SgRNA highly targeting IFNGR1 gene, and composition and application thereof

Abstract

According to the sgRNA highly targeting the IFNGR1 gene, the composition and the application of the sgRNA, the sgRNA provided by the invention can highly target the IFNGR1 gene, complete knockout of IFNGR1 on mixed cloning is realized through combined application of three sgRNAs, and the process and the time of constructing an IFNGR1 knockout cell line are greatly shortened and shortened.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to sgRNA highly targeting the IFNGR1 gene, a composition thereof, and applications thereof. Background Art

[0002] IFNγ receptor subunit 1 (IFNGR1) is a key component of the IFNγ signaling pathway. Its surface expression is essential for full activation of this pathway. Knockout of IFNGR1 is a classic approach to studying the signaling function of human IFN-γ / IFN-γR. The IFNγ signaling pathway is primarily associated with various biological responses related to host defense and immune surveillance. IFNγ leads to the upregulation of major histocompatibility complex (MHC) molecules and the upregulation of MHC I and II antigen processing and presentation machinery.

[0003] However, the existing IFNGR1 knockout cell line construction process is cumbersome and monoclonal screening is time-consuming. There is currently a lack of a simple, fast and efficient construction method. Summary of the Invention

[0004] In view of this, in order to make up for the deficiencies of the prior art, the present invention is proposed.

[0005] Unless otherwise indicated, embodiments of the present invention employ conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, which are within the skill of the art (Sambrook, Fritsch, and Maniatis (1989) Molecular Cloning: A Laboratory Manual, 2nd ed.; Ausubel et al., eds. (1987) Current Protocols in Molecular Biology); MacPherson et al., eds. (1995) Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach); Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual; Freshney, ed. (1987) Animal Cell Culture).

[0006] In a first aspect, the present invention provides a guide RNA (sgRNA) that is highly targeted to the human IFNGR1 gene, wherein the sgRNA targets the third constitutive exon region.

[0007] In some embodiments, the sgRNA is any one of sgRNA2, sgRNA3, and sgRNA4. The nucleotide sequence of sgRNA2 is as shown in SEQ ID NO.2 or has at least 90% homology to the nucleotide sequence shown in SEQ ID NO.2, the nucleotide sequence of sgRNA3 is as shown in SEQ ID NO.3 or has at least 90% homology to the nucleotide sequence shown in SEQ ID NO.3, and the nucleotide sequence of sgRNA4 is as shown in SEQ ID NO.4 or has at least 90% homology to the nucleotide sequence shown in SEQ ID NO.4.

[0008] The term "sgRNA" stands for "single-guide RNA (sgRNA)," which includes trRNA (also known as tracrRNA) and crisprRNA (crRNA). sgRNA is designed based on a target site on a target gene, containing sequences sufficient to synergize with the endonuclease Cas9, guiding Cas9-mediated DNA double-strand breaks at the target site. In this case, the target gene is the human IFNGR1 gene.

[0009] In some embodiments, the nucleotide sequence of sgRNA2 is as shown in SEQ ID NO.2 or has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the nucleotide sequence shown in SEQ ID NO.2.

[0010] In some embodiments, the nucleotide sequence of the sgRNA3 is as shown in SEQ ID NO. 3 or has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the nucleotide sequence shown in SEQ ID NO. 3.

[0011] In some embodiments, the nucleotide sequence of the sgRNA4 is as shown in SEQ ID NO.4 or has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the nucleotide sequence shown in SEQ ID NO.4.

[0012] In some embodiments, the sgRNA comprises a modified sgRNA.

[0013] In some embodiments, the modified sgRNA comprises one or more modified nucleotides at any one or more of the upper stem region, the hairpin 1 region, the hairpin 2 region, between the hairpin 1 region and the hairpin 2 region, the lower stem region, the 5' end, and / or the 3' end.

[0014] In some embodiments, the sgRNA comprises a 3' end modification in the 3' terminus. In some embodiments, the sgRNA comprises a modification of at least two of the last four nucleotides at the 3' terminus of the 3' terminus.

[0015] In some embodiments, the sgRNA comprises a 5' end modification in the 5' terminus. In some embodiments, the sgRNA comprises a modification of at least two of the first four nucleotides at the 5' terminus of the 5' terminus.

[0016] In some embodiments, the sgRNA comprises a 3' end modification in the 3' end and a 5' end modification in the 5' end.

[0017] In some embodiments, the sgRNA comprises modifications of at least two of the last four nucleotides at the 3' end of the 3' terminus and at least two of the first four nucleotides at the 5' end of the 5' terminus.

[0018] In some embodiments, these modifications include, but are not limited to, one or more of 2'-O-methyl (2'-O-Me), 2'-O-(2-methoxyethyl) (2'-O-moe), 2'-fluoro (2'-F), phosphorothioate (PS) bonds between nucleotides, GC substitutions, reversed abasic bonds between nucleotides, and their equivalents.

[0019] In some embodiments, the sgRNA comprises a PS bond between at least two of the last four nucleotides at the 3' end of the 3' terminus and / or at least two of the first four nucleotides at the 5' end of the 5' terminus.

[0020] In some embodiments, the sgRNA comprises a modification in the nexus region.

[0021] The second aspect of the present invention provides an sgRNA composition that is highly targeted to the human IFNGR1 gene, wherein the sgRNA composition comprises at least two of sgRNA2, sgRNA3, and sgRNA4 described in the first aspect of the present invention.

[0022] In some embodiments, the sgRNA composition includes sgRNA2, sgRNA3, and sgRNA4 described in the first aspect of the present invention.

[0023] The third aspect of the present invention provides a recombinant vector, which comprises the sgRNA described in the first aspect of the present invention or the sgRNA composition described in the second aspect of the present invention.

[0024] In the present invention, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is connected. Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules comprising one or more free ends, no free ends (e.g., circular); nucleic acid molecules comprising DNA, RNA, or both; and other various polynucleotides known in the art. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, in which virally derived DNA or RNA sequences are present in a vector for packaging as a virus (e.g., retrovirus, replication-defective retrovirus, adenovirus, replication-defective adenovirus, and adeno-associated virus). Viral vectors also include polynucleotides carried by viruses for transfection into host cells.

[0025] In some embodiments, the plasmid includes but is not limited to pLCNICK, pCmv-BEACON1, pGL3-U6-sgRNA-ccdB-EF1α-Puro.

[0026] In some embodiments, the viral vector includes, but is not limited to, retrovirus, adenovirus, adeno-associated virus, herpes virus, poxvirus, baculovirus, papillomavirus, papovavirus, and bacteriophage.

[0027] In some embodiments, the vector is selected from a plasmid.

[0028] The fourth aspect of the present invention provides a kit, comprising the sgRNA according to the first aspect of the present invention, the sgRNA composition according to the second aspect of the present invention, or the recombinant vector according to the third aspect of the present invention.

[0029] In some embodiments, the kit further includes, but is not limited to, one or more of Cas9 nuclease, delivery reagent, positive control, negative control, buffer, diluent, instructions, and PCR primers.

[0030] In the present invention, the Cas9 nuclease can be in the form of plasmid DNA encoding Cas9, in vitro transcribed Cas9 mRNA, or purified Cas9 protein.

[0031] The term "Cas9 protein" refers to the main protein element of the CRISPR / Cas9 system, which forms a complex with crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA) to form an activated endonuclease or nickase. The CRISPR / Cas9 system is an RNA-guided genetic engineering tool that can cause target-specific double-strand breaks in DNA. These breaks can stimulate endogenous DNA repair mechanisms, including NHEJ and homologous recombination (HDR). NHEJ is a low-fidelity DNA repair process. During the repair and reconnection of broken DNA, random bases are inserted or lost, resulting in changes in splicing.

[0032] In addition, the Cas9 protein may include not only wild-type Cas9, but also inactivated Cas9 (dCas9) or Cas9 variants such as Cas9 nickase. Among them, the inactivated Cas9 may be RFN comprising a FokI nuclease domain bound to dCas9 or dCas9 bound to a transcription activator or repressor domain; the Cas9 nickase may be D10ACas9 or H840ACas9, but is not limited thereto.

[0033] The Cas9 nuclease of the present invention is not limited in its source. For example, the Cas9 nuclease can be derived from, but not limited to, Streptococcus pyogenes (Spy), Streptococcus dysgalactiae, Streptococcus canis, Streptococcus equi, Streptococcus iniae, Streptococcus phocae, Streptococcus pseudoporcinus, Streptococcus oralis, Streptococcus pseudoporcinus, Streptococcus infantis, Streptococcus pyogenes, ... In some embodiments, the present invention relates to a method for the synthesis of a novel nucleic acid sequence of the present invention. The method comprises the following steps: a) a nucleic acid sequence of the present invention wherein the nucleic acid sequence of the present invention is cleaved from a nucleic acid sequence of the present invention; b) a nucleic acid sequence of the present invention wherein the nucleic acid sequence of the present invention is cleaved from a nucleic acid sequence of the present invention; c) a nucleic acid sequence of the present invention wherein the nucleic acid sequence of the present invention is cleaved from a nucleic acid sequence of the present invention; and c) a nucleic acid sequence of the present invention wherein the nucleic acid sequence of the present invention is cleaved from a nucleic acid sequence of the present invention; and d) a nucleic acid sequence of the present invention wherein the nucleic acid sequence of the present invention is cleaved from a nucleic acid sequence of the present invention; and d) a nucleic acid sequence of the present invention wherein the nucleic acid sequence of the present invention is cleaved from a nucleic acid sequence of the present invention; In other embodiments, the Cas9 nuclease is from Staphylococcus.In other embodiments, the Cas9 nuclease is from Staphylococcus aureus, S. simiae, S. auricularis, S. carnosus, S. condimenti, S. massiliensis, S. piscifermentans, S. simulans, S. capitis, S. caprae, S. epidermidis, Staphylococcus aureus (S.epidermidis), Staphylococcus saccharolyticus (S.saccharolyticus), Staphylococcus devriesei (S.devriesei), Staphylococcus haemolyticus (S.haemolyticus), Staphylococcus hominis (S.hominis), Staphylococcus agnettis (S.agnetis), Staphylococcus chromogenes (S.chromogenes), Staphylococcus felis (S.felis), Staphylococcus delphini (S.delphini), Staphylococcus hyicus (S.hyicus), Staphylococcus intermedius (S.intermedius), Staphylococcus luturi (S.lut rae), Staphylococcus microti, Staphylococcus muscae, Staphylococcus pseudointermedia, Staphylococcus rostri, Staphylococcus schleiferi, Staphylococcus lugdunensis, Staphylococcus arlettae, Staphylococcus cohnii, Staphylococcus equorum, Staphylococcus gallinarum, Staphylococcus kloosii, S. leei, and Nipo Staphylococcus aureus, Staphylococcus succinus, Staphylococcus xylosus, Staphylococcus fleurettii, Staphylococcus lentus, Staphylococcus sciuri, S. stepanovicii, Staphylococcus vitulinus, Staphylococcus simulans, Staphylococcus pasteuri, Staphylococcus warneri, or variants thereof.

[0034] In some embodiments, the delivery agent includes agents used in electroporation, calcium phosphate, liposomes, DEAE dextran, microinjection, and viral infection.

[0035] In some embodiments, the positive control includes a positive control sgRNA and target sequence, an sgRNA designed for a site known to have high editing efficiency in commonly used cell lines (e.g., HEK293) (e.g., the AAVS1 safe harbor site), and / or a plasmid / synthetic DNA fragment containing the target sequence. This is used to confirm Cas9 activity, transfection efficiency, and the effectiveness of the editing detection method.

[0036] In some embodiments, the negative control includes a scrambled sgRNA that does not target any location in the genome or an sgRNA targeting a gene from an unrelated species, which is used to assess the background of off-target effects and nonspecific toxicity / reactions that may be caused by Cas9 itself.

[0037] In some embodiments, the buffer includes, but is not limited to, a dedicated buffer for maintaining Cas9 enzyme activity and stability (if Cas9 protein is provided).

[0038] In some embodiments, the diluent includes, but is not limited to, nuclease-free water.

[0039] The fifth aspect of the present invention provides a method for constructing human IFNGR1 gene knockout cells, wherein the method delivers the recombinant vector of the third aspect of the present invention into cells to obtain human IFNGR1 gene knockout cells.

[0040] In some embodiments, the method of delivery includes but is not limited to electroporation, calcium phosphate method, liposome method, DEAE dextran method, microinjection, viral infection.

[0041] In some embodiments, the method comprises the steps of:

[0042] (1) Construct the recombinant vector described in the third aspect of the present invention.

[0043] (2) Transfect the recombinant vector and Cas9 expression vector in step (1) into cells.

[0044] In some embodiments, (1) comprises the steps of:

[0045] a. Amplify a fragment containing any one or more of sgRNAs among sgRNA2, sgRNA3, and sgRNA4 described in the first aspect of the present invention.

[0046] b. Connect the fragments of any one or more of sgRNAs among sgRNA2, sgRNA3, and sgRNA4 to the connection vector.

[0047] c. Transform and identify the ligation product.

[0048] In a specific embodiment, the ligation vector is pGL3-U6-sgRNA-ccdB-EF1α-Puro.

[0049] In a specific embodiment, sgRNA2, sgRNA3, and sgRNA4 are ligated to a ligation vector.

[0050] The sixth aspect of the present invention provides a human IFNGR1 gene knockout cell, which is constructed using the method described in the fifth aspect of the present invention.

[0051] In some embodiments, the cells include cells having a human IFNGR1 gene.

[0052] In some embodiments, the cells include, but are not limited to, immune cells, tissue cells, cancer cells, and stem cells.

[0053] In some embodiments, the cancer includes but is not limited to adrenal cortical carcinoma, bladder urothelial carcinoma, breast cancer, cervical squamous cell carcinoma, endocervical adenocarcinoma, bile duct cancer, colon adenocarcinoma, lymphoid tumors, esophageal cancer, glioblastoma multiforme, head and neck squamous cell carcinoma, renal chromophobe cell carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, leukemia, brain low-grade glioma, hepatocellular carcinoma, mesothelial cell carcinoma, ovarian cancer, pancreatic cancer, pheochromocytoma and paraganglioma, prostate cancer, rectal cancer, malignant sarcoma, melanoma, gastric cancer, testicular germ cell tumor, thyroid cancer, thymic cancer, endometrial cancer, uterine sarcoma, uveal melanoma, myeloma, lymphoma, lung cancer, sarcoma, anal cancer, melanoma, retinoblastoma.

[0054] In some embodiments, the cancer is selected from pancreatic cancer.

[0055] In a specific embodiment, the pancreatic cancer cells include but are not limited to PANC-1 and AsPC-1.

[0056] A seventh aspect of the present invention provides any of the following applications:

[0057] (1) Use of the sgRNA described in the first aspect of the present invention, the sgRNA composition described in the second aspect of the present invention, the recombinant vector described in the third aspect of the present invention, or the kit described in the fourth aspect of the present invention in knocking out the human IFNGR1 gene;

[0058] (2) Use of the sgRNA described in the first aspect of the present invention, the sgRNA composition described in the second aspect of the present invention, the recombinant vector described in the third aspect of the present invention, or the kit described in the fourth aspect of the present invention in preparing a product for knocking out the human IFNGR1 gene;

[0059] (3) Use of the sgRNA described in the first aspect of the present invention, the sgRNA composition described in the second aspect of the present invention, the recombinant vector described in the third aspect of the present invention, or the kit described in the fourth aspect of the present invention in constructing cells in which the human IFNGR1 gene is knocked out;

[0060] (4) Use of the human IFNGR1 gene knockout cells described in the sixth aspect of the present invention in studying the biological function of IFNγ receptor or IFN-γ / IFN-γR signal transduction.

[0061] The present invention has the following advantages and beneficial effects:

[0062] The present invention provides sgRNAs that are highly targeted to the IFNGR1 gene, compositions thereof, and uses thereof. The sgRNAs provided by the present invention are capable of highly targeting the IFNGR1 gene, and by combining three sgRNAs, complete knockout of IFNGR1 in mixed clones is achieved, greatly reducing the process and time for constructing IFNGR1 knockout cell lines. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 is a schematic diagram of the list of IFNGR1 splice variants;

[0064] Figure 2 This is a diagram of the sgRNA design pattern;

[0065] Figure 3 It is a schematic diagram of the sgRNA pattern;

[0066] Figure 4 is a schematic diagram of sequencing results;

[0067] Figure 5 is a schematic diagram of the results of IFNGR1 knockout;

[0068] Figure 6 is a schematic diagram of the results of IFNGR1 knockout;

[0069] Figure 7 This is a schematic diagram of the protein pathway detection results. DETAILED DESCRIPTION

[0070] The present invention will be further described below with reference to the following embodiments. The following description is merely a preferred embodiment of the present invention and does not limit the present invention in any other form. Any person skilled in the art may utilize the above disclosed technical content to make equivalent embodiments with equivalent variations. Any simple modification or equivalent variation of the following embodiments made in accordance with the technical essence of the present invention without departing from the content of the present invention shall fall within the scope of protection of the present invention.

[0071] Example 1 Design and synthesis of sgRNA targeting the human IFNGR1 gene

[0072] The UCSC Genome Browser Home website was used to retrieve 19 splice variants of the human IFNGR1 gene (201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, and 219). Figure 1 As shown. Among them, 8 splice variants (201, 202, 207, 209, 210, 211, 212, 213) can encode proteins. Four sgRNAs were designed for the constitutive exon regions of the 8 splice variants encoding proteins, of which sgRNA1 is located in the first constitutive exon region and sgRNA2-4 are located in the third constitutive exon region. The schematic diagram of the location of the four sgRNAs is shown in Figure 2 The sgRNA sequences are shown in Table 1.

[0073] Table 1 Four sgRNA sequences targeting the IFNGR1 gene

[0074]

[0075] Using the pGL3-U6-sgRNA-ccdB-EF1α-Puro vector as a template, multiple sgRNA primer sequences containing three sgRNAs were designed (as shown in Table 2, where the underlines indicate the positions of the three sgRNAs in the primer sequences). Figure 3 Schematic diagram of the sgRNA model.

[0076] Table 2 Primers for multiple sgRNA primer sequence design

[0077]

[0078] sgRNA2, sgRNA3, and sgRNA4 were cloned in tandem into the pGL3-U6-sgRNA-ccdB-EF1α-Puro vector to construct a multiple sgRNA recombinant plasmid targeting the IFNGR1 gene. Each sgRNA contained its own complete expression cassette in this recombinant plasmid. The specific steps were as follows: 200 ng of each sgRNA-containing DNA fragment, 200 ng of the pGL3-U6-sgRNA-ccdB-EF1α-Puro vector, 3 μl of 5× T4 DNA ligase buffer, 1 μl of T4 DNA ligase, 1 μl of Esp3 I, and deionized water to 20 μl. Reaction conditions included 10 cycles of 37°C for 10 min, 16°C for 15 min, followed by 37°C for 20 min, and 85°C for 10 s. After the reaction, 10 μl of the ligation product was added to 100 μl of DH5α competent cells for subsequent transformation, single clone extraction, and sequencing verification. In the recombinant plasmid, each sgRNA has a complete expression frame and can be transcribed independently without interference from the expression of other sgRNAs. The individual sgRNA recombinant plasmids of sgRNA1, sgRNA2, sgRNA3, and sgRNA4 were constructed simultaneously. The sequencing results confirmed that we have successfully constructed multiple sgRNA recombinant plasmids targeting the IFNGR1 gene. The positive clones with correct sequencing will be used for subsequent experiments. The sequencing results are as follows Figure 4 shown.

[0079] Example 2 Human IFNGR1 Knockout and Verification

[0080] 1. Experimental Methods

[0081] 1. Transfection of sgRNA recombinant plasmid into pancreatic cancer PANC-1 cells and AsPC-1 cells

[0082] (1) 12-24 h before transfection, PANC-1 cells and AsPC-1 cells were plated at 2×10 5 / well were seeded in 6-well cell culture plates;

[0083] (2) Replace the preheated culture medium with 2 mL per well 1 h before transfection;

[0084] (3) Prepare liposome dilution solution for transfection: Based on a mass-to-volume ratio of 1:3 between the plasmid to be transfected and Lippofectamine 2000, add 7.5 μl of Lippofectamine 2000 liposomes to 100 μl of 1× Opti-MEM medium, gently pipette to mix, and let stand at room temperature for 5 min.

[0085] Prepare plasmid dilution solution: calculate the volume of Cas9 expression vector and sgRNA recombinant plasmid at a mass ratio of 2:1, add a total mass of 2.5 μg of plasmid to 100 μL of 1× Opti-MEM medium, and mix gently by pipetting;

[0086] Add the liposome dilution to the plasmid dilution, pipette and mix thoroughly, and let it stand at room temperature for 20 min;

[0087] Evenly add 200 μL of transfection complex to the corresponding wells of a 6-well plate, shake gently, and transfer to a cell culture incubator for culture;

[0088] (4) 24 h after transfection, the liquid in the 6-well plate was replaced with fresh culture medium; at the same time, 10 pg / mL of blasticidin and 2 μg / mL of Puro were added for drug screening, and the protein was extracted 14 days after drug screening.

[0089] 2. Cell protein extraction and concentration determination

[0090] 2.1 Cell protein extraction

[0091] (1) Prepare lysis buffer: Mix 1×RIPA, 25×PI, 100×phosphatase inhibitor, and 100×PMSF in a ratio of 100:4:1:1 and place on ice until ready to use;

[0092] (2) Remove the cultured cells from the incubator, aspirate the culture medium, carefully add 1 mL of pre-cooled 1× PBS along the wall of the culture dish, gently shake the culture dish to wash the cells, and aspirate the supernatant as much as possible;

[0093] (3) Pipette and mix the lysis solution, add 60 μL of lysis solution to each well of the 6-well plate, and shake well so that the cells at the bottom of the plate can fully contact the lysis solution;

[0094] (4) Place the 6-well plate on ice. Use a clean cell scraper to scrape the bottom of the dish in a "Z" shape for each cell type, carefully scraping every corner. After scraping, tilt the bottom of the dish and wait for 5 minutes to allow the cell proteins and lysate to gather in one corner. Then, aspirate the cell-containing lysate into the corresponding labeled EP tube.

[0095] (5) After 30 seconds of Votex, place on ice for 5 minutes, repeat three times (prepare two timers), and prepare another set of EP tubes and place them on ice for pre-cooling;

[0096] (6) Centrifuge in a refrigerated centrifuge at 4°C, 12,000 g, for 15 min. Remove the sample and place on ice. Place the supernatant in a pre-cooled EP tube, label it, and store it in a -20 or -80°C refrigerator.

[0097] 2.2 Protein concentration detection

[0098] Dilute the protein standard to 2 mg / mL. Select 9 wells of a 96-well plate and add 25 μL of ddHO to each well, diluting the sample to 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03625, 0.018125, and 0 mg / mL. (Add 25 μL of the standard to the first well, mix thoroughly by pipetting, and add 25 μL of the mixture to the next well, continuing through the eighth well. After mixing, discard 25 μL from the eighth well and do not add to the ninth well.)

[0099] (2) Prepare the total amount of BCA working solution: Mix the A and B reagents in the BCA protein quantification kit at a ratio of 50:1;

[0100] (3) Add BCA working solution: Add 200 μL of BCA working solution to each well and incubate at 37°C for 30 min;

[0101] (4) Measure absorbance using a microplate reader: circle the wells to be measured, measure the absorbance of each well at 562 nm, and draw a BCA standard concentration-absorbance curve based on the template;

[0102] (5) After calculating the protein concentration, calculate the protein volume according to 20-30 μg per well, adjust the volume to the same level with RIPA lysis buffer, add 5× loading buffer and mix well, and finally denature the protein sample using a small PCR instrument at 99°C for 15 min.

[0103] 2. Experimental Results

[0104] 1. Western Blot was used to detect the knockout efficiency of IFNGR1 gene in pancreatic cancer cell lines. The results showed that four single sgRNAs targeting IFNGR1 could not achieve complete knockout of IFNGR1. The knockout efficiency of sgRNA1 in pancreatic cancer cell line PANC-1 was less than 10%. Only when sgRNA2+sgRNA3+sgRNA4 were used in combination could complete knockout of IFNGR1 be achieved. Similar results were obtained in pancreatic cancer cell lines PANC-1 and AsPC-1 (e.g. Figure 5 shown).

[0105] 2. Select PANC-1 IFNGR1 KO The cells were sorted for monoclonal isolation and the knockout efficiency of IFNGR1 was detected by flow cytometry. Figure 6 As can be seen from the results, PANC-1 constructed by this method IFNGR1 KO Mixed clones can replace single clones (1#~8#) to achieve complete knockout of IFNGR1.

[0106] 3. Treat PANC-1 cells and PANC-1 with IFN-γ cytokine at a concentration of 10 ng / mL. IFNGR1 KOAfter 12 hours, the expression and activation of JAK1, JAK2 and STAT1 were detected by Western Blot. Figure 7 As shown in the results, IFN-γ treatment of PANC-1 cells activated the JAK-STAT pathway, an important downstream signaling pathway of IFN-γR. However, after successful knockout of IFN-γR1 expression, the activation of JAK / STAT signaling was significantly inhibited.

[0107] In summary, it is difficult to achieve complete knockout of IFNGR1 using a single sgRNA, and different sgRNAs have different knockout efficiencies. The present invention provides a vector for knocking out the human IFNGR1 gene; a pancreatic cancer cell line PANC-1 with IFNGR1 knockout was successfully constructed. IFNGR1 KO and AsPC-1 IFNGR1 KO The combined use of sgRNA2, sgRNA3, and sgRNA4 enabled complete knockout of IFNGR1 in mixed clones, and JAK-STAT signaling pathway transduction disorders were detected in IFN-γR1 knockout cell lines. In summary, this invention significantly reduces the process and time required to construct IFNGR1 knockout cell lines, providing essential research tools for functional analysis of human IFN-γ / IFN-γR signaling.

[0108] The above embodiments are only provided for understanding the method and core concept of the present invention. It should be noted that, without departing from the principles of the present invention, a number of improvements and modifications may be made to the present invention by a person skilled in the art, and such improvements and modifications shall fall within the scope of protection of the claims of the present invention.

Claims

1. An sgRNA highly targeting the human IFNGR1 gene, characterized in that: The sgRNA targets the third constitutive exon region; Preferably, the sgRNA is any one of sgRNA2, sgRNA3, and sgRNA4; wherein the nucleotide sequence of the sgRNA2 is as shown in SEQ ID NO.2 or has at least 90% homology to the nucleotide sequence shown in SEQ ID NO.2, the nucleotide sequence of the sgRNA3 is as shown in SEQ ID NO.3 or has at least 90% homology to the nucleotide sequence shown in SEQ ID NO.3, and the nucleotide sequence of the sgRNA4 is as shown in SEQ ID NO.4 or has at least 90% homology to the nucleotide sequence shown in SEQ ID NO.

4.

2. The sgRNA highly targeting human IFNGR1 gene according to claim 1, characterized in that The sgRNA includes a modified sgRNA; Preferably, the modified sgRNA comprises one or more modified nucleotides at any one or more of the upper stem region, hairpin 1 region, hairpin 2 region, between hairpin 1 region and hairpin 2 region, lower stem region, 5' end and / or 3' end; Preferably, the modification comprises one or more of 2'-O-methyl, 2'-O-(2-methoxyethyl), 2'-fluoro, phosphorothioate bonds between nucleotides, GC substitutions, reversed abasic bonds between nucleotides, and equivalents thereof.

3. An sgRNA composition highly targeting the human IFNGR1 gene, the sgRNA composition comprising at least two of sgRNA2, sgRNA3, and sgRNA4 according to claim 1; Preferably, the sgRNA composition comprises sgRNA2, sgRNA3 and sgRNA4 according to any one of claims 1-2.

4. A recombinant vector, characterized in that The recombinant vector comprises the sgRNA according to any one of claims 1 to 2 or the sgRNA composition according to claim 3; Preferably, the vector comprises a plasmid or a viral vector; Preferably, the vector is selected from a plasmid.

5. A kit, characterized in that The kit comprises the sgRNA according to any one of claims 1 to 2, the sgRNA composition according to claim 3, or the recombinant vector according to claim 4; Preferably, the kit further comprises one or more of Cas9 nuclease, delivery reagent, positive control, negative control, buffer, diluent, instruction manual, and PCR primers.

6. A method for constructing human IFNGR1 gene knockout cells, characterized in that: The method described above delivers the recombinant vector described in claim 4 into cells to obtain cells with human IFNGR1 gene knockout; Preferably, the delivery method includes electroporation, calcium phosphate method, liposome method, DEAE dextran method, microinjection, and viral infection.

7. The method according to claim 6, characterized in that The method comprises the following steps: (1) constructing the recombinant vector according to claim 4; (2) Transfect the recombinant vector and Cas9 expression vector in step (1) into cells.

8. The method according to claim 7, characterized in that (1) comprising the steps of: a. amplifying a fragment containing any one or more sgRNAs of sgRNA2, sgRNA3, and sgRNA4 according to any one of claims 1 to 2; b. Connecting the fragments of any one or more sgRNAs among sgRNA2, sgRNA3, and sgRNA4 to the connection vector; c. transforming and identifying the ligation product; Preferably, the ligation vector is pGL3-U6-sgRNA-ccdB-EF1α-Puro; Preferably, step b comprises connecting sgRNA2, sgRNA3 and sgRNA4 to a connection vector.

9. A human IFNGR1 gene knockout cell, characterized in that: The cell is constructed using the method according to any one of claims 6 to 8; Preferably, the cells include cells having human IFNGR1 gene; Preferably, the cells include immune cells, tissue cells, cancer cells, and stem cells; Preferably, the cancer comprises adrenal cortical carcinoma, bladder urothelial carcinoma, breast cancer, cervical squamous cell carcinoma, endocervical adenocarcinoma, bile duct cancer, colon adenocarcinoma, lymphoid tumors, esophageal cancer, glioblastoma multiforme, head and neck squamous cell carcinoma, renal chromophobe cell carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, leukemia, brain low-grade glioma, hepatocellular carcinoma, mesothelial cell carcinoma, ovarian cancer, pancreatic cancer, pheochromocytoma and paraganglioma, prostate cancer, rectal cancer, malignant sarcoma, melanoma, gastric cancer, testicular germ cell tumor, thyroid cancer, thymic cancer, endometrial cancer, uterine sarcoma, uveal melanoma, myeloma, lymphoma, lung cancer, sarcoma, anal cancer, melanoma, retinoblastoma; Preferably, the cancer is selected from pancreatic cancer; Preferably, the pancreatic cancer cells include PANC-1 and AsPC-1.

10. Applications as described in any of the following: (1) Use of the sgRNA according to any one of claims 1 to 2, the sgRNA composition according to claim 3, the recombinant vector according to claim 4, or the kit according to claim 5 in knocking out the human IFNGR1 gene; (2) Use of the sgRNA according to any one of claims 1 to 2, the sgRNA composition according to claim 3, the recombinant vector according to claim 4, or the kit according to claim 5 in preparing a product for knocking out the human IFNGR1 gene; (3) Use of the sgRNA according to any one of claims 1 to 2, the sgRNA composition according to claim 3, the recombinant vector according to claim 4, or the kit according to claim 5 in constructing cells for knocking out the human IFNGR1 gene; (4) Use of the human IFNGR1 gene knockout cells according to claim 9 in studying the biological function of IFNγ receptor or IFN-γ / IFN-γR signal transduction.

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