SgRNA of targeted ASCT2 gene and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to sgRNA of a targeted ASCT2 gene and application of the sgRNA. Two efficient knockout targets are provided for the human ASCT2 gene, and knockout effect detection and identification show that the two sgRNAs can effectively knock out the ASCT2 gene on a 293T cell. The 293T cells without the ASCT2 gene are high in stability, formation of syncytial bodies induced by BaEV envelope glycoprotein can be remarkably inhibited, cell source impurities in lentivirus preparation liquid are greatly reduced, the titer of BaEV envelope lentivirus is increased by 5 times, and the method has practical application value.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to sgRNA targeting the ASCT2 gene and applications thereof. Background Art

[0002] ASCT2 (Alanine-Serine-Cysteine ​​Transporter 2; SLC1A5) is a Na+ transporter encoded by the SLC1A5 gene. + The ASCT2 gene is located at 19q13.3 and contains 8 exons. In the NCBI and Ensemble databases, three transcripts of the human gene from different translation start points were reported. The first variant, NM_005628, represents the longest transcript, consisting of 2882 nucleotides and 8 exons. This transcript encodes a peptide of 541 amino acids. The second variant, NM_001145144, consists of 1750 nucleotides, and its 5'UTR is different from that of variant NM_005628. In NM_001145144, translation starts downstream of the third exon, producing a shorter peptide of 313aa. The third subtype, NM_001145145, has 1872 nucleotides and lacks the first exon. It presents a different translation start point at 5' and encodes a peptide of 339 amino acids. ASCT2 represents a key player in cell biology due to its broad expression and substrate specificity adapted to different cellular needs.

[0003] Studies have reported that ASCT2 can be used as a receptor by a group of membrane protein pseudotyped lentiviruses to infect human cells, including RD114, RDPro, RD114-TR, BaEV, BaEV-TR, and BaEVRLess. Therefore, in order to avoid excessive fusion of human cells induced by lentivirus during lentivirus synthesis, ASCT2 can be knocked out in cells to eliminate lentivirus-induced cell fusion. Yi-Jun Lan et al. The invention discloses the use of cells with ASCT2 knocked out to obtain high-titer BaEVRless-LV. However, the experiment found that when ASCT2 in cells is knocked out using CRISPR / Cas9 technology, sgRNA affects the knockout efficiency of the gene, and also affects the stability of the cells after the gene knockout, causing the cells to undergo morphological and intrinsic changes during the subculture process, affecting the packaging of the lentivirus. In view of this, it is necessary to provide an sgRNA that can make the cells still have high stability after the ASCT2 gene is knocked out, and can effectively and stably package high-titer lentivirus. Summary of the invention

[0004] To achieve the above object, the present invention provides an sgRNA, whose nucleotide sequence is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0005] The present invention also provides an RNP complex, which is composed of an sgRNA having a nucleotide sequence as shown in SEQ ID NO.1 or SEQ ID NO.2 and a Cas9 protein.

[0006] The present invention also provides a method for preparing the aforementioned RNP complex, comprising the following steps:

[0007] The sgRNA with the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.2 is mixed and incubated with the Cas9 protein to obtain;

[0008] The molar mass ratio of the sgRNA to the Cas9 protein is 100 pmol:10-20 μg, preferably 100 pmol:16 μg;

[0009] The incubation temperature is 20-25°C, preferably 25°C.

[0010] The present invention also provides a use of the aforementioned RNP complex in preparing a cell line with ASCT2 gene knockout.

[0011] Furthermore, the cell line includes 293T cells.

[0012] The present invention also provides a method for constructing a cell line with ASCT2 gene knockout, which comprises the following steps:

[0013] The aforementioned RNP complex was electroporated into 293T cells, and then monoclonal screening was performed. Monoclonal cells were taken and identified by immunofluorescence staining using ASCT2 antibody.

[0014] The present invention also provides an ASCT2 gene knockout cell line, which is constructed according to the above method.

[0015] The present invention also provides a use of the aforementioned cell line in preparing BaEV pseudotype lentivirus.

[0016] Finally, the present invention provides a method for preparing BaEV pseudotype lentivirus, comprising the following steps:

[0017] Take the aforementioned cell line and culture it to a confluence of 70-80%, transfect it with shuttle plasmid pCDH-GFP, packaging plasmid pSpax2 and envelope plasmid pCMV-BaEV-Rless, and then culture it for 48-72 hours, and take the cell culture supernatant.

[0018] Furthermore, the mass ratio of the shuttle plasmid pCDH-GFP, the packaging plasmid pSpax2 and the envelope plasmid pCMV-BaEV-Rless is 1.2:1.2:1.

[0019] The beneficial effects of the present invention are:

[0020] (1) The present invention provides two highly efficient knockout targets for the human ASCT2 gene.

[0021] (2) The knockout effect detection and identification of the present invention showed that the knockout of ASCT2 gene in 293T cells can be effectively achieved. The 293T cells with ASCT2 gene knocked out have high stability, can significantly inhibit the formation of syncytia induced by BaEV envelope glycoprotein, greatly reduce the cell-derived impurities in the lentivirus preparation solution, and increase the titer of BaEV envelope lentivirus by 5 times, which has practical application value.

[0022] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0023] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Immunofluorescence staining was used to detect the targeted knockout effects of two ASCT2 gene knockout cell lines, ASCT2sgRNA-1-1# and ASCT2sgRNA-2-4#.

[0025] Figure 2 Fluorescence imaging of different cells after 48 hours of lentiviral packaging.

[0026] Figure 3 Fluorescence imaging of different cells after 72 hours of lentiviral packaging.

[0027] Figure 4Titer detection results after lentiviral concentrates prepared from different cells were infected with 293T WT cells. DETAILED DESCRIPTION

[0028] The raw materials, reagents and equipment used in the specific embodiments of the present invention are all purchased from commercial sources.

[0029] Example 1 Study on targeted knockout of ASCT2 gene in 293T cells

[0030] The targeting sgRNA was designed based on the longest transcript NM_005628 of the human ASCT2 gene in the NCBI database, and the sequence was synthesized after modification.

[0031] After digesting the 293T cells in good condition, wash them with DPBS and resuspend the cells; at the same time, incubate the Cas9 protein, sgRNA, and RNP (mix sgRNA and Cas9 protein at a ratio of 100 pmol / 16 μg and incubate at room temperature for 10 min), and prepare the electroporation solution; resuspend the 293T cells with the prepared electroporation solution, blow gently to evenly mix, take an appropriate amount of cell suspension, add it to the RNP, blow and pipet gently once, transfer it to the bottom of the electrode cup, ensure that the sample solution covers the bottom of the electroporation hole, cover the lid, and put it into the instrument for electroporation.

[0032] After electroporation, the cells were resuspended in culture medium containing 10% fetal bovine serum and placed in a cell culture incubator at 37°C and 5% CO2 for 4 days. The electroporated 293T cells were then screened for monoclonal proliferation using the limiting dilution method. Well-growing monoclonal cells were selected and immunofluorescence staining was performed using ASCT2 antibodies to test the knockout effect of the ASCT2 gene.

[0033] The results of ASCT2 gene knockout in monoclonal cells are shown in Figure 1 , Figure 1 There are two 293T cell lines with ASCT2 gene knockout, ASCT2-sgRNA-1-1# and ASCT2-sgRNA-2-4#. These two cell lines are targeted for knockout by ASCT2-sgRNA-1 (SEQ ID NO.1): 5'-CAGCGCCACACCAAA GACGA-3' and ASCT2-sgRNA-2 (SEQ ID NO.2): 5'-TCCCACGGAGGCGTTGATGG-3'.

[0034] The following experimental examples further illustrate the beneficial effects of the two ASCT2 gene knockout 293T cell lines obtained in Example 1 in preparing BaEV pseudotyped lentivirus:

[0035] Experimental Example 1 Verification of lentiviral packaging in 293T cells with ASCT2 gene knockout

[0036] 1. Lentivirus packaging

[0037] 1. 293T WT cells, ASCT2-sgRNA-1-1#22nd generation cells, and ASCT2-sgRNA-2-421st generation cells were inoculated into 15 cm culture dishes and cultured in 10% fetal bovine serum (Gibco) culture medium to a confluence of 70-80% at the time of transfection; 1 hour before transfection, the cell culture medium was replaced with Opti-MEM medium;

[0038] 2. Take shuttle plasmid (pCDH-GFP), packaging plasmid (pSpax2), and envelope plasmid (pCMV-BaEV-Rless) at a mass ratio of 1.2:1.2:1, mix 25 μg of the above three plasmids, add Opti-MEM medium to 500 μl, mix carefully, and let stand for 5 minutes. This is called solution A.

[0039] 3. Dilute PEIpro to 500 μl with Opti-MEM medium (the ratio of PEIpro to the total mass of the three plasmids is 1:1), mix carefully, and let stand for 5 minutes, which is recorded as liquid B;

[0040] 4. Slowly add liquid B to liquid A, slowly mix, and let stand for 10 minutes to obtain a mixed solution; slowly add the mixed solution to the cells replaced with Opti-MEM medium, gently mix, and start transfection; replace with 10% fetal bovine serum (Gibco) medium 12 hours after transfection, observe the cell state at 48h and 72h after transfection, harvest the cell culture supernatant at 48h and replace with fresh medium containing 10% fetal bovine serum, and only harvest the cell culture supernatant at 72h. The harvested supernatant was centrifuged at low speed and filtered with a 0.45μm filter to obtain 48h lentivirus and 72h lentivirus, respectively, and the lentivirus was concentrated using a 300KDa ultrafiltration centrifuge tube, and the concentrate was stored at -80℃ for standby use for lentivirus titer detection.

[0041] Observation results are shown in Figures 2-3 . Figure 2 This is the cell status at 48h after transfection, from which it can be seen that knocking out the ASCT2 gene significantly inhibited the formation of syncytia. Figure 3 This is the cell status 72 hours after transfection. Knockout of the ASCT2 gene significantly inhibited the formation of syncytia, and the cells were still well attached 72 hours after transfection.

[0042] 2. Lentivirus titer detection

[0043] 1. Count 293T cells after digestion and adjust the density to 2.0×10 5 / ml, inoculated into 12-well plates, 1ml in each well.

[0044] 2. Make 10-fold gradient dilutions of the virus solution in a 1.5ml centrifuge tube. The dilution method is: prepare 5 1.5ml tubes for each virus, add 135μl of culture medium to each tube, add 15μl of virus stock solution to the first tube, mix well, then pipette 15μl and add to the second tube to mix well. And so on, make 5 dilutions. After the cells in the well plate adhere to the wall, discard the original culture medium in the 12-well plate and replace it with 0.9ml of new complete culture medium. Add 100μl of diluted virus solution to each well and mark it. Repeat 3 wells for each dilution, continue to culture for 48h, and then replace with fresh culture medium.

[0045] 3. The lentivirus itself expresses green fluorescent protein. After 72 hours of infection, the cells were digested and the percentage of cells expressing green fluorescence was detected by flow cytometry to calculate the lentivirus titer.

[0046] Lentivirus titer calculation formula:

[0047] Virus infection titer (TU / ml) = number of cells × fluorescence percentage × 10 3 / Volume of virus stock solution (μl); Lentivirus titer test results are shown in Figure 4 At 48h, the titer of lentivirus prepared by the two gene knockout 293T cells was higher than that of WT cells. At 72h, almost no lentivirus prepared by WT cells was obtained, but the titer of lentivirus of the two gene knockout cells was significantly increased compared with 48h. Compared with WT cells, the total titer of lentivirus of the two gene knockout cells at 48h+72h increased by 5 times.

[0048] In summary, the two sgRNAs for efficiently knocking out targets provided by the present invention for the human ASCT2 gene can effectively knock out the ASCT2 gene in 293T cells. The 293T cells with ASCT2 gene knocked out have high stability, can significantly inhibit the formation of syncytia induced by BaEV envelope glycoprotein, greatly reduce cell-derived impurities in the lentivirus preparation solution, and greatly increase the titer of BaEV envelope lentivirus, which has practical application value.

Claims

1. A sgRNA, characterized in that: The nucleotide sequence is shown in SEQ ID NO.1 or SEQ ID NO.

2.

2. An RNP complex, characterized in that: It is composed of sgRNA and Cas9 protein, the nucleotide sequence of which is shown in SEQ ID NO.1 or SEQ ID NO.

2.

3. A method for preparing the RNP complex according to claim 2, characterized in that: The steps include: The sgRNA with a nucleotide sequence as shown in SEQ ID NO.1 or SEQ ID NO.2 is mixed and incubated with the Cas9 protein to obtain; The molar mass ratio of the sgRNA to the Cas9 protein is 100 pmol:10-20 μg, preferably 100 pmol:16 μg; The incubation temperature is 20-25°C, preferably 25°C.

4. Use of the RNP complex according to claim 2 in preparing a cell line with ASCT2 gene knockout.

5. The use according to claim 4, characterized in that: The cell lines include 293T cells.

6. A method for constructing a cell line with ASCT2 gene knockout, characterized in that: The steps include: The RNP complex described in claim 2 is electrotransferred into 293T cells, and then monoclonal screening is performed, and monoclonal cells are taken for immunofluorescence staining and identification using ASCT2 antibody to obtain the obtained product.

7. A cell line with ASCT2 gene knockout, characterized in that: The invention is constructed according to the method of claim 6.

8. Use of the cell line according to claim 7 in preparing BaEV pseudotyped lentivirus.

9. A method for preparing a BaEV pseudotyped lentivirus, characterized in that: The steps include: Take the cell line described in claim 8 and culture it until the confluence is 70-80%, transfect the shuttle plasmid pCDH-GFP, packaging plasmid pSpax2 and envelope plasmid pCMV-BaEV-Rless, culture it for another 48-72 hours, and take the cell culture supernatant.

10. The method according to claim 9, characterized in that: The mass ratio of the shuttle plasmid pCDH-GFP, the packaging plasmid pSpax2 and the envelope plasmid pCMV-BaEV-Rless is 1.2:1.2:1.

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