant donor cells and oocytes as nuclear transplant recipient cells; the cloned embryos are transferred into the pig uterus for pregnancy, and African swine fever resistant transgenic pigs with gene knockout are obtained.

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

[0023] The present invention uses CRISPR / Cas9 technology to knock out the African swine fever receptor APP gene, construct an APP knockout pig fibroblast cell line, and screen positive clones as nuclear transplant donor cells, and oocytes as nuclear transplant recipient cells; the cloned embryos are transferred into the pig uterus for pregnancy, and cloned pigs resistant to African swine fever after gene knockout are obtained. After testing, the APP gene knockout cell line and the gene knockout pig were identified, and it was found that the APP gene was not expressed in the pig body, and the pig did not die after being infected with African swine fever. It shows that APP is resistant to African swine fever virus after knockout, which lays the foundation for breeding research against African swine fever. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] 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.

[0025] Figure 1 This 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 the internal reference gene;

[0027] Figure 3The T7E1 enzyme digestion method in Example 2 of the present invention is used to identify the cleavage of the genome by the PX459 plasmid on pig fibroblasts; wherein the test group is the genome of pig fibroblasts transfected with the px459 plasmid, and the WT group is the genome of wild-type pig fibroblasts; maker is a standard DNA molecule;

[0028] Figure 4 This is a diagram showing the Western blot test results of newborn piglets in Example 4 of the present invention;

[0029] Figure 5 The results of detecting the ASFV positive rate in Example 4;

[0030] Figure 6 The infection efficiency of ASFV was detected by qPCR in Example 5. DETAILED DESCRIPTION

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0036] The inventors found in experimental research that the expression of APP protein in primary alveolar macrophages infected with African swine fever virus changed significantly, such as Figure 2 As shown in the figure, APP-HA plasmid with HA tag and E248R-FLAG plasmid with FLAG tag were constructed and co-transfected into 293T cells. Through immunoprecipitation experiments, it was found that APP protein interacted with African swine fever virus protein pE248R, which indicated that APP gene might be the key gene for African swine fever virus infection. Therefore, further in-depth research on APP gene was carried out.

[0037] The present invention adopts CRISPR / Cas9 gene knockout technology to construct a CRISPR / Cas9 targeting vector, and co-transforms the PX459-APP vector into porcine fibroblasts to obtain positive cell clones, in which the APP gene is not expressed, and ASFV resistance is obtained when the African swine fever virus receptor APP gene is not expressed.

[0038] Example 1 Construction of porcine APP gene knockout vector

[0039] S1. Use the CRISPR / Cas9 target design tool (https: / / benchling.com / ) to select the CRISPR / Cas9 system knockout target. To maximize the frameshift mutation of the gene and show the phenotype of the silent gene, select the 20bp sequence of the exon target shared by the gene subtypes as the knockout target. The selected target should be 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 by the present invention are as follows:

[0041] The two knockout targets of the porcine APP gene (NCBI: XM_005670302.3) are shown in Table 1. Complementary oligonucleotides were synthesized by Shanghai Bioengineering according to the sgRNA sequence, where lowercase letters represent restriction sites.

[0042] Table 1 Oligonucleotides

[0043]

[0044] S2. Construction of targeting vector

[0045] The oligonucleotides in Table 1 were used for the construction process: 37°C for 30 min, 95°C for 5 min, and then 25°C for 10 min, and then immediately placed on ice to anneal the oligonucleotides; PX459 vector (map see Figure 1), BbsI endonuclease, T4 ligase mixture, 37℃ 5min; 25℃ 5min, after six cycles, the sgRNA double-stranded template is connected to the PX459 vector. Transformation and plating are performed by conventional transformation methods. After a single colony grows, several colonies are picked for expansion and sequencing. The sequencing verification is correct, indicating that the present invention successfully constructs a CRISPR-Cas9 targeting vector.

[0046] Table 2 Reaction system and reaction process

[0047]

[0048] S3. Expansion culture of positive single colonies

[0049] The specific steps are:

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

[0051] (b) To expand the culture, transfer the overnight culture into a flask containing 100 mL of LB medium at a volume ratio of 1:500, and then culture at 37°C and 250 rpm for 16 h.

[0052] S4.px459 plasmid endotoxin extraction

[0053] According to the method provided in the plasmid endotoxin extraction kit, the px459 plasmid was extracted and used for cell transfection.

[0054] S5. Cell Transfection

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

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

[0057] (B) Mix the plasmid and Lipo3000 reagent. Dilute PX459 and Lipo3000 reagent to appropriate concentrations according to the instructions of the kit. Then, mix equal volumes of plasmid and Lipo3000 reagent and gently shake the tube to mix thoroughly.

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

[0059] (D) Cell transfection: The pretreated cells were washed twice with cell culture medium to allow the cells to adhere to the culture dish. Then, the culture medium containing the mixture was added to allow the cells to contact the mixture of plasmid and Lipo3000 reagent, avoiding the generation of bubbles.

[0060] (E) Removal of transfection solution: cells were washed with medium containing 10% fetal bovine serum to remove unadsorbed plasmid and Lipo3000 reagent.

[0061] (f) Cell culture: Add the washed cells into fresh culture medium and continue to culture. According to the experimental needs, add the corresponding screening agent and inducer for later screening.

[0062] Example 2 Screening and identification of positive cell monoclones

[0063] S1. Screening of positive monoclonal cells

[0064] The PX459 plasmid is resistant to puromycin, so after transfection, puromycin is used for screening until no cells survive in the blank control. Genomic DNA (gDNA) is extracted from some cells, and some cells continue to be cultured. Primers are designed using the genomic DNA sequence of NCBI (UCSC, Ensemble, etc.) as a template. The primers are located about 200-300bp upstream and downstream of the sgRNA target position. Using the extracted gDNA as a template, PCR amplification is performed to obtain a PCR product of about 400-600bp. Gel electrophoresis is used to detect the specificity of the PCR product. If the PCR product is a single band, it is sent for sanger sequencing, and the sequencing primer is the PCR primer. If there are mixed bands, redesign primers with strong specificity, or increase the annealing temperature. The latter is effective in some cases.

[0065] S2. Establishment of permanent cell lines

[0066] The cells that continue to be cultured after the above puromycin screening are gradiently diluted to obtain single cells and then continue to be cultured, extract gDNA, and select the cells to be edited by PCR and Sanger sequencing, such as gene knockout or precise editing such as single base editing. After obtaining the cells, expand the culture and partially freeze them.

[0067] S3. Detection of targeting efficiency

[0068] According to the method provided on the genome extraction kit, the cell genome was extracted; the extracted genome and the wild-type porcine fibroblast genome (WT group) were used as templates, and PCR was performed with KOD DNA polymerase to amplify a 528bp fragment. The primers used were F (as shown in SEQ ID NO.5): 5'-GCTGAATGAGTGCAGTAAAATAGTGGTTAC-3' and R (as shown in SEQID NO.6): 5'-ACACACTAGATAAATTCATTCTGAGGCAGG-3'. The amplification conditions were 94℃, 2min; 90℃, 30sec; 60℃, 30sec; 65℃, 60sec; 70℃, 6min; 45 cycles, and the results were observed by 2.0% agarose electrophoresis, and then the PCR products were recovered and the concentration was measured. 400ng of the recovered product was taken for annealing, and the temperature was programmed to decrease from 95℃ to 4℃. The annealed product was digested with T7E1 enzyme in the following system: 10 μL annealed product, 2 μL NEB buffer 2, 0.5 μL T7E1, and 20 μL ddH2O. After the digestion was completed, the results were observed by 1% agarose gel electrophoresis. The results showed (see Figure 3 ), which can cut the genome in porcine fibroblasts.

[0069] Example 3 Preparation and identification of ASFV-resistant cloned pigs

[0070] S1. Preparation of ASFV-resistant cloned pigs

[0071] The positive cells that successfully underwent homologous recombination obtained in Example 2 were used as nuclear transplant donor cells, and the oocytes of pre-pubertal sows matured in vitro were used as nuclear transplant recipient cells. The nuclear transplant donor cells were transplanted into the enucleated oocytes, and cloned embryos were constructed through electrical fusion and activation. The cloned embryos with good morphology were selected and surgically transplanted into the uterus of multiparous sows (6 heads) in natural estrus for pregnancy. The surgical embryo transplantation steps were conventional anesthesia with Shutai, supine binding on the surgical frame, and a surgical incision of about 8 cm in length was made in the midline of the abdomen to expose the ovaries, fallopian tubes and uterus. The embryo transfer tube was inserted about 5 cm along the fimbria of the fallopian tube, and the embryos (more than 400 pieces / head) were transplanted to the junction of the ampulla of the fallopian tube and the isthmus. B-type ultrasound was used to detect pregnancy 30 days after embryo transplantation.

[0072] S2. Genetic identification of newborn piglets

[0073] 1g to 5g of tissue was collected from the ears of newborn pigs and immediately frozen with liquid nitrogen, stored at -80°C, and sent to Shanghai Bioengineering Company with sufficient dry ice for sequencing and identification. The results showed that 4 out of 6 piglets were positive.

[0074] S3. Western blot detection of newborn piglets

[0075] Take 1-5g of cloned piglet ear and tail tissues, and 1-5g of common commercial pig ear and tail tissues as negative controls. Cut the tissue into small pieces and add 1.5mL of lysis solution (PMSF is added to the lysis solution a few minutes before use, so that the final concentration of PMSF is 10μM). Homogenize with a glass homogenizer until fully lysed. After fully lysed, centrifuge and take the supernatant to obtain the total protein of each tissue. Measure the concentration with the BCA protein concentration determination kit. Take 20μg of total protein each and perform electrophoresis using 10% SDS-PAGE gel, 100V, 30min; 120V, 1.5h. After electrophoresis, use a Bio-Rad wet transfer membrane transfer instrument at 350mA for 80min. After the transfer, the membrane was blocked with 5% skim milk powder for 2 hours, then the APP primary antibody (1:1000 dilution) was added for incubation overnight, the membrane was washed with TBST for 3×10 minutes, and then the HRP-labeled goat anti-rabbit secondary antibody (1:1000 dilution) was incubated for 2 hours, the membrane was washed with TBST for 3×10 minutes, and finally BCL color was developed. The results show (see Figure 4 ), APP protein was not expressed in the cloned newborn piglets No. 1, 2, 3, and 5.

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

[0077] ASFV resistance detection was performed in an "aerosol" infection mode. Three ASFV-resistant cloned pigs and three healthy commercial pigs were kept in the same pig house without biosafety protection for 3 consecutive months to observe the resistance differences between the two types of pigs under natural infection conditions. Blood was collected once a week and the ASFV positive rate was detected using the Mingrida ASFV detection kit (veterinary medicine product number 010688870). Figure 5 As shown, the test results showed that positive cases of porcine ASFV were detected in ordinary healthy commercial pigs, while no ASFV infection was detected in APP gene knockout cloned pigs.

[0078] Example 5 APP gene knockout pig PAMs cell collection ASFV resistance detection

[0079] After euthanizing the APP gene knockout pig, the chest cavity was opened, the lung tissue was completely removed, and the porcine alveolar macrophages (PAMs) were collected by rinsing with PBS. They were cultured in an incubator at 37°C and 5% CO2. After the cells adhered to the wall, the ASFV infection test was performed, and the infection dose was MOI = 1. After 24 hours of infection, the cells were washed three times with PBS, the cell mRNA was extracted, and the infection efficiency of ASFV was detected by qPCR. Figure 6 As shown, the results showed that compared with normal pig PAMs, the ASFV infection efficiency of PAMs cells of APP gene knockout cloned pigs was significantly decreased, 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 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 sgRNA targeting APP gene knockout, characterized in that: The nucleotide sequence of the sgRNA is shown in SEQ ID NO.1-2 or SEQ ID NO.3-4.

2. A DNA molecule encoding the sgRNA according to claim 1.

3. A recombinant vector containing the sgRNA according to claim 1. A transgenic cell comprising the recombinant vector according to claim 3.

5. A method for constructing a cell line with APP gene knockout, characterized in that: The method comprises the step of knocking out the APP gene in the cell line using the CRISPR / Cas9 system, wherein the nucleotide sequence of the sgRNA of the CRISPR / Cas9 system is shown in SEQ ID NO.1-2 or SEQ ID NO.3-4.

6. The construction method according to claim 5, characterized in that: The cell lines include porcine fibroblasts and porcine alveolar macrophages.

7. A cell line with APP gene knocked out, characterized in that: It is prepared by the construction method described in claim 5 or 6.

8. Use of the sgRNA as claimed in claim 1 in constructing a cell line for knocking out the APP gene.

9. Use of the cell line according to claim 7 in any of the following: (1) Application in the preparation of APP gene function research models; (2) Application in the preparation of transgenic pigs resistant to African swine fever; (3) Application in breeding for resistance to African swine fever.

10. A method for preparing transgenic pigs resistant to African swine fever, characterized in that: The following steps are involved: A cell line is constructed using the construction method described in claim 5 or 6, positive cell clones are screened, and then cloned embryos are obtained using the positive cell clones as nuclear transplant donor cells and oocytes as nuclear transplant recipient cells; the cloned embryos are transferred into the pig uterus for pregnancy, and transgenic pigs resistant to African swine fever after gene knockout are obtained.

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