Method for knocking out SIRPA allele and application thereof
Using CRISPR/Cas9 technology and specific sgRNA, the SIRPA allele was efficiently knocked out, solving the problem of the lack of SIRPA gene-edited cell lines in the existing technology. This provides an efficient SIRPA allele knockout method, which can be applied to the preparation of U937 cells.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-08
- Publication Date
- 2026-03-13
AI Technical Summary
Currently, there are no known methods for creating cell lines with the SIRPA gene knocked out, nor are there any applications of these cell lines in xenotransplantation research. Furthermore, there is a lack of accurate humanized cell models for predicting human diseases.
Using CRISPR/Cas9 technology, cells were co-transfected with specific sgRNA (such as SIRPA-gRNA3) and Cas9 protein, and cell lines with efficient SIRPA allele knockout were obtained through enrichment, culture, positive cell line screening and sequencing.
This study achieved a high knockout rate of 95% for the SIRPA allele and a cell viability of 88.6%, reducing the cost of cell line construction and providing a method for preparing U937 cells with the SIRPA allele knocked out.
Smart Images
Figure CN121653124A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of gene editing technology, specifically relating to a method for... SIRPA Methods and applications of allele knockout. Background Technology
[0002] Signal regulatory protein α (SIRPA), also known as CD172a or Src homology 2 domain containing protein tyrosine phosphatase substrate-1 (SHPS-1), is a receptor-type transmembrane glycoprotein belonging to the immunoglobulin superfamily. Its extracellular domain contains three Ig-like domains (including one V-like domain and two C1-like domains) to bind ligands, while its intracellular domain has two tyrosine phosphorylation sites at the C-terminus, which can bind to and activate protein tyrosine phosphatases SHP-1 and SHP-2 to exert their signal transduction function. This protein is mainly expressed on the surface of phagocytes such as macrophages and neutrophils, as well as some dendritic cells, mast cells, hematopoietic stem cells, and other myeloid cells. It is also present on the surface of neurons and some T cells and natural killer cells.
[0003] SIRPA has diverse biological functions. It participates in immune regulation, activating phagocytosis by binding to pathogen ligands to clear pathogens, and is involved in various immune responses such as cytokine release and inflammatory responses. It can also serve as a substrate for various activated tyrosine kinases such as EGFR and PDGFR, participating in the negative regulation of receptor tyrosine kinase-coupled signaling pathways. After binding to its ligand CD47, it promotes tyrosine kinase phosphorylation of immunoreceptor tyrosine inhibitory motifs (ITIMs) in the cytoplasm, thereby recruiting and activating tyrosine phosphatases SHP-1 and SHP-2 to exert signal regulation effects. It also regulates cell adhesion and migration, affecting cell localization and distribution in tissues, and plays a role in embryonic development and tissue repair. In addition, it is mainly expressed on the surface of macrophages, and after binding to CD47 on other cells, it transmits inhibitory signals to macrophages, inhibiting phagocytosis of target cells, helping the body to distinguish between "self" and "non-self", and maintaining immune homeostasis.
[0004] SIRPA is closely associated with a variety of diseases. In cancer, tumor cells often overexpress CD47 to activate SIRPA, thereby inhibiting macrophages from destroying cancer cells and helping tumor cells evade the immune system. Therefore, blocking the interaction between the two has become an important strategy for cancer immunotherapy. In autoimmune diseases, such as systemic lupus erythematosus, abnormal expression and function of SIRPA may lead to the immune system attacking its own tissues and participate in the occurrence and development of the disease. In inflammatory diseases, SIRPA affects the occurrence, development and resolution of inflammation by regulating the function of immune cells. For example, myeloid cell-specific SIRPA deficiency can reduce inflammation and inhibit atherosclerosis.
[0005] However, no knockout has been found yet. SIRPA There are no reports on gene cell line methods and their application in xenotransplantation research, and so far there are none. SIRPA Humanized cells, which are used in gene editing to more accurately predict human diseases, serve as models, and therefore... SIRPA Gene editing has enabled the reporting of immune studies on macrophages in xenotransplantation. Summary of the Invention
[0006] The purpose of this invention is to provide a method for SIRPA The methods and applications of allele knockout have the advantage of high knockout efficiency.
[0007] This invention provides a method for SIRPA Allele knockout sgRNA, wherein the sgRNA includes: SIRPA-gRNA3; The nucleotide sequence of the SIRPA-gRNA3 is shown in SEQ ID NO. 3.
[0008] This invention provides a CRISPR / Cas9 vector for SIRPA allele knockout, wherein the CRISPR / Cas9 vector contains the sgRNA described in the above technical solution.
[0009] This invention provides a method for SIRPA allele knockout, comprising the following steps: The sgRNA and Cas9 protein described in the above technical solution are mixed and co-transfected into the cells to be edited to obtain transfected cells; The transfected cells were sequentially enriched, cultured, screened for positive cell lines, and sequenced to obtain... SIRPA Allele knockout cell lines.
[0010] Preferably, the cells to be edited include U937 cells.
[0011] Preferably, the mass ratio of sgRNA to CRISPR / Cas9 is 1:1.
[0012] Preferably, the primers used for screening positive cell lines include: SIRPA-F and SIRPA-R; The nucleotide sequence of the SIRPA-F is shown in SEQ ID NO. 4; The nucleotide sequence of the SIRPA-R is shown in SEQ ID NO. 5.
[0013] This invention provides the application of the sgRNA described in the above technical solution, or the CRISPR / Cas9 vector, or the method described, for preparing knockout RNA. SIRPA U937 cells with alleles and / or enhanced SIRPA Allele knockout rate.
[0014] Beneficial effects: This invention provides an sgRNA for SIRPA allele knockout, the sgRNA comprising: SIRPA-gRNA3; the nucleotide sequence of SIRPA-gRNA3 is shown in SEQ ID NO. 3, and the sgRNA's site of action is located on the SIRPA gene. Based on CRISPR / Cas9 technology, this invention achieves SIRPA allele knockout through a specific sgRNA, with a knockout rate as high as 95%, and the resulting... SIRPA The survival rate of allele-edited U937 cells was as high as 88.6%. Therefore, the technical solution of this invention can be used to prepare knockout cells. SIRPA U937 cells with alleles, improving SIRPA Allele knockout rate reduced SIRPA The construction cost of allele knockout cell lines and their practical application value. Attached Figure Description
[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0016] Figure 1 The following are the SIRPA knockout sequencing results of U937 cells provided by this invention; Figure 2 Flow cytometry was used to detect the SIRPA protein knockout status in SIRPA knockout cells provided by this invention. Figure 3 The present invention provides a Western blot assay for SIRPA protein knockout in SIRPA knockout cells. Figure 4 The development of SIRPA gene-edited U937 cells provided by this invention. Detailed Implementation
[0017] In this invention, unless otherwise specified, the equipment, reagents and methods used are all conventional selections.
[0018] To further illustrate the present invention, the solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.
[0019] Example 1: SIRPA gene-editing cell line and application research 1.1 Design of sgRNA targeting the SIRPA gene and verification of its knockout efficiency Referring to the human SIRPA (Gene ID: 140885) gene sequence information in NCBI, the SIRPA gene sequence was searched and downloaded from the NCBI database. Three sequences were designed and selected for SIRPA gene knockout (sgRNA was synthesized by Suzhou Genewise Biotechnology Co., Ltd.).
[0020] Table 1. sgRNA sequence information of the SIRPA gene.
[0021] Before transfection, U937 cells were seeded in 60 mm culture dishes and transfected using an electroporation kit (Lonza, VPI-1002). The CAS protein used in the experiment was purchased from IDT (Alt-R Sp Cas9 Nuclease V3-GFP).
[0022] Collect U937 cells in good growth condition, centrifuge at 1200 rpm for 5 min, discard the supernatant, and obtain the cells; Resuspend cells in 100 μL of electroporation buffer, repeating 5 × 10⁶ times per cell. 5 Mix the transfection components shown in Table 2 with the cells and transfect them according to the transfection combination and dosage shown in Table 2. After mixing, transfer the mixture to an electroporation cuvette and perform electroporation and electroporation sequentially using a Lonza nuclear transfection instrument. Then, seed the cells into 100 mm cell culture dishes for culture (the culture medium is 1640 solution containing 20% FBS).
[0023] Table 2 Transfection components and dosage
[0024] 24 hours after transfection, cells were collected, cellular DNA was isolated and identified by PCR (product size 697 bp). The knockout gene SIRPA was amplified by PCR using primers. Identification primers were designed flanking the SIRPA gene editing site sequence to amplify the target gene sequence, which was then sequenced to identify the knockout efficiency and positive cell lines. The sequences are shown in Table 3. Table 3 PCR Primer Information
[0025] The PCR amplification system consisted of: 0.5 μL of upstream primer (10 pmol / μL), 0.5 μL of downstream primer (10 pmol / μL), 2 μg of genomic DNA, 10 μL of KoDFx Buffer, 0.5 μL of FxKoD, and sterile distilled water to a final volume of 20 μL.
[0026] PCR reaction conditions: 95℃ for 2 min; (98℃ for 30 s, 60℃ for 30 s, 68℃ for 20 s) × 35 cycles; 68℃ for 5 min; store at 16℃.
[0027] The PCR products were purified and recovered, and TA cloned and ligated into the T vector. The ligation products were transformed into DH5α competent cells for culture. Each sgRNA treatment colony was considered a treatment. Twenty single colonies from each treatment were selected for sequencing. The sequencing results were compared with the wild-type gene sequence to calculate the knockout efficiency of each sgRNA. The results are shown in Table 4.
[0028] Table 4 SIRPA-F-gRNA knockout efficiency
[0029] As can be seen from the data in Table 3, SIRPA-F-gRNA3 had the highest knockout efficiency, and subsequent experiments were conducted.
[0030] 1.2 Cell transfection screening and monoclonal identification U937 cells were co-transfected with SIRPA-F-gRNA3 and Cas9, which have the highest knockout efficiency. The number of co-transfected cells was counted, and one cell per well was seeded into a 96-well plate using the extreme dilution method. After culturing for about 12 days, 10 single clones were picked for DNA extraction and knockout gene identification. The results showed that 8 of the single clones were SIRPA knockout cell clones.
[0031] Cells with better growth status (SIRPAKO (-7bp / -28bp), i.e., cell lines numbered 1 and 2) were selected for sequencing. The results are shown in [Figure 1]. Figure 1 (exist Figure 1 In the diagram, Allele1 represents the sequencing results of cell number 1; Allele2 represents the sequencing results of cell number 2.
[0032] Cells numbered 1 were analyzed by flow cytometry, with wild-type SIRPA as the control group, to illustrate the SIRPA protein knockout status. Results are shown below. Figure 2 (exist Figure 2 In the image, the left side shows the results of the SIRPAKO group, and the right side shows the results of the control group; blue represents unstained cells, and red represents antibody-stained cells. Combination Figure 2 The results showed that, compared with the wild-type control group, no significant SIRPA protein expression was detected in the SIRPAKO group, indicating that the SIRPA protein in this cell line had been successfully knocked out.
[0033] Western blotting was performed on cells numbered 1 to assess the SIRPA protein knockout status, with wild-type SIRPA serving as the control group. Results are shown below. Figure 3 (The image on the left shows the results of the SIRPAKO group, and the image on the right shows the results of the control group.) Combination Figure 3 The results showed that, compared with the wild-type control group, no significant SIRPA protein expression was detected in the SIRPAKO group, indicating that the SIRPA protein in this cell line had been successfully knocked out.
[0034] Combination Figure 1-3 It can be seen that the obtained SIRPAKO cell line successfully produced frameshift mutations at the DNA level, and flow cytometry and Western blotting experiments confirmed that the gene was also successfully knocked out at the cellular protein level.
[0035] 1.3 Passage culture of SIRPA gene-edited U937 cells When the U937 (SIRPAKO (-7bp / -28bp) cell line reaches the passage conditions, collect the original liquid culture medium and transfer it to a freshly sterilized 15mL centrifuge tube. Centrifuge at 1200rpm for 5min to gather the cells to the bottom of the tube, thoroughly remove the supernatant, wash once with DPBS (Dubor's phosphate buffer) preheated to 37.5℃, transfer to a freshly sterilized 15mL centrifuge tube, centrifuge at 1200rpm for 5min to gather the cells to the bottom of the tube, thoroughly remove the supernatant, add 10mL of complete culture medium (1640 solution containing 20% FBS), aspirate to form a single-cell suspension, and centrifuge at 5×10⁻⁶. 6 SIRPAKO gene-edited U937 cells were passaged in 10cm cell culture dishes and cultured in an incubator at 37.5℃, saturated humidity, and 5% CO2. Cell growth after passage is shown in the table below. Figure 4 (exist Figure 4 In the figure, Figure A shows the cell growth of U937 cells after passage; Figure B shows the cell growth of SIRPA gene-edited U937 cells after passage.
[0036] Combination Figure 4 It can be seen that the morphology and state of the two types of cells did not change significantly, and gene knockout had no significant effect on cell growth and passage.
[0037] In summary, this invention, based on CRISPR / Cas9 technology, achieves a knockout efficiency of over 90% for the SIRPA allele using specific sgRNAs, thereby improving the SIRPA gene knockout efficiency and the success rate of constructing SIRPA allele knockout cell lines, while reducing the construction cost of SIRPA allele knockout cell lines, demonstrating its practical application value.
[0038] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.
Claims
1. An sgRNA for SIRPA allele knockout, characterized in that, The sgRNA includes: SIRPA-gRNA3; The nucleotide sequence of the SIRPA-gRNA3 is shown in SEQ ID NO.
3.
2. A CRISPR / Cas9 vector for SIRPA allele knockout, characterized in that, The CRISPR / Cas9 vector contains the sgRNA as described in claim 1.
3. A method for SIRPA allele knockout, characterized in that, Includes the following steps: The sgRNA and Cas9 described in claim 1 were mixed and co-transfected into the cells to be edited to obtain transfected cells; The transfected cells were sequentially enriched, cultured, screened for positive cell lines, and sequenced to obtain... SIRPA Allele knockout cell lines.
4. The method according to claim 3, characterized in that, The cells to be edited include: U937 cells.
5. The method according to claim 3, characterized in that, The mass ratio of sgRNA to Cas9 protein is 1:
1.
6. The method according to claim 3, characterized in that, Primers used for screening positive cell lines include: SIRPA-F and SIRPA-R; The nucleotide sequence of the SIRPA-F is shown in SEQ ID NO. 4; The nucleotide sequence of the SIRPA-R is shown in SEQ ID NO.
5.
7. The application of the sgRNA of claim 1, the CRISPR / Cas9 vector of claim 2, or the method of any one of claims 3-6, characterized in that, Used to prepare knockout SIRPA U937 cells with alleles and / or enhanced SIRPA Allele knockout rate.