Gene editing system for targeted knockout of HLA-G isomer gene and application

Abstract

The invention provides a gene editing system for targeted knockout of HLA-G isomer genes and application, and belongs to the technical field of gene engineering. The invention provides sgRNA of an exon 1, an exon 2, an exon 3, an exon 4, an exon 5, an intron 2 and an intron 4 of a targeted HLA-G gene. The sgRNA respectively comprises a targeted sequence and a complementary sequence. According to the invention, the sgRNA is annealed to form a double-chain fragment, and the double-chain fragment is inserted into a gene editing vector, so that the CRISPR / Cas9 gene editing vector combination aiming at the whole HLA-G gene, the membrane-bound HLA-G1-G4 gene, the soluble HLA-G5-G7 gene and the novel HLA-G gene is constructed. The CRISPR / Cas9 gene editing vector combination is respectively packaged by lentivirus, so that different HLA-G isomer genes can be accurately knocked out or the HLA-G genes, including novel isomers, can be completely knocked out.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and in particular relates to a gene editing system and application for targeted knockout of HLA-G isoform genes. Background Art

[0002] Human leukocyte antigen-G (HLA-G) belongs to the non-classical HLA class I molecule. The gene is located in the 6p21.3 region of chromosome 6 and is about 6.0 kb in length. After selective splicing, the initial transcript of HLA-G produces at least 7 messenger RNAs (mRNAs), which encode Figure 1 At least seven protein isoform molecules are shown, including four membrane-bound HLA-G1 to -G4 and three soluble HLA-G5 to -G7, as well as new HLA-G isoforms.

[0003] exist Figure 1 During the protein translation process shown in the figure, the first exon of HLA-G mRNA encodes signal peptides (SP), the second, third and fourth exons encode the α1, α2 and α3 domains of the extracellular region, respectively, and the fifth exon encodes the transmembrane region (TM). The sixth exon encodes the intracellular segment of the HLA-G molecule containing only 6 amino acid residues. Since exon 6 contains a termination codon (TGA), the seventh and eighth exons correspond to the 3′-untranslated region (3′UTR) of the HLA-G gene.

[0004] HLA-G1 is encoded by full-length HLA-G mRNA, which is similar to HLA class I antigens in structure, and contains α1, α2, and α3 domains. HLA-G2 lacks the α2 domain. HLA-G3 lacks the α2 and α3 domains. HLA-G4 lacks the α3 domain. The extracellular domains of HLA-G5 and HLA-G6 are the same as those of HLA-G1 and HLA-G2, respectively, but they are encoded by HLA-G mRNA containing intron 4. Because intron 4 contains a stop codon (TAA), the encoded protein molecule lacks the transmembrane region encoded by exon 5, forming a soluble HLA-G molecule. Since the mRNA encoding HLA-G7 contains a stop codon (TAA) in intron 2, the extracellular region only contains the α1 domain and two amino acid residues encoded by intron 2. The molecular weights of HLA-G1 to -G7 isomers are approximately 39kD, 31kD, 23kD, 30kD, 37kD, 27kD and 17kD.

[0005] Compared with the classic HLA I molecules, HLA-G has a unique promoter region, limited gene polymorphism, strict tissue distribution, and a single type of antigen presentation. Under physiological conditions, HLA-G molecules are restrictedly distributed in immune-exempt tissues, such as extravillous trophoblast cells at the maternal-fetal interface, thymus, cornea, pancreatic islets, etc. Clinically, HLA-G is found to be specifically upregulated in pathological tissues such as tumors, organ transplantation, autoimmune diseases, inflammation, viral infections, etc.; and the level of HLA-G expression is closely related to the clinical stage, prognosis, and survival time of tumor patients. Functionally, HLA-G is an important immune tolerance molecule in the body, which can inhibit the function of immune cells or induce the production of regulatory cells, allowing tumor cells to escape the host's immune surveillance, thereby leading to tumorigenesis.

[0006] In recent years, HLA-G has gradually been considered as a new type of immune checkpoint molecule. It is inferred that the combination of HLA-G checkpoint inhibitors with chemotherapy, radiotherapy or other immune target drugs will bring better therapeutic effects to patients with recurrent metastatic tumors. At present, antibodies or CAR-T drugs developed for HLA-G are still in the early stages of research. As for the function of HLA-G, most studies focus on the full-length membrane-bound molecule HLA-G1 and its soluble molecule HLA-G5. There are few studies on several other HLA-G isoforms, but they are closely related to tumor progression, such as renal cancer and gastric cancer. According to different studies, the role of HLA-G isoforms in tumorigenesis is still controversial. In addition, in different tumor tissue cells, it was found that there is a wide range of heterogeneity in the expression of different HLA-G isoforms. Therefore, different HLA-G isoforms may have specific biological functions and their clinical significance, making it urgent to study the molecular functions of HLA-G isoforms other than HLA-G1 and -G5.

[0007] At present, except for the reported biological functions of HLA-G1 and -G5 isoforms, the functions and significance of HLA-G2, -G3, -G4, -G6, -G7 isoforms, including the new HLA-G isoforms, have not yet been elucidated. However, there are no reports on the successful precise knockout of different HLA-G isoform genes by any technology. Summary of the invention

[0008] The present invention provides a gene editing system and application for targeted knockout of HLA-G isoform genes, which can accurately knock out different HLA-G isoform genes or completely knock out the entire HLA-G gene, thereby achieving the purpose of accurately knocking out a specific gene region of HLA-G.

[0009] The present invention provides an sgRNA combination for knocking out an HLA-G isoform gene, comprising a target sequence and a complementary sequence designed for the gene structure of the HLA-G isoform gene respectively;

[0010] The gene structure includes exon 1, exon 2, exon 3, exon 4, exon 5, intron 4 and intron 2.

[0011] Preferably, the nucleotide sequences of the target sequence and the complementary sequence are shown in SEQ ID No.1 to SEQ ID No.14, respectively.

[0012] The present invention provides a CRISPR / Cas9 vector combination for targeted knockout of HLA-G isoform genes, including a CRISPR / Cas9 vector constructed by connecting a target sequence designed for each gene structure in the above-mentioned sgRNA combination and a double-stranded sequence formed after annealing of a complementary sequence with a gene editing vector.

[0013] Preferably, the gene editing vector comprises the pLentiCRISPRV2 gene recombination plasmid.

[0014] The present invention provides a kit for knocking out HLA-G isoform genes, comprising the above-mentioned sgRNA combination, or the above-mentioned CRISPR / Cas9 vector combination.

[0015] Preferably, when the kit is used to knock out the entire HLA-G gene, the CRISPR / Cas9 vector combination includes: pLentiCRISPRV2-E1, pLentiCRISPRV2-E2, pLentiCRISPRV2-E3, pLentiCRISPRV2-E4, pLentiCRISPRV2-E5, pLentiCRISPRV2-I2 and pLentiCRISPRV2-I4;

[0016] Among them, pLentiCRISPRV2-E1 is constructed by connecting the double-stranded sequence formed by annealing the target sequence designed for exon 1 and the complementary sequence to the pLentiCRISPRV2 vector;

[0017] pLentiCRISPRV2-E2 was constructed by connecting the double-stranded sequence formed by annealing the target sequence designed for exon 2 and the complementary sequence to the pLentiCRISPRV2 vector;

[0018] pLentiCRISPRV2-E3 was constructed by connecting the double-stranded sequence formed by annealing the target sequence designed for exon 3 and the complementary sequence to the pLentiCRISPRV2 vector;

[0019] pLentiCRISPRV2-E4 was constructed by connecting the double-stranded sequence formed by annealing the target sequence designed for exon 4 and the complementary sequence to the pLentiCRISPRV2 vector;

[0020] pLentiCRISPRV2-E5 was constructed by connecting the double-stranded sequence formed by annealing the target sequence designed for exon 5 and the complementary sequence to the pLentiCRISPRV2 vector;

[0021] pLentiCRISPRV2-I2 was constructed by connecting the double-stranded sequence formed by annealing the target sequence designed for intron 2 and the complementary sequence to the pLentiCRISPRV2 vector;

[0022] pLentiCRISPRV2-I4 was constructed by connecting the double-stranded sequence formed by annealing the target sequence designed for intron 4 and the complementary sequence to the pLentiCRISPRV2 vector.

[0023] Preferably, the vector combination for knocking out the membrane-bound HLA-G1 gene using the kit includes: pLentiCRISPRV2-E1, pLentiCRISPRV2-E2, pLentiCRISPRV2-E3, pLentiCRISPRV2-E4 and pLentiCRISPRV2-E5;

[0024] The vector combination for knocking out the membrane-bound HLA-G2 gene using the kit includes: pLentiCRISPRV2-E1, pLentiCRISPRV2-E2, pLentiCRISPRV2-E4 and pLentiCRISPRV2-E5;

[0025] The vector combination for knocking out the membrane-bound HLA-G3 gene using the kit includes: pLentiCRISPRV2-E1, pLentiCRISPRV2-E2 and pLentiCRISPRV2-E5;

[0026] The vector combination for knocking out the membrane-bound HLA-G4 gene using the kit includes: pLentiCRISPRV2-E1, pLentiCRISPRV2-E2, pLentiCRISPRV2-E3 and pLentiCRISPRV2-E5;

[0027] The vector combination for knocking out the soluble HLA-G5 gene using the kit includes: pLentiCRISPRV2-E1, pLentiCRISPRV2-E2, pLentiCRISPRV2-E3, pLentiCRISPRV2-E4 and pLentiCRISPRV2-I4;

[0028] The vector combination for knocking out the soluble HLA-G6 gene using the kit includes: pLentiCRISPRV2-E1, pLentiCRISPRV2-E2, pLentiCRISPRV2-E4 and pLentiCRISPRV2-I4;

[0029] The vector combination for knocking out the soluble HLA-G7 gene using the kit includes: pLentiCRISPRV2-E1, pLentiCRISPRV2-E2 and pLentiCRISPRV2-I2.

[0030] Preferably, the vector combination for knocking out the HLA-G gene lacking α1 using the kit includes: pLentiCRISPRV2-E3, pLentiCRISPRV2-E4 and pLentiCRISPRV2-E5;

[0031] The vector combination for knocking out the HLA-G gene lacking α1 and α2 by using the kit includes: pLentiCRISPRV2-E4 and pLentiCRISPRV2-E5.

[0032] The present invention also provides a CRISPR / Cas9 lentiviral kit for targeted knockout of HLA-G isoform genes, including a CRISPR / Cas9 lentiviral system for targeted knockout of different HLA-G isoform genes;

[0033] The preparation method of the CRISPR / Cas9 lentiviral system comprises packaging each CRISPR / Cas9 vector in the CRISPR / Cas9 vector combination using a lentiviral packaging plasmid, and transfecting human cells to obtain a CRISPR / Cas9 lentiviral system for targeted knockout of different HLA-G isoform genes.

[0034] The present invention also provides the use of the above-mentioned sgRNA combination, the above-mentioned CRISPR / Cas9 vector combination, the above-mentioned kit or the above-mentioned CRISPR / Cas9 lentiviral kit in the preparation of a drug with HLA-G as a therapeutic target.

[0035] Beneficial effects: The present invention designs a small guide RNA (sgRNA) based on the human HLA-G gene sequence, which can accurately target the 1st exon (SP), 2nd exon (α1), 3rd exon (α2), 4th exon (α3), 5th exon (TM), 2nd intron and 4th intron of the HLA-G gene.

[0036] The present invention forms double-stranded fragments by annealing target sequences and complementary sequences of various structures of the HLA-G gene, and inserts the double-stranded fragments into a gene editing vector to construct a CRISPR / Cas9 gene editing vector combination targeting the whole HLA-G gene, membrane-bound HLA-G1 to -G4 genes, soluble HLA-G5 to -G7 genes and a novel HLA-G gene.

[0037] The present invention combines the efficient transfection characteristics of lentiviral vectors and the efficient gene editing characteristics of CRISPR / Cas9 technology, and utilizes lentivirus to package the CRISPR / Cas9 gene editing vector combination separately, so as to achieve accurate knockout of different HLA-G isoform genes or complete knockout of HLA-G genes, including new isoforms. The present invention is of great significance for exploring the functions of different HLA-G isoform molecules in the future, promoting the research and development of HLA-G immunosuppressants and their evaluation in immunotherapy effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 A diagram of the sgRNA combination design scheme for precise knockout of HLA-G isoforms. A: The full-length HLA-G gene contains 8 exons and 7 introns. B: HLA-G transcripts contain 4 membrane-bound, 3 soluble, and 4 potential new isoforms. C: sgRNA combination scheme for targeted knockout of different HLA-G isoforms.

[0039] Figure 2 This is a result diagram of predicting the target sequence of HLA-G gene knockout based on the score and targeting efficiency;

[0040] Figure 3 This is a diagram showing the basic principle of using CRISPR / Cas9 to target and knock out specific HLA-G gene sequences;

[0041] Figure 4 is the plasmid map of the pLentiCRISPRV2 vector;

[0042] Figure 5 The sequencing result diagram shows that the sgRNA1 sequence was successfully inserted into the pLentiCRISPRV2 plasmid;

[0043] Figure 6Schematic diagram of determining editing effect by agarose electrophoresis after T7E1 digestion, where: M is DNA marker, lane 1 is the positive result of HLA-G gene knockout, and lane 2 is the negative result;

[0044] Figure 7 Schematic diagram of positive clones of JEG-3 cells selected by bleomycin;

[0045] Figure 8 This is a partial map of the genomic DNA sequencing of the JEG-3 stable cell line after infection with the virus solution;

[0046] Fig. 9 This is the result of detecting the expression of HLA-G mRNA at the cell transcription level by qPCR technology; JEG-3-KO-E1 indicates that only exon 1 is edited; JEG-3-KO-E1~E5 indicates that the HLA-G1 sgRNA combination scheme is used to edit exons 1~5; the red arrow indicates that the expression of HLA-G1 mRNA is not detected and its gene is completely knocked out;

[0047] Fig.10 This is a result diagram of the expression of total HLA-G protein at the cellular translation level detected by Western blot; in the figure, 1 represents JEG-3KO-control, 2 represents JEG-3KO-E1 (for exon 1), 3 represents JEG-3KO-E2, 4 represents JEG-3KO-E3, 5 represents JEG-3KO-E4, 6 represents JEG-3KO-E5, 7 represents JEG-3KO-I4, and 8 represents JEG-3KO-E1~E5; channel 8 shows that no HLA-G protein was detected, indicating that its gene has been completely knocked out, and the red arrow indicates the residual HLA-G isoform;

[0048] Fig.11 This is the result of flow cytometry detection of HLA-G protein expression on the cell membrane surface; the red arrow indicates that the expression of HLA-G on the cell membrane surface is significantly reduced, and its protein has been successfully knocked out;

[0049] Fig.12 The results of Western blot detection of the expression of HLA-G isoform proteins at the translation level in each JAR cell; Figure A: the expression results of each HLA-G isoform protein in JAR cells overexpressing HLA-G1 to -G7 respectively; Figure B: after the implementation of the sgRNA combination scheme for targeted knockout of different HLA-G isoforms of the present invention, different HLA-G isoform genes can be accurately knocked out. DETAILED DESCRIPTION

[0050] The present invention provides an sgRNA combination for knocking out an HLA-G isoform gene, comprising a target sequence and a complementary sequence designed for the gene structure of the HLA-G isoform gene respectively;

[0051] The gene structure includes exon 1 (E1), exon 2 (E2), exon 3 (E3), exon 4 (E4), exon 5 (E5), intron 4 (I4) and intron 2 (I2).

[0052] The HLA-G isoform gene of the present invention has Figure 1 In one embodiment, the sgRNA is designed using the website https: / / asia.ensembl.org / index.html, Human is selected, Gene ID: 3135 is entered, the transcript sequence of HLA-G is displayed, the exon of HLA-G is found, and the exon sequence of HLA-G is pasted into the text box of http: / / crispor.tefor.net / , and Submit is clicked for prediction. The result is as follows Figure 2 As shown. According to the prediction results, several sequences with high scores and targeting efficiencies were compared on the ensembl website. Based on the database optimization results, the optimal target sequences of 7 sgRNAs were finally determined, namely sgRNA1, sgRNA2, sgRNA3, sgRNA4, sgRNA5, sgRNA6 and sgRNA7 (5'-3'). In Table 1, F represents the target sequence and R represents the complementary sequence.

[0053] Table 1 sgRNA combination sequences of the present invention

[0054]

[0055] The present invention provides a CRISPR / Cas9 vector combination for targeted knockout of HLA-G isoform genes, including a CRISPR / Cas9 vector constructed by connecting a target sequence designed for each gene structure in the above-mentioned sgRNA combination and a double-stranded sequence formed after annealing of a complementary sequence with a gene editing vector.

[0056] The gene editing vector of the present invention needs to carry HIV elements and sticky ends of the BsmBI-v2 enzyme. In the embodiment, the pLentiCRISPRV2 vector is used as the vector backbone, and the efficient sgRNA and target site sequences shown in Table 1 that specifically target exon 1 (E1), exon 2 (E2), exon 3 (E3), exon 4 (E4), exon 5 (E5), intron 2 (I2), and intron 4 (I4) of HLA-G are respectively integrated. Specifically, the HLA-G gene is used as the editing target gene, and 7 CRISPR / Cas9 vectors for targeted knockout of the HLA-G gene are established: pLentiCRISPRV2-E1, pLentiCRISPRV2-E2, pLentiCRISPRV2-E3, pLentiCRISPRV2-E4, pLentiCRISPRV2-E5, pLentiCRISPRV2-I2, and pLentiCRISPRV2-I4.

[0057] The construction methods of the seven CRISPR / Cas9 vectors described in the present invention are the same. First, a pair of target sequences and complementary sequences are annealed at 95°C for 5 min and then gradually cooled to room temperature at 5°C / min for 1 h. The pLentiCRISPRV2 vector is linearized by BsmBI-v2 enzyme and then subjected to T4 digestion with the diluted double-stranded DNA according to the following system. DNA ligation reaction was performed to construct the above-mentioned CRISPR / Cas9 vector, wherein pLentiCRISPRV2-E1 was constructed by connecting the double-stranded sequence formed by annealing the target sequence designed for exon 1 and the complementary sequence to the pLentiCRISPRV2 vector; pLentiCRISPRV2-E2 was constructed by connecting the double-stranded sequence formed by annealing the target sequence designed for exon 2 and the complementary sequence to the pLentiCRISPRV2 vector; pLentiCRISPRV2-E3 was constructed by connecting the double-stranded sequence formed by annealing the target sequence designed for exon 3 and the complementary sequence to the pLentiCRISPRV2 vector; pLentiCRISPRV2-E4 was constructed by connecting the double-stranded sequence formed by annealing the target sequence designed for exon 3 and the complementary sequence to the pLentiCRISPRV2 vector; The target sequence designed for exon 4 and the complementary sequence were annealed to form a double-stranded sequence, which was then connected to the pLentiCRISPRV2 vector to obtain the construct; pLentiCRISPRV2-E5 was constructed by connecting the target sequence designed for exon 5 and the complementary sequence to form a double-stranded sequence, which was then connected to the pLentiCRISPRV2 vector to obtain the construct; pLentiCRISPRV2-I2 was constructed by connecting the target sequence designed for intron 2 and the complementary sequence to form a double-stranded sequence, which was then connected to the pLentiCRISPRV2 vector to obtain the construct; and pLentiCRISPRV2-I4 was constructed by connecting the target sequence designed for intron 4 and the complementary sequence to form a double-stranded sequence, which was then connected to the pLentiCRISPRV2 vector to obtain the construct.

[0058] The present invention provides a kit for knocking out HLA-G isoform genes, comprising the above-mentioned sgRNA combination, or the above-mentioned CRISPR / Cas9 vector combination.

[0059] The sgRNA combination or the above-mentioned CRISPR / Cas9 vector combination of the present invention can accurately target the HLA-G isoform gene, thereby achieving the knockout of the HLA-G isoform gene. In one embodiment of the present invention, when the entire HLA-G gene is knocked out, the CRISPR / Cas9 vector combination includes: pLentiCRISPRV2-E1, pLentiCRISPRV2-E2, pLentiCRISPRV2-E3, pLentiCRISPRV2-E4, pLentiCRISPRV2-E5, pLentiCRISPRV2-I2 and pLentiCRISPRV2-I4. In one embodiment of the present invention, when the membrane-bound HLA-G1 gene is knocked out, the vector combination includes: pLentiCRISPRV2-E1, pLentiCRISPRV2-E2, pLentiCRISPRV2-E3, pLentiCRISPRV2-E4 and pLentiCRISPRV2-E5. In one embodiment of the present invention, when the membrane-bound HLA-G2 gene is knocked out, the vector combination includes: pLentiCRISPRV2-E1, pLentiCRISPRV2-E2, pLentiCRISPRV2-E4 and pLentiCRISPRV2-E5. In one embodiment of the present invention, when the membrane-bound HLA-G3 gene is knocked out, the vector combination includes: pLentiCRISPRV2-E1, pLentiCRISPRV2-E2 and pLentiCRISPRV2-E5. In one embodiment of the present invention, when the membrane-bound HLA-G4 gene is knocked out, the vector combination includes: pLentiCRISPRV2-E1, pLentiCRISPRV2-E2, pLentiCRISPRV2-E3 and pLentiCRISPRV2-E5. In one embodiment of the present invention, when the soluble HLA-G5 gene is knocked out, the vector combination includes: pLentiCRISPRV2-E1, pLentiCRISPRV2-E2, pLentiCRISPRV2-E3, pLentiCRISPRV2-E4 and pLentiCRISPRV2-I4. In one embodiment of the present invention, when the soluble HLA-G6 gene is knocked out, the vector combination includes: pLentiCRISPRV2-E1, pLentiCRISPRV2-E2, pLentiCRISPRV2-E4 and pLentiCRISPRV2-I4. In one embodiment of the present invention, when the soluble HLA-G7 gene is knocked out, the vector combination includes: pLentiCRISPRV2-E1, pLentiCRISPRV2-E2 and pLentiCRISPRV2-I2.

[0060] The kit of the present invention can be used to knock out the novel HLA-G gene (Tronik-Le Roux D, Daouya M, Jacquier A, Schenowitz C, Desgrandchamps F, Rouas-Freiss N, Carosella ED. The HLA-G immune checkpoint: a new immuno-stimulatory role for the α1-domain-deleted isoform. Cell Mol Life Sci. 2022, 79 (6): 310.). For example, in one embodiment of the present invention, when the HLA-G (α1-deficient) gene is knocked out, the vector combination includes: pLentiCRISPRV2-E3, pLentiCRISPRV2-E4 and pLentiCRISPRV2-E5; when the HLA-G (α1-deficient and α2-deficient) gene is knocked out, the vector combination includes: pLentiCRISPRV2-E4, pLentiCRISPRV2-E5.

[0061] The present invention also provides a CRISPR / Cas9 lentiviral kit for targeted knockout of HLA-G isoform genes, including a CRISPR / Cas9 lentiviral system for targeted knockout of different HLA-G isoform genes;

[0062] The preparation method of the CRISPR / Cas9 lentiviral system comprises packaging each CRISPR / Cas9 vector in the CRISPR / Cas9 vector combination using a lentiviral packaging plasmid, and transfecting human cells to obtain a CRISPR / Cas9 lentiviral system for targeted knockout of different HLA-G isoform genes.

[0063] The present invention does not specifically limit the method for lentiviral packaging. For example, the lentiviral packaging plasmids selected in the embodiment are pLP / VSVG, pLP1 and pLP2, which are then mixed with the pLentiCRISPRV2 recombinant plasmid constructed as described above, and transfection is prepared according to the instructions of the Lip3000 transfection kit to obtain the CRISPR / Cas9 lentiviral system.

[0064] The present invention also provides the use of the above-mentioned sgRNA combination, the above-mentioned CRISPR / Cas9 vector combination, the above-mentioned kit or the above-mentioned CRISPR / Cas9 lentiviral kit in the preparation of a drug with HLA-G as a therapeutic target.

[0065] In the embodiment of the present invention, the above-mentioned sgRNA 1-7 combination and CRISPR / Cas9 lentiviral system can be used as follows Figure 3The complete knockout of the entire HLA-G gene shown in the figure will not leave any HLA-G isoforms, which improves the accuracy of the research data; at the same time, the different sgRNA combinations used can accurately target and knock out any HLA-G isoform gene, including membrane-bound HLA-G1~-G4 and soluble HLA-G5~-G7, providing research tools for future research on the functions of various HLA-G isoform molecules. The sgRNA combination design used in the present invention, in addition to knocking out 7 common HLA-G isoforms, is suitable for efficient and accurate knockout of HLA-G isoform genes in a variety of cells, thereby providing a powerful tool for further studying the functions of different HLA-G isoforms. In addition, the sgRNA involved in the present invention is expected to be used in the fields of tumor treatment, organ transplantation, reproduction, etc., and has a wide range of application prospects.

[0066] To further illustrate the present invention, the gene editing system and application for targeted knockout of HLA-G isoform gene provided by the present invention are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0067] Example 1 Construction of a plasmid for precise knockout of HLA-G isoform genes based on CRISPR / Cas9 technology

[0068] 1. sgRNA and primer design

[0069] The sgRNA oligonucleotide sequences shown in Table 1 were synthesized.

[0070] 2. Synthesize sgRNA oligonucleotide sequences

[0071] 2.1 According to the structure of pLentiCRISPRV2 vector (purchased from Hunan Fenghui Biotechnology Co., Ltd.), the BsmBI-v2 restriction site was determined. The results are as follows Figure 4 The above target sequence and complementary sequence were added to the sticky ends of the vector BsmBI-v2 enzyme and synthesized by BGI.

[0072] 3. Vector construction

[0073] 3.1 Dilute each target sequence and complementary sequence to 200 μmol / L, and prepare a 50 μL system: sgRNA-F 5 μL, sgRNA-R 5 μL, 10×T4 Ligase buffer 5 μL and the rest of water. The annealing program is 95℃ for 5 min, and then gradually cool down to room temperature for 1 h at 5℃ / min.

[0074] 3.2 The pLentiCRISPRV2 vector was linearized by BsmBI-v2 enzyme. Reaction conditions: 55°C, 15 min, and then inactivated at 80°C, 20 min.

[0075] 50μL enzyme digestion system: BsmBI-v2 1μL, pLentiCRISPRV2 1μg, 10×NEB buffer 3.15μL and the rest water.

[0076] 3.3 The linear vector was mixed with the diluted double-stranded DNA for T4 DNA ligation reaction. The reaction conditions were: 4°C overnight.

[0077] 10μL ligation system: 1μL T4 ligase buffer, 1μL T4 DNAligase, 30ng pLentiCRISPRV2 linearized vector, 1μL 10nM double-stranded product, and the balance water.

[0078] 4. Transformation of competent cells

[0079] 4.1 Mix the ligation product obtained in the previous step with 50 μL Stable3 competent cells by blowing gently and let it stand on ice for 30 minutes.

[0080] 4. Heat shock at 242℃ for 90s and place on ice again for 3min.

[0081] 4.3 Add 900 μL of liquid culture medium and culture at 37°C constant temperature shaker at 150 rpm for 45 min.

[0082] 4. Centrifuge at 3000 rpm for 3 min, discard most of the supernatant, blow gently to mix the precipitate, and then apply it to a 100 μg / mL Amp-resistant solid culture medium plate.

[0083] 4. Culture at a constant temperature of 537℃ for 12 to 16 hours.

[0084] 5. Plasmid extraction

[0085] 5.1 Pick several monoclonal colonies for colony PCR and run gel verification.

[0086] 5.2 Select the successfully verified colonies and incubate them at 37℃ for 12 to 16 hours.

[0087] 5.3 Use the endotoxin-free plasmid extraction kit to extract the plasmid.

[0088] 6. Sequencing verification

[0089] The plasmid was sent to BGI for sequencing. The results were as follows Figure 5 As shown, the sgRNA was correctly inserted into the pLentiCRISPRV2 vector. Figure 5This is a partial sequencing map of the pLentiCRISPRV2-E1 (sgRNA1) recombinant plasmid. Sequence alignment confirmed that sgRNA1 was successfully inserted into the pLentiCRISPRV2 vector. The sequencing primer is shown in SEQ ID No. 15: pLentiCRISPRV2-U6: 5'-ATGGACTATCATATGCTTACCGTA-3'.

[0090] Example 2 Example 1 Construction of recombinant plasmid gene editing effect

[0091] 1. HEK 293T cell culture

[0092] HEK293T cells were revived according to conventional methods, cultured in a 37°C, 5% CO2 incubator using high-glucose DMEM + 10% inactivated fetal bovine serum, grown adherently, and passaged every 2 to 3 days.

[0093] 2. Transient transfection of HEK 293T cells

[0094] 2.1 HEK293T cells were seeded into 6-well plates in a single-cell dispersed state and cultured in a 37°C, 5% CO2 incubator for 24 hours. Transfection could be performed after the HEK293T cell confluence reached 80% on the day of transfection.

[0095] 2.2 The pLentiCRISPRV2 recombinant plasmid constructed in Example 1 was transfected into HEK 293T cells using Lip3000 under conventional conditions and cultured at 37° C. and 5% CO 2 .

[0096] 3. T7E1 digestion to evaluate editing effect

[0097] 3.1 Extract genomic DNA from HEK 293T cells before and after transfection, and amplify the target fragment using specific primers designed for HLA-G and high-fidelity DNA polymerase.

[0098] HLA-G(-154)F (SEQ ID No. 16): 5'-GTGTCGGGTCCTTCTTCCTG-3';

[0099] HLA-G(+2351)R(SEQ ID No.17):5'-TGTCCTTAGCCAGGGACCTT-3';

[0100] 3.2 The target fragment was amplified by PCR according to the following reaction conditions: 95℃ for 5 min; 95-85℃, decreasing by 2℃ per second; 85-25℃, decreasing by 0.1℃ per second; stored at 4℃.

[0101] 19μL system: 5μL PCR product, 2μL 10×T7 Buffer and the balance water.

[0102] 3.3 Add 1 μL T7E1 enzyme to the above 19 μL annealed system and incubate at 37°C for 30 min; heat at 85°C for 15 min to terminate the enzyme cleavage reaction.

[0103] 3.4 The digested products were subjected to agarose gel electrophoresis. Figure 6 As shown, the T7E1 enzyme can recognize and cut the mismatch sites in the DNA double helix, indicating that the HLA-G gene has been successfully edited.

[0104] Example 3 Example 1 Construction of a lentiviral system for recombinant plasmids

[0105] 1. Lip3000 transfection method to package lentivirus

[0106] 1.1 One day before transfection (Day-1): HEK293T cells in good condition were seeded into a 10 cm cell culture dish and cultured in a 37°C, 5% CO2 incubator overnight.

[0107] 1.2 On the day of transfection (Day 0): HEK293T cells were growing well, with a cell density of 60% to 80%. Two hours before transfection, the HEK293T cells were cultured in DMEM medium containing 10% fetal bovine serum at room temperature.

[0108] 1.3 Prepare lentiviral packaging plasmids (pLP / VSVG, pLP1 and pLP2, purchased from Hunan Fenghui Biotechnology Co., Ltd.) and the constructed pLentiCRISPRV2 recombinant plasmid, and prepare the DNA-transfection reagent complex according to the instructions of the Lip3000 transfection kit.

[0109] EP tube A: 500 μL Opti-MEM+32 μL Lip3000, let stand for 5 min;

[0110] EP tube B: 500μL Opti-MEM+32μLP3000+8μg recombinant plasmid+4μgpLP / VSVG+4μg pLP1+4μgpLP2. Mix liquid A and liquid B, let stand for 15 minutes, and then add dropwise into a 10cm HEK293T cell culture dish.

[0111] 1.4 On the second day of transfection (Day 1), replace the HEK 293T cells with fresh culture medium that has been placed at room temperature.

[0112] 1.5pLentiCRISPRV2 vector contains GFP green fluorescent protein. The transfection efficiency was determined by observing the cell fluorescence under a fluorescence microscope. The cell supernatant (virus solution) was collected on Day 2 (48h) and Day 3 (72h).

[0113] 1.6 Virus concentration: Filter the collected virus solution with a 0.22μm filter, add 4℃ pre-cooled Biyuntian virus concentration reagent, and mix at low speed on a shaker at 4℃ for 12 to 16 hours.

[0114] Centrifuge at 1.74°C, 3500g for 1h, carefully remove the supernatant, resuspend the virus pellet with 1 / 10 the initial volume of virus resuspension solution containing virus protection reagent, and titer the resuspended virus solution or store it at -80°C.

[0115] 2. Virus titer determination

[0116] 2.1 The HEK293T cells in good condition were subcultured at a rate of 1.5×10 4 / well, spread on a 96-well plate, set 8 gradient wells for each type of lentivirus to be tested, and culture in a 37°C, 5% CO2 incubator overnight.

[0117] 2.2 Prepare 8 sterile EP tubes, add 180 μL of fresh complete culture medium to each EP tube, take 20 μL of the virus solution to be tested and add it to the first EP tube, mix well, take 20 μL of the virus dilution solution and add it to the second EP tube, and so on, until the dilution is reached to the last tube.

[0118] 2.3 Aspirate the original culture medium in the 96-well plate, add 100 μL of diluted virus solution to each well, mark it, and culture it in a 37°C, 5% CO2 incubator for 72 hours.

[0119] 2.4 Observe the number of fluorescent cells in each well under a fluorescence microscope or use a flow cytometer to detect the positive rate. The calculation formula is: virus titer (TU / mL) = (number of green fluorescent cells (cells) × virus solution dilution multiple) / virus solution volume (mL). The unit of virus titer is: TU / mL, which represents the number of virus particles with transduction function in each milliliter of virus solution. The virus volume required for the experiment (μL) = number of cells × MOI / virus titer. Calculate the 14μL of virus concentrate required for cells inoculated into a 24-well plate.

[0120] Example 4 Preparation and Identification of HLA-G Gene Knockout Human Choriocarcinoma Cell JEG-3

[0121] According to the sgRNA combination in Example 1, a gene editing vector combination was constructed to knock out the HLA-G gene: pLentiCRISPRV2-E1, pLentiCRISPRV2-E2, pLentiCRISPRV2-E3, pLentiCRISPRV2-E4, pLentiCRISPRV2-E5, pLentiCRISPRV2-I2 and pLentiCRISPRV2-I4.

[0122] 1. Infect JEG-3 cells (HLA-G positive expression strain, purchased from ATCC cell bank) with the virus solution packaged in Example 3

[0123] 1.1 Day 1: Subculture the HLA-G positive JEG-3 cells in good condition, spread an appropriate amount of cells on a 24-well plate, and culture them in a 37°C, 5% CO2 incubator overnight so that the cell density reaches 30% to 45% on the next day.

[0124] 1.2 Day 2 Aspirate the original culture medium in the 24-well plate, add an appropriate volume of virus concentrate and fresh cell culture medium to each well, place in a 37°C, 5% CO2 incubator for continued culture, and replace with fresh cell culture medium after 24 hours.

[0125] 1.3 Observe the cell fluorescence and density under a fluorescence microscope. After 48 hours of infection with the virus solution, add 50 μg / mL bleomycin for screening until the cells have positive clones and obtain stable cell lines. The results are as follows: Figure 7 As shown, a positively selected monoclonal cell line (containing GFP fluorescent protein) was obtained.

[0126] 2. Sequencing methods to identify editing effects

[0127] 2.1 DNA identification primers (SEQ ID No. 16 and SEQ ID No. 17) were designed based on the HLA-G genome sequence and synthesized by BGI.

[0128] 2.2 After the positive cell clones obtained above were expanded and cultured, their genomic DNA was extracted and PCR amplified using HLA-G DNA identification primers.

[0129] 50 μL PCR amplification system: 25 μL 2× high-fidelity enzyme, 1 μL DNA, 1.5 μL KO-F, 1.5 μL KO-R, and the balance water;

[0130] PCR amplification program: pre-denaturation at 95°C for 5 min; denaturation at 94°C for 1 min, annealing at 60°C for 1 min, extension at 72°C for 2 min 45 s, 35 cycles; and extension at 72°C for another 10 min.

[0131] 2.3 The PCR product was connected to the PUC118 plasmid, transformed, and the plasmid was extracted in small quantities and then PCR and electrophoresis were performed to confirm the fragment size. Then, it was sent to BGI for forward and reverse sequencing identification. The results were as follows Figure 8 As shown, the HLA-G gene fragment is missing, indicating that the sgRNA has a significant editing effect.

[0132] 3. Detection of HLA-G mRNA expression in stable cell lines using qPCR technology

[0133] 3.1 Extract total cellular RNA before and after virus infection, and reverse transcribe the RNA into cDNA.

[0134] 3.2 Design qPCR primers for HLA-G isoform transcripts and perform fluorescence quantitative PCR to detect the expression of HLA-G mRNA in cells. Fig. 9 As shown, HLA-G expression was significantly decreased.

[0135] qHLA-G F (SEQ ID No. 18): 5'-CCGAACCCTCTTCCTGCTGC-3';

[0136] qHLA-G R (SEQ ID No. 19): 5'-CACGGCGGCGTGAAATACC-3';

[0137] 4. Detection of HLA-G protein expression in stable cell lines by Western blot

[0138] 4.1 Extract total cell protein before and after virus infection and perform BCA protein quantification experiment.

[0139] 4.2 The expression of HLA-G total protein was detected by Western blot. The results are as follows Fig.10 As shown, HLA-G expression was significantly decreased.

[0140] 5. Flow cytometry was used to detect the expression of HLA-G protein in stable cell lines.

[0141] The expression of HLA-G on the cell membrane surface before and after virus infection was detected by flow cytometry. Fig.11 As shown, HLA-G expression was significantly decreased.

[0142] Example 5 Preparation and identification of cells with precise knockout of HLA-G isoform genes

[0143] sgRNA combination plan:

[0144] HLA-G1: pLentiCRISPRV2-E1, pLentiCRISPRV2-E2, pLentiCRISPRV2-E3, pLentiCRISPRV2-E4 and pLentiCRISPRV2-E5;

[0145] HLA-G2: pLentiCRISPRV2-E1, pLentiCRISPRV2-E2, pLentiCRISPRV2-E4 and pLentiCRISPRV2-E5;

[0146] HLA-G3: pLentiCRISPRV2-E1, pLentiCRISPRV2-E2 and pLentiCRISPRV2-E5;

[0147] HLA-G4: pLentiCRISPRV2-E1, pLentiCRISPRV2-E2, pLentiCRISPRV2-E3 and pLentiCRISPRV2-E5;

[0148] HLA-G5: pLentiCRISPRV2-E1, pLentiCRISPRV2-E2, pLentiCRISPRV2-E3, pLentiCRISPRV2-E4 and pLentiCRISPRV2-I4;

[0149] HLA-G6: pLentiCRISPRV2-E1, pLentiCRISPRV2-E2, pLentiCRISPRV2-E4 and pLentiCRISPRV2-I4;

[0150] HLA-G7: pLentiCRISPRV2-E1, pLentiCRISPRV2-E2 and pLentiCRISPRV2-I2;

[0151] 1. Example 3 The virus solution packaged with lentivirus was used to infect JAR cells (HLA-G negative expression strain, purchased from ATCC cell bank) that overexpress HLA-G1 to -G7 isoforms. The JAR cells that overexpress HLA-G1 to -G7 isoforms have been disclosed in the article (Xu Huihui. Cloning and expression of membrane-bound HLA-G1 to -G4 molecules and their effects on NK cell killing function. Chinese Journal of Microbiology and Immunology, 2010, (11): 982-986; Xu Huihui. Cloning and expression of soluble HLA-G5 to -G7 molecules and their effects on NK cell killing function. Modern Immunology, 2010, (6): 477-481.)

[0152] The experimental techniques and operating procedures used are basically the same as those in Example 4.

[0153] 2. Western blot was used to detect the expression level of HLA-G isoform protein in stable cell lines.

[0154] 2.1 Extract total cell protein before and after virus infection and perform BCA protein quantification experiment.

[0155] 2.2 The expression of HLA-G total protein was detected by Western blot. The results are as follows Fig.12 As shown, HLA-G isoforms were significantly decreased.

[0156] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A sgRNA combination for knocking out an HLA-G isoform gene, characterized in that: Including target sequences and complementary sequences designed for the gene structures of HLA-G isoform genes respectively; The gene structure includes exon 1, exon 2, exon 3, exon 4, exon 5, intron 4 and intron 2.

2. The sgRNA combination according to claim 1, characterized in that The nucleotide sequences of the target sequence and the complementary sequence are shown in SEQ ID No. 1 to SEQ ID No. 14, respectively.

3. A CRISPR / Cas9 vector combination for targeted knockout of HLA-G isoform genes, characterized in that: The invention comprises a CRISPR / Cas9 vector constructed by connecting a double-stranded sequence formed by annealing a target sequence designed for each gene structure in the sgRNA combination of claim 1 or 2 and a complementary sequence with a gene editing vector.

4. The CRISPR / Cas9 vector combination according to claim 3, characterized in that: The gene editing vector includes the pLentiCRISPRV2 gene recombination plasmid.

5. A kit for knocking out HLA-G isoform genes, characterized in that: Comprising the sgRNA combination of claim 1 or 2, or the CRISPR / Cas9 vector combination of claim 3 or 4.

6. The kit according to claim 5, characterized in that When the kit is used to knock out the entire HLA-G gene, the CRISPR / Cas9 vector combination includes: pLentiCRISPRV2-E1, pLentiCRISPRV2-E2, pLentiCRISPRV2-E3, pLentiCRISPRV2-E4, pLentiCRISPRV2-E5, pLentiCRISPRV2-I2 and pLentiCRISPRV2-I4; Among them, pLentiCRISPRV2-E1 is constructed by connecting the double-stranded sequence formed by annealing the target sequence designed for exon 1 and the complementary sequence to the pLentiCRISPRV2 vector; pLentiCRISPRV2-E2 was constructed by connecting the double-stranded sequence formed by annealing the target sequence designed for exon 2 and the complementary sequence to the pLentiCRISPRV2 vector; pLentiCRISPRV2-E3 was constructed by connecting the double-stranded sequence formed by annealing the target sequence designed for exon 3 and the complementary sequence to the pLentiCRISPRV2 vector; pLentiCRISPRV2-E4 was constructed by connecting the double-stranded sequence formed by annealing the target sequence designed for exon 4 and the complementary sequence to the pLentiCRISPRV2 vector; pLentiCRISPRV2-E5 was constructed by connecting the double-stranded sequence formed by annealing the target sequence designed for exon 5 and the complementary sequence to the pLentiCRISPRV2 vector; pLentiCRISPRV2-I2 was constructed by connecting the double-stranded sequence formed by annealing the target sequence designed for intron 2 and the complementary sequence to the pLentiCRISPRV2 vector; pLentiCRISPRV2-I4 was constructed by connecting the double-stranded sequence formed by annealing the target sequence designed for intron 4 and the complementary sequence to the pLentiCRISPRV2 vector.

7. The kit according to claim 5 or 6, characterized in that The vector combination for knocking out the membrane-bound HLA-G1 gene using the kit includes: pLentiCRISPRV2-E1, pLentiCRISPRV2-E2, pLentiCRISPRV2-E3, pLentiCRISPRV2-E4 and pLentiCRISPRV2-E5; The vector combination for knocking out the membrane-bound HLA-G2 gene using the kit includes: pLentiCRISPRV2-E1, pLentiCRISPRV2-E2, pLentiCRISPRV2-E4 and pLentiCRISPRV2-E5; The vector combination for knocking out the membrane-bound HLA-G3 gene using the kit includes: pLentiCRISPRV2-E1, pLentiCRISPRV2-E2 and pLentiCRISPRV2-E5; The vector combination for knocking out the membrane-bound HLA-G4 gene using the kit includes: pLentiCRISPRV2-E1, pLentiCRISPRV2-E2, pLentiCRISPRV2-E3 and pLentiCRISPRV2-E5; The vector combination for knocking out the soluble HLA-G5 gene using the kit includes: pLentiCRISPRV2-E1, pLentiCRISPRV2-E2, pLentiCRISPRV2-E3, pLentiCRISPRV2-E4 and pLentiCRISPRV2-I4; The vector combination for knocking out the soluble HLA-G6 gene using the kit includes: pLentiCRISPRV2-E1, pLentiCRISPRV2-E2, pLentiCRISPRV2-E4 and pLentiCRISPRV2-I4; The vector combination for knocking out the soluble HLA-G7 gene using the kit includes: pLentiCRISPRV2-E1, pLentiCRISPRV2-E2 and pLentiCRISPRV2-I2.

8. The kit according to claim 5 or 6, characterized in that: The vector combination for knocking out the HLA-G gene lacking α1 using the kit includes: pLentiCRISPRV2-E3, pLentiCRISPRV2-E4 and pLentiCRISPRV2-E5; The vector combination for knocking out the HLA-G gene lacking α1 and α2 by using the kit includes: pLentiCRISPRV2-E4 and pLentiCRISPRV2-E5.

9. A CRISPR / Cas9 lentiviral kit for targeted knockout of HLA-G isoform genes, characterized in that: Includes a CRISPR / Cas9 lentiviral system for targeted knockout of different HLA-G isoform genes; The method for preparing the CRISPR / Cas9 lentiviral system comprises packaging each CRISPR / Cas9 vector in the CRISPR / Cas9 vector combination of claim 3 or 4 using a lentiviral packaging plasmid, and transfecting human cells to obtain a CRISPR / Cas9 lentiviral system for targeted knockout of different HLA-G isoform genes.

10. Use of the sgRNA combination of claim 1 or 2, the CRISPR / Cas9 vector combination of claim 3 or 4, the kit of any one of claims 5 to 8, or the CRISPR / Cas9 lentiviral kit of claim 9 in the preparation of a drug with HLA-G as a therapeutic target.

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