A porcine genome-wide crisper / cas9 knockout cell library susceptible to prrsv and construction method and application thereof

Abstract

The present application relates to the technical field of cell engineering, and particularly relates to a PRRSV-susceptible pig whole-genome CRISPR / Cas9 knockout cell library, a construction method thereof and an application thereof; the present application uses the principle that CD163 molecules are responsible for PRRSV uncoating and genome release and determine the degree of susceptibility of target cells to viruses, first carries out CD163 gene modification on immortalized pig alveolar macrophages, obtains a monoclonal cell strain stably expressing CD163, and then uses a pig whole-genome CRISPR / Cas9 lentivirus library to knockout the modified immortalized pig alveolar macrophages, thereby constructing a pig whole-genome CRISPR / Cas9 knockout cell library which is susceptible to PRRSV, can be cultured for a long time in vitro and has good homogeneity, and which has important significance for realizing systematic screening of host key factors at the whole-genome level in view of PRRSV replication dependence; and the construction method is simple in process, stable in product performance and good in operability, and is suitable for large-scale construction of the PRRSV-susceptible pig whole-genome CRISPR / Cas9 knockout cell bank.

Description

Technical Field

[0001] This invention relates to the field of cell engineering technology, mainly to the field of knockout cell library construction technology, specifically to a PRRSV-susceptible porcine whole-genome CRISPR / Cas9 knockout cell library and its construction method and application. Background Technology

[0002] Porcine reproductive and respiratory syndrome (PRRS) is a severe immunosuppressive infectious disease caused by Porcine reproductive and respiratory syndrome virus (PRRSV), resulting in enormous economic losses to the global pig industry. Clinically, the disease is characterized by two main features: reproductive disorders in pregnant sows and respiratory damage in pigs of all ages. PRRS is widespread in the global pig industry, posing a serious threat to pig health, farming efficiency, meat safety, human health, and environmental pollution.

[0003] Current prevention and control measures for this disease mainly include vaccination and biosafety management. However, due to the complex interactions between the PRRSV virus and its host, its high genomic variability, and its antibody-dependent enhancement effect, vaccine development and disease control face significant challenges. Currently, effective vaccines and specific drugs for the prevention and treatment of PRRSV are still lacking.

[0004] Besides vaccines and drugs, a more effective approach is to modify key host genes using genetic methods to breed disease-resistant pigs through disease-resistant breeding strategies. CRISPR / Cas9 whole-genome knockout library screening technology can systematically evaluate the impact of each gene knockout on viral replication across the entire genome, providing a powerful screening tool for discovering new host factors and elucidating the interaction mechanisms between the virus and the host. Therefore, systematically screening host factors related to PRRSV replication across the entire genome is of great significance for elucidating viral pathogenesis mechanisms and developing novel antiviral strategies.

[0005] Currently, although whole-genome CRISPR-Cas9 gene knockout technology has been successfully applied to related research in animals such as pigs and chickens, and resistance genes related to swine influenza virus and Japanese encephalitis virus have been screened based on this technology, systematic whole-genome screening of key host factors dependent on PRRSV replication has not been sufficiently reported. This seriously affects the discovery of important host genes related to PRRSV replication and the study of the interaction mechanism between the virus and the host, which is detrimental to the development of new drugs and vaccines for PRRS prevention and control, as well as the breeding of disease-resistant pig breeds.

[0006] Existing research has shown that PRRSV can only infect cells of monocyte and macrophage lineages in its natural host, such as porcine alveolar macrophages (PAMs). In particular, primary PAMs are the optimal cells for PRRSV infection. However, PAMs are difficult to obtain (requiring isolation from live pig lungs) and cannot be passaged in vitro, making them unsuitable for convenient preservation or long-term use. Furthermore, the phenotype of isolated porcine monocytes is heterogeneous, hindering their widespread application in PRRSV research. Therefore, providing a PRRSV-susceptible, long-term in vitro passaged, and homogeneous porcine whole-genome CRISPR / Cas9 knockout cell bank is of great significance for achieving genome-wide systematic screening of key host factors that PRRSV replication depends on. Summary of the Invention

[0007] The purpose of this invention is to address the aforementioned deficiencies in existing porcine whole-genome CRISPR / Cas9 knockout cell libraries by providing a PRRSV-susceptible porcine whole-genome CRISPR / Cas9 knockout cell library, its construction method, and its applications.

[0008] To achieve the above-mentioned objectives, this invention provides a method for constructing a PRRSV-susceptible porcine whole-genome CRISPR / Cas9 knockout cell library, comprising the following steps:

[0009] S1: Design and synthesize cloning primers for the porcine CD163 gene, and amplify the CD163 coding sequence fragment from the cDNA of primary porcine alveolar macrophages; S2: The CD163 coding sequence fragment was cloned into the lentiviral overexpression vector pCDH-CMV-MCS-EF1a-mCherry-Hygro to construct the pCDH-CD163 recombinant plasmid. S3: HEK293T cells were co-transfected with pCDH-CD163 recombinant plasmid, psPAX2 plasmid and VSVG plasmid and packaged to obtain CD163 lentivirus; S4: Immortalized porcine alveolar macrophages were infected with CD163 lentivirus, and after screening with hygromycin and monoclonalization, a monoclonal cell line stably expressing CD163 was obtained. S5: After infecting a monoclonal cell line stably expressing CD163 with a porcine whole-genome CRISPR / Cas9 lentiviral library, the cells were screened with puromycin to obtain a porcine whole-genome CRISPR / Cas9 knockout cell library susceptible to PRRSV.

[0010] This invention discloses a method for constructing a PRRSV-susceptible porcine whole-genome CRISPR / Cas9 knockout cell library. Utilizing the principle that the CD163 molecule is responsible for PRRSV uncoating and genome release, determining the susceptibility of target cells to the virus, the method involves first modifying immortalized porcine alveolar macrophages with the CD163 gene to obtain a stable CD163-expressing monoclonal cell line. Then, a porcine whole-genome CRISPR / Cas9 lentiviral library is used to knock out the modified immortalized porcine alveolar macrophages, thereby constructing a PRRSV-susceptible, long-term in vitro passaged, and homogeneous porcine whole-genome CRISPR / Cas9 knockout cell library. This method is of great significance for achieving systematic screening of PRRSV replication-dependent host key factors at the whole-genome level. Furthermore, the method is simple, produces stable products, and is highly operable, making it suitable for large-scale construction of PRRSV-susceptible porcine whole-genome CRISPR / Cas9 knockout cell libraries.

[0011] In step S3, preferably, the mass ratio of the pCDH-CD163 recombinant plasmid, psPAX2 plasmid, and VSVG plasmid is 4:(3-2):2; more preferably, the mass ratio is 4:3:2. The preferred plasmid ratio results in higher transfection efficiency and a greater yield of the target product.

[0012] In step S4, preferably, the immortalized porcine alveolar macrophages are 3D4 / 21 cells; these cells have good passage stability and are suitable for large-scale application.

[0013] Preferably, the concentration of hygromycin is 500 μg / mL; the preferred concentration of hygromycin results in high screening efficiency and more stable and higher expression of CD163 in the obtained cell line.

[0014] In step S5, preferably, the porcine whole-genome CRISPR / Cas9 lentiviral library contains 123,951 sgRNAs targeting 20,661 genes.

[0015] Preferably, the infection MOI is 0.3; a preferred MOI results in higher infection efficiency and better effect.

[0016] Preferably, polybrene is added at a final concentration of 5-8 μg / mL during infection; more preferably, polybrene is added at a final concentration of 8 μg / mL; this can further improve infection efficiency.

[0017] Preferably, the concentration of puromycin is 5 μg / mL; this preferred concentration of puromycin allows for faster and better screening results.

[0018] Preferably, the screening time is 48-72 hours after infection; the screening duration is 3-5 days; more preferably, the screening time is 72 hours after infection; the screening duration is 3 days.

[0019] To further achieve the above-mentioned objectives, the present invention also provides a PRRSV-susceptible pig whole-genome CRISPR / Cas9 knockout cell bank, which is constructed by the above method. This cell bank has the advantages of PRRSV susceptibility, long-term in vitro passage culture, and good homogeneity. It also achieves permanent loss of gene function and is closer to the natural environment of PRRSV in vivo infection, which is beneficial to enhance the specificity of PRRSV resistance gene screening results.

[0020] To achieve the above-mentioned objectives, this invention further provides the application of a PRRSV-susceptible pig whole-genome CRISPR / Cas9 knockout cell library in screening for PRRSV resistance genes.

[0021] Preferably, the method for screening PRRSV resistance genes is as follows: (1) The PRRSV virus was used to infect the knockout cell bank with a lethal infection multiplicity MOI (MOI=0.1) as the experimental group, while a control group was set up with a single clone cell line that stably expressed CD163 without knockout. (2) After all the cells in the control group died, the surviving cells in the experimental group were collected, cultured on a large scale, and the first round of screening cells were obtained; (3) Repeat steps (1) and (2) for the first round of screening cells; after a total of 3 rounds of screening, collect the surviving cells, which are the target cells; (4) Genomic DNA was extracted from the knockout cell bank and the control group cells respectively, and PCR amplification was performed using universal primers targeting the sgRNA backbone region to obtain sgRNA fragments from the knockout cell bank and the control group cells. (5) Next-generation sequencing was performed on the sgRNA fragments of the knockout cell bank and the control group cells respectively, and the sequencing data were compared and enriched. The sgRNA fragments that were significantly enriched in the target cells (p-value < 0.001, log2FC ≥ 1) were selected as the target sgRNAs. The corresponding gene of the target sgRNA was selected as the PRRSV resistance gene (the host gene necessary for PRRSV replication) obtained by screening.

[0022] In step (1), preferably, the control group refers to immortalized porcine alveolar macrophages that have not undergone knockout treatment.

[0023] In step (4), preferably, the universal primer for the sgRNA backbone region is LentiCRISPRv2-F / R.

[0024] Preferably, the sgRNA fragment has approximately 215 base pairs.

[0025] In step (5), preferably, the next-generation sequencing of sgRNA fragments is performed using an Illumina high-throughput sequencing library; the sequencing data is compared and enriched using MAGeCK software; the preferred sequencing and analysis methods are highly efficient and accurate.

[0026] Beneficial effects of this invention: 1. This invention provides a method for constructing a PRRSV-susceptible porcine whole-genome CRISPR / Cas9 knockout cell bank. This method can construct a porcine whole-genome CRISPR / Cas9 knockout cell bank that is PRRSV-susceptible, can be passaged in vitro for a long time, and has good homogeneity. This is of great significance for achieving systematic screening of PRRSV replication-dependent host key factors at the whole-genome level.

[0027] 2. This invention provides a method for constructing a PRRSV-susceptible pig whole-genome CRISPR / Cas9 knockout cell library. The process is simple, the product performance is stable, and the operation is good. It is suitable for the large-scale construction of a PRRSV-susceptible pig whole-genome CRISPR / Cas9 knockout cell library. Attached Figure Description

[0028] Figure 1 The results of the susceptibility verification of the monoclonal cell lines that stably express CD163 in Example 1 of the present invention are as follows: (A is the map of the pCDH-CD163 recombinant plasmid; B is the relative expression level of the CD163 gene in each monoclonal cell line detected by RT-qPCR; C is the fluorescence microscopy observation results of each monoclonal cell line after 48 h of PRRSV-GFP infection (scale bar is 300 μm)).

[0029] Figure 2 The quality assessment results of the PRRSV-susceptible pig whole genome CRISPR / Cas9 knockout cell bank in Example 1 of this invention are shown in Figure A (statistics of the number of sgRNAs contained in each gene in the cell bank; Figure B is a distribution map of the total sgRNA count for each gene).

[0030] Figure 3 This is a scatter plot of the PRRSV disease resistance genes screened in Example 2 of the present invention.

[0031] Figure 4 The diagram shows the analysis results of PRRSV resistance genes screened in Example 2 of this invention (A is the GO analysis diagram of resistance genes enriched to P<0.001; B is the KEGG pathways analysis diagram of resistance genes enriched to P<0.001).

[0032] Figure 5 The figure shows the effect of SETD1B, TARDBP, and SLC35B2 on PRRSV infection in the interference cell line in Example 2 of the present invention (A is the mRNA expression level of SETD1B, TARDBP, and SLC35B2 in the interference cell line detected by RT-qPCR; B is the fluorescence microscopy observation results of PRRSV-GFP infection 48 h later (scale bar is 300 μm); C is the mRNA expression level of PRRSV-N detected by RT-qPCR). Detailed Implementation

[0033] The technical solutions in some embodiments of this disclosure will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this disclosure, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments provided in this disclosure are within the scope of protection of this disclosure.

[0034] The main reagents used in the following examples and their sources are shown in Table 1.

[0035] Table 1. Main reagents and their sources

[0036] The main instruments used in the following examples and their sources are shown in Table 2.

[0037] Table 2. Main Instruments and Their Sources

[0038] Example 1 A method for constructing a PRRSV-susceptible pig whole-genome CRISPR / Cas9 knockout cell library includes the following steps: S1: Cloning primers for the porcine CD163 gene were designed and synthesized to amplify the CD163 coding sequence fragment from cDNA of primary porcine alveolar macrophages. Specifically, CD163-specific cloning primers were designed using SnapGene software (CD163-F: catagaagattctagagATGGTGCTACTTGAAGACTCTGGA; CD163-R: gcggccgcggatccTCATTGTACTTCAGAGTGGTCTCCTGAGG); the CD163 coding sequence fragment was amplified by PCR using PrimeSTAR® Max DNA polymerase from TAKARA, with cDNA from primary PAM cells as a template. The reaction solution was prepared as follows: PrimeSTAR® Max DNA Polymerase, 25 μL; cDNA, 1 μL; forward and reverse primers, 1 μL each; ddH2O, 22 μL. The PCR temperature program was set as follows: 98℃, 10s; 55℃, 5s; 72℃, 30s, for a total of 35 cycles. After the cycle is complete, maintain the temperature at 72°C for 5 minutes, and then hold at 12°C for the last time.

[0039] S2: The CD163 coding sequence fragment was cloned into the lentiviral overexpression vector pCDH-CMV-MCS-EF1a-mCherry-Hygro to construct the pCDH-CD163 recombinant plasmid. Specifically: For the pCDH-CMV-MCS-EF1a-mCherry-Hygro vector, double digestion with Nhe I and BamHI was performed; the digestion system was as follows: pCDH-CMV-MCS-EF1a-mCherry-Hygro vector, 1 μg; Nhe I, 1 μL; BamHI, 1 μL; 10×QuickCut Buffer, 5 μL; ddH2O to a final volume of 50 μL. First, digest the enzyme in a metal bath at 30℃ for 10 min, then digest it at 37℃ for 15 min. The reaction solution was prepared as follows: 157.57 ng of the inserted CD163 fragment, 200 ng of the vector, 4 μL of 5×Reaction Buffer, 1 μL of NovoRec® Plus Recombinase, and ddH2O to a final volume of 20 μL. The ligation system was placed in a metal bath and reacted at 50℃ for 10 min to obtain the ligation product.

[0040] 10 µL of the ligation product was mixed with 100 µL of DH5α competent cells and incubated sequentially on ice for 30 min, followed by heat shock at 42°C for 90 s, and then on ice for 5 min. 700 µL of antibiotic-free LB liquid medium was then added, and the mixture was incubated at 37°C with shaking at 180 rpm for 1 h. After centrifugation at 4000 rpm, the supernatant was discarded, and the bacterial pellet was collected and evenly spread onto a solid plate containing ampicillin. The plate was inverted and incubated at 37°C for 14 h. Single colonies were picked from the solid medium and transferred to 15 mL of ampicillin-containing liquid medium. The mixture was incubated at 37°C with shaking at 180 rpm for 16 h. The bacterial cells were collected by centrifugation at 4000 rpm for 5 min. Plasmid extraction was then performed to obtain the pCDH-CD163 recombinant plasmid.

[0041] S3: HEK293T cells were co-transfected with pCDH-CD163 recombinant plasmid, psPAX2 plasmid, and VSVG plasmid to obtain CD163 lentivirus. Specifically, frozen HEK293T cells were removed and immediately thawed in a 37°C water bath. After thawing, the cells were transferred to clean centrifuge tubes, 1 mL of culture medium (DMEM (+)) was added, and the cells were centrifuged at 1000 r / min for 5 minutes. The supernatant was discarded, and the cells were resuspended in fresh culture medium. The cells were seeded into 60 mm culture dishes and mixed using a cross-hatching method. The dishes were then incubated at 37°C in a 5% CO2 incubator. Cells were passaged according to cell density. The culture medium was discarded, and the cells were washed with PBS and discarded. 1 mL of 0.25% trypsin was added, and the cells were digested for 3 minutes. When the cells became rounded and began to detach from the bottom of the dish, an equal volume of serum-containing culture medium was added to neutralize the cells. The cells were collected into centrifuge tubes and centrifuged at 1000 r / min for 5 minutes. The supernatant was discarded. Resuspend cells in fresh culture medium; seed into new culture dishes or plates; resuscitate HEK293T cells up to passage 3, culture until confluence reaches 80% or higher, then replace the culture medium with DMEM (-) for starvation treatment. Follow the instructions at 1×10⁻⁶ cells per cell. 6 The calculations were performed based on approximately 1 μg of total plasmid transfected into cells. 6 μg of plasmid was transfected into cells in a 60 mm culture dish. The required mass of each plasmid was calculated according to the ratio of pCDH-CD163 recombinant plasmid: psPAX2:VSVG = 4:3:2.

[0042] Prepare EP tubes containing twice the number of plasmids to be transfected. Add 200 μL of Opti-MEM medium to each tube, dividing them into two groups. One group is named "PEI tubes," with 18 μL of PEI added and gently mixed. The other group is named "plasmid tubes," with helper plasmid and pCDH-CD163 recombinant plasmid added sequentially and mixed thoroughly. After mixing, let stand for 5 min. Carefully and gently add the liquid from the plasmid tubes to the PEI tubes, gently mix, and let stand for 20 min. Then, change the medium in a 60 mm culture dish to DMEM (+), and carefully and evenly drop the above-mentioned liquid onto the surface of the medium, minimizing shaking, and continue culturing the cells. After 12 h, replace with fresh medium. Continue culturing for 48 h, collect the supernatant, centrifuge at 2000 r / min for 5 min, and filter through a 0.45 μm filter to obtain a suspension containing CD163 lentivirus. S4: Immortalized porcine alveolar macrophages were infected with CD163 lentivirus, and after screening with hygromycin and monoclonalization, a monoclonal cell line stably expressing CD163 was obtained.

[0043] Specifically, the cryopreserved immortalized porcine alveolar macrophages (3D4 / 21) were removed and immediately thawed in a 37°C water bath. After thawing, the cells were transferred to a clean centrifuge tube, and 1 mL of culture medium (RPMI 1640 (+)) was added. The cells were centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and the cells were resuspended in fresh culture medium. The cells were seeded into 60 mm culture dishes and mixed using a cross-hatching method. The dishes were then incubated at 37°C in a 5% CO2 incubator. The cells were passaged according to the cell density. The culture medium was discarded first, and the cells were washed with PBS and discarded. 1 mL of 0.25% trypsin was added, and the cells were digested for 3 minutes. When the cells became rounded and began to detach from the bottom of the dish, an equal volume of serum-containing culture medium was added to neutralize the cells. The cells were collected into a centrifuge tube, centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The cells were resuspended in fresh culture medium and seeded into new culture dishes or culture plates.

[0044] Cells were infected with a virus particle suspension at a ratio of 1:1 to RPMI 1640 (+) (3D4 / 21). After 24 hours of infection, the virus suspension was replaced with RPMI 1640 (+). Once the cells recovered, they were treated with 500 μg / mL hygromycin for 24 hours for selection. After two rounds of selection, the mixed cells were subjected to single-clone selection to obtain the stable CD163-expressing monoclonal cell line with the highest infection efficiency (3D4 / 21-CD163-10).

[0045] Monoclonal cell line susceptibility verification: Dilute contaminated cells to an appropriate density and add them to 96-well plates at appropriate volumes. Observe under a microscope to ensure that each well contains only a single cell. Immediately add 100 μL of culture medium to each single-cell well and continue culturing the cells to 12-well plates. Collect cells according to the passage procedure and perform RT-qPCR to detect CD163 gene expression (e.g., Figure 1As shown in the figure, the cell line with the highest PRRSV infection efficiency was selected as the stable CD163 expression cell line (3D4 / 21-CD163-10).

[0046] S5: After infecting a stable CD163-expressing cell line (3D4 / 21-CD163-10) with a porcine whole-genome CRISPR / Cas9 lentiviral library, puromycin was used for selection to obtain a PRRSV-susceptible porcine whole-genome CRISPR / Cas9 knockout cell library. Specifically, the cell count was 2 × 10⁻⁶. 8 3D4 / 21-CD163-10 cells were inoculated at a density of 40% into T175 flasks and infected with a library of lentivirus (MOI=0.3), with polybrene added to a final concentration of 8 μg / mL. After 24 h, the medium was replaced with fresh RPMI 1640 medium. After 48 h, 5 μg / mL puromycin was added for screening. The medium was changed every 48 h, and 5 μg / mL puromycin was added for further screening to obtain a PRRSV-susceptible porcine whole-genome CRISPR / Cas9 knockout cell bank.

[0047] Quality assessment of PRRSV-susceptible pig whole-genome CRISPR / Cas9 knockout cell bank: Genomic DNA was extracted from the knockout cell bank, and the sgRNA region was amplified by PCR for high-throughput sequencing. Results showed (e.g.) Figure 2 As shown in the figure, the cell bank coverage reached 88.54%, the proportion of undetected genes was less than 1‰, and the total number of sgRNAs corresponding to each gene was evenly distributed, indicating that the quality of the PRRSV-susceptible pig whole genome CRISPR / Cas9 knockout cell bank is qualified.

[0048] Example 2 A method for screening PRRSV resistance genes using a PRRSV-susceptible pig whole-genome CRISPR / Cas9 knockout cell library, comprising the following steps: (1) PRRSV virus was used to infect a knockout cell bank as the experimental group with an MOI of 0.1, while a control group was set up with 3D4 / 21-CD163-10. The cells of the experimental group and the control group were seeded in 6-well plates and cultured until 70%~80% confluence. Then, they were infected with PRRSV with an MOI of 0.1. 600 μL of serum-free medium was added to each well and incubated at 37°C for 2 h. The old medium was then discarded and replaced with RPMI 1640 medium with a serum concentration of 2% for another 48 h before the samples were collected for detection.

[0049] (2) After all the cells in the control group died, the surviving cells in the experimental group were collected, cultured on a larger scale, and the first round of screening cells were obtained.

[0050] (3) Repeat steps (1) and (2) for the first round of screening cells; after three rounds of screening, collect the surviving cells, which are the target cells.

[0051] (4) Genomic DNA was extracted from the knockout cell bank and the control group cells respectively, and PCR amplification was performed using universal primers targeting the sgRNA backbone region to obtain sgRNA fragments from the knockout cell bank and the control group cells.

[0052] (5) Next-generation sequencing was performed on sgRNA fragments from the knockout cell bank and the control group, and the sequencing data were compared and enriched using MAGeCK software. sgRNA fragments significantly enriched in the target cells were selected as target sgRNAs, and the corresponding genes of the target sgRNAs were used as the selected PRRSV resistance genes. The results showed that sgRNAs were significantly enriched in multiple pathways, including DNA damage response, extrinsic apoptosis pathway, inflammatory response, and EBV infection. The top 12 genes were FCGRT, TMEM41B, B2M, MAPK14, CD163, SETD1B, TARDBP, C4H1orf43, UBE2M, PLEKHN1, SLC35B2, and THBS1 (as shown in Table 3). Figure 3 and Figure 4 (As shown).

[0053] Table 3. Genome-wide positive screening of key host factors for PRRSV replication

[0054] (6) Design and construct specific shRNA lentiviral interference vectors for the screened PRRSV resistance genes (such as SETD1B, TARDBP, and SLC35B2); knock down the expression of the above genes (SETD1B, TARDBP, and SLC35B2) in 3D4 / 21-CD163-10 cells respectively; infect the knockdown cell line and control cell line with PRRSV-GFP, and observe the expression level of PRRSV N gene by fluorescence microscopy and RT-qPCR after 48 hours.

[0055] Table 4 Gene knockdown primer sequences

[0056] Specifically: shRNA primers were designed using porcine SETD1B, TARDBP, and SLC35B2 gene sequences (gene knockdown primers are shown in Table 4). The synthesized shRNA primers needed to be annealed. The annealing system was as follows: 1 μL each of upstream and downstream primers; 1 μL of 10×T4 Buffer; and 7 μL of ddH2O. After mixing, a gradient cooling annealing program was performed: 95℃ for 3 min; then cooled to 85℃ at a rate of 2℃ / s and held for 1 min; then cooled to 75℃ at a rate of 0.3℃ / s and held for 1 min; subsequently, the temperature was further reduced to 25℃ following the same cooling rate and time as the 85℃ to 75℃ stage; finally, the temperature was stored at 4℃.

[0057] For the U6 vector, double digestion with BamHI and EcoRI was performed. The linearized vector was recovered; 100 ng of the linearized vector; 1.5 μL of annealing primer; 2 μL of 10×T4 buffer; 1 μL of T4 ligase; and ddH2O to a final volume of 20 μL were added. After mixing, the mixture was incubated in a metal bath at 22°C for 30 min to complete the ligation.

[0058] After transformation, plasmid extraction, and sequencing, lentiviruses were packaged. 3D4 / 21-CD163-10 cells were revived and cultured for up to three passages before viral infection. Cells were infected with a virus particle suspension at a 1:1 ratio of RPMI 1640 (+), with 8 μg / mL polybrene added. After 24 h of infection, the medium was replaced with RPMI 1640 (+). Once the cells recovered, 5 μg / mL Puro was added for selection. The Puro-containing medium was changed every 48 h until all cells in the control group died. The surviving transfected cell lines sh-SETD1B, sh-TARDBP, and sh-SLC35B2 were cultured on a large scale, and RNA samples were collected to detect interference efficiency. RT-qPCR confirmed knockdown efficiencies of 70%, 60%, and 90%, respectively.

[0059] The above-mentioned knockout cell lines and control cell lines were infected with PRRSV-GFP. The specific steps were as follows: Cells were seeded in 6-well plates and cultured until 70%–80% confluence. Then, PRRSV was injected at 0.1 MOI. 600 μL of serum-free medium was added to each well, and the cells were incubated at 37°C for 2 h. The old medium was then discarded and replaced with RPMI 1640 medium containing 2% serum. After 48 h, fluorescence was observed and PRRSV N gene expression was detected. The results showed that compared with the control group, the PRRSV infection efficiency of sh-SETD1B, sh-TARDBP, and sh-SLC35B2 cells was significantly reduced (e.g., ...). Figure 5 (As shown).

[0060] In the description of this specification, specific features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments or examples.

[0061] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for constructing a PRRSV-susceptible porcine whole-genome CRISPR / Cas9 knockout cell library, characterized in that, Includes the following steps: S1: Design and synthesize cloning primers for the porcine CD163 gene, and amplify the CD163 coding sequence fragment from the cDNA of primary porcine alveolar macrophages; S2: The CD163 coding sequence fragment was cloned into the lentiviral overexpression vector pCDH-CMV-MCS-EF1a-mCherry-Hygro to construct the pCDH-CD163 recombinant plasmid. S3: HEK293T cells were co-transfected with pCDH-CD163 recombinant plasmid, psPAX2 plasmid and VSVG plasmid and packaged to obtain CD163 lentivirus; S4: Immortalized porcine alveolar macrophages were infected with CD163 lentivirus, and after screening with hygromycin and monoclonalization, a monoclonal cell line stably expressing CD163 was obtained. S5: After infecting a monoclonal cell line stably expressing CD163 with a porcine whole-genome CRISPR / Cas9 lentiviral library, the cells were screened with puromycin to obtain a porcine whole-genome CRISPR / Cas9 knockout cell library susceptible to PRRSV.

2. The construction method according to claim 1, characterized in that, In step S3, the mass ratio of the pCDH-CD163 recombinant plasmid, psPAX2 plasmid, and VSVG plasmid is 4:(3-2):

2.

3. The construction method according to claim 1, characterized in that, In step S4, the concentration of the hygromycin is 500 μg / mL.

4. The construction method according to claim 1, characterized in that, In step S5, the infection multiplicity is 0.

3.

5. The construction method according to claim 1, characterized in that, In step S5, polybrene is added at a final concentration of 5-8 μg / mL during infection.

6. The construction method according to claim 1, characterized in that, In step S5, the concentration of puromycin is 5 μg / mL.

7. The construction method according to claim 1, characterized in that, In step S5, the screening time is 48-72 hours after infection; the screening duration is 3-5 days.

8. A PRRSV-susceptible porcine whole-genome CRISPR / Cas9 knockout cell bank, characterized in that, The knockout cell library is constructed using the construction method described in any one of claims 1-7.

9. The application of the PRRSV-susceptible porcine whole-genome CRISPR / Cas9 knockout cell library as described in claim 8 in screening for PRRSV resistance genes.

10. The application according to claim 9, characterized in that, The method for screening PRRSV resistance genes includes: (1) The PRRSV virus was used to infect the knockout cell bank with lethal infection multiples as the experimental group, while the control group was set up with a single clone cell line that stably expressed CD163 without knockout. (2) After all the cells in the control group died, the surviving cells in the experimental group were collected, cultured on a large scale, and the first round of screening cells were obtained; (3) Repeat steps (1) and (2) for the first round of screening cells; after three rounds of screening, collect the surviving cells, which are the target cells; (4) Genomic DNA was extracted from the knockout cell bank and the control group cells respectively, and PCR amplification was performed using universal primers targeting the sgRNA backbone region to obtain sgRNA fragments from the knockout cell bank and the control group cells. (5) Next-generation sequencing was performed on the sgRNA fragments of the knockout cell bank and the control group cells respectively, and the sequencing data were compared and enriched. The sgRNA fragments that were significantly enriched in the target cells were selected as the target sgRNAs. The corresponding genes of the target sgRNAs were selected as the PRRSV resistance genes obtained through screening.

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