CN117384952A - Construction method of Raji-Luc cell line with CD19 gene knocked out and cell line thereof - Google Patents
Construction method of Raji-Luc cell line with CD19 gene knocked out and cell line thereof Download PDFInfo
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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Abstract
The invention provides a construction method of a Raji-Luc cell line with a CD19 gene knocked out, which comprises the following steps: designing a plurality of sgRNA sequences targeting CD19, constructing a plurality of PB-CRISPR-CD19sgRNA plasmids by the plurality of sgRNA sequences, respectively electrically transfecting Raji-Luc cells by the plurality of PB-CRISPR-CD19sgRNA plasmids, detecting the CD19 knockout efficiency of the Raji-Luc cells after the electric transfection by a flow cytometer, determining the cell growth state by utilizing cell culture, screening out the optimal sgRNA sequences, electrically transfecting Raji-Luc cells in batches by the PB-CRISPR-CD19sgRNA plasmids constructed by the optimal sgRNA sequences, and screening out stable knocked-out monoclonal Raji-Luc cell strains. The Raji-Luc cell with the CD19 gene knocked out constructed by the invention can simulate the condition of low or even no expression of antigen after tumor recurrence, and the expression of luciferase gene enables subsequent researchers to observe and detect tumors for a long time in an in vivo environment.
Description
Technical Field
The invention relates to the technical field of cells, in particular to a construction method of a Raji-Luc cell line for CD19 gene knockout and a cell line thereof.
Background
Lymphomas are one of the most common hematological neoplasms, and are largely classified into non-hodgkin lymphoma (NHL) and Hodgkin Lymphoma (HL). Wherein, the non-Hodgkin's lymphoma accounts for 90% of all lymphomas, the incidence rate of B cell non-Hodgkin's lymphoma is three times that of T cell lymphomas, and the B cell non-Hodgkin's lymphoma can be arranged in the first 10 in common malignant tumors in China.
CD19 is a surface protein expressed on all B cell lines except plasma cells, malignant B cells and follicular dendritic cells (follicular dendritic cells, FDCs), a member of the immunoglobulin (Ig) superfamily, located on chromosome 16 short arm (16p11.2). CD19 is expressed throughout the maturation and eventual differentiation of B cells into plasma cells. It is an important membrane antigen involved in B cell proliferation, differentiation, activation and antibody production, and can promote BCR signaling. There are studies showing that CD19 expression is normal to high levels in cancer cells in most B cell malignancy patients. Thus, CD19 is one of the most important target antigens in B cell malignancies, the most promising target for developing chimeric antigen receptor CAR-T cells against non-hodgkin's lymphoma and B cell leukemia.
Chimeric antigen receptor gene modified T (CAR-T) cell therapies are designed to modify T cells to express a fusion protein comprising an antigen recognition domain, a co-stimulatory domain and a T cell activating domain, and the modified CAR-T cells can specifically recognize and eliminate tumor cells expressing a target antigen, which is known as a hope for future tumor therapy. In the current research of CAR-T related targets, the research on CD19 is also most perfect and mature. There are six CAR-T cell products approved for sale by the united states Food and Drug Administration (FDA), four of which target CD19. However, about 60% of cancer cells in patients who relapse after treatment show reduced or complete loss of CD19 expression. Clinical studies indicate that one patient with B-cell acute lymphoblastic leukemia (B-cell Acute Lymphoblastic Leukemia, B-ALL) had complete remission after 19 days of anti-CD 19CAR-T cell infusion, but had recurrent and eventually died at 261 days after treatment. Researchers found that all leukemic cells following relapse in this patient abnormally expressed anti-CD 19CAR and no expression of CD19 could be detected. Thus, although CAR-T cells are significantly effective in treating B-cell malignancies, tumor recurrence is often seen after treatment, and the primary cause of tumor recurrence is related to antigen escape.
Burkitt Lymphoma (BL) is an aggressive B-cell lymphoma, which is a non-hodgkin lymphoma. In 1963, pulveraft RJV isolated Raji cells from Burkitt lymphoma of the left maxilla of an 11 year old black boy established serial passage cells of the first human hematopoietic system. CD19 and CD38 of Raji cells are expressed in double positive and are often used as target cells for non-Hodgkin's lymphoma related studies. The Raji-Luc CD19KO cell is constructed by a PB transposon system and a CRISPR cas9 technology, and a foundation is laid for the follow-up exploration of how to solve the problem of tumor recurrence caused by antigen immune escape of a CAR-T cell therapy.
Disclosure of Invention
In order to solve the technical problems, the invention provides a construction method of a Raji-Luc cell line with CD19 gene knockout, which comprises the following steps:
step 1: designing a plurality of sgRNA sequences targeting CD 19;
step 2: constructing a plurality of PB-CRISPR-CD19sgRNA plasmids by using the plurality of sgRNA sequences in the step 1;
step 3: electrotransfecting Raji-Luc cells with the plurality of PB-CRISPR-CD19sgRNA plasmids of the step 2 respectively;
step 4: detecting the CD19 knockout efficiency of the Raji-Luc cells obtained in the step 3 after electrotransfection by using a flow cytometer, determining the growth state of the cells by using cell culture, and screening out the optimal sgRNA sequence;
step 5: the PB-CRISPR-CD19sgRNA plasmid constructed by the optimal sgRNA sequence is used for carrying out batch electrotransfection on Raji-Luc cells;
step 6: screening the stable knocked-out monoclonal Raji-Luc cell strain.
In one embodiment, the optimal sgRNA sequence is SEQ ID No.1: GCTAGGTCCGAAACATTCCAC.
In one embodiment, in step 6, the electrotransformed Raji-Luc CD19 cells are taken, resuspended in staining buffer, and stained in the dark with a flow antibody; after the dyeing is finished, detecting the transfection efficiency of the cells by using a flow cytometer, carrying out flow sorting, and screening the stably knocked-out monoclonal Raji-Luc cell strain by using a cell limit dilution method after sorting.
In one embodiment, a Raji-Luc cell line with CD19 gene knockout obtained by constructing the above method is provided.
In one embodiment, there is provided a sgRNA specifically targeting the CD19 gene, the targeting sequence of which is SEQ ID No.1: GCTAGGTCCGAAACATTCCAC.
In one embodiment, there is provided a CRISPR-specific targeting CD19 gene-based sgRNA targeting sequence of SEQ ID No.1: GCTAGGTCCGAAACATTCCAC.
In one embodiment, there is provided the use of the above sgrnas for the knockout of the CD19 gene or for the preparation of a CD19 gene knockout cell line.
In one embodiment, a kit for knocking out an sgRNA gene is provided, which is characterized in that the kit comprises the sgRNA described above, or a targeting vector for targeting the knocked-out sgRNA gene; the targeting vector for targeted knocking out of the sgRNA gene comprises the coding sequences of the sgRNA and CRISPR protein genes.
In one embodiment, a gene knockout vector is provided comprising the sgRNA sequence described above.
In recent years, anti-CD 19CAR-T cells have achieved rapid and long lasting significant efficacy in the treatment of relapsed or refractory acute B-lymphocyte leukemia and non-hodgkin's lymphoma. However, during clinical treatment of CAR-T cells, a portion of patients develop a phenomenon that initially produces a high response rate, but fails to treat in subsequent treatments. In 2022, 6 patients received the first treatment of allogeneic CAR-T cell therapy with PD-1 knockout CB-010 in the clinical study of the calibou company (NCT 04637763), with 3 patients showing relapse within 6 months after treatment. The study shows that the main reason for the failure of the treatment is due to the partial or complete loss of target antigen expression by tumor cells, i.e. the phenomenon of antigen escape. This phenomenon has forced us to explore how CAR-T cells can still kill tumor cell antigens after they are lost or find more suitable alternative targets.
The invention constructs PB-CRISPR-CD19sgRNA plasmid by design, and constructs the CD19 knocked-out Raji-Luc cell by adopting an electrotransfection method. And obtaining the stable knocked-out monoclonal cell strain through flow separation and screening. And a luciferase chemiluminescence method is adopted to verify that the luciferase expression of two groups of monoclonal cells of Raji-Luc CD19KO and Raji-Luc cells has no obvious difference, and CD19CAR-T cells cannot be activated to kill the cells. The Raji-Luc CD19KO cell constructed by the invention can simulate the condition of low or even no expression of antigen after tumor recurrence, and the expression of luciferase gene enables subsequent researchers to observe and detect tumors for a long time in an in vivo environment. The method provides a cell model for subsequently improving the antigen escape phenomenon in the treatment process of the CAR-T cells and treating the Hodgkin lymphoma, and provides assistance for further improving the treatment capacity of the CAR-T, improving the cure rate of lymphoma patients and reducing the tumor recurrence rate.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a flow chart of an experiment of the present invention;
FIG. 2 is a schematic representation of the PB-CRISPR-CD19sgRNA construction of the present invention;
FIG. 3 is a graph of the result of electrotransfection of different sgRNAs of the present invention;
FIG. 4 is a graph showing the results of viability and fold cell proliferation of different sgRNA electrotransfected cells of the present invention;
FIG. 5 is a graph showing the results of flow assay for Raji-Luc CD19KO monoclonal cell surface protein expression;
FIG. 6 is a graph showing the results of luciferase assay from Raji-Luc CD19KO cell line;
FIG. 7 is a graph of the results of Raji-Luc CD19 KO2 and 20 monoclonal sequencing;
FIG. 8 is a graph of transduction efficiency results of flow assays for CD19CAR-T and CD38 CAR-T, FIG. 8a is a schematic of CD19CAR and CD38 CAR structures, and FIG. 8b is a graph of transduction efficiency results for CD19CAR-T and CD38 CAR-T;
FIG. 9 is a graph showing the results of detection of killing efficiency of CAR-T cells on Raji-Luc and Raji-Luc CD19KO cells, wherein FIG. 9a is a graph showing the results of Raji-Luc cells, and FIG. 9b is a graph showing the results of Raji-Luc CD19KO cells.
Detailed Description
In order that those skilled in the art will better understand the technical solutions of the present application, the present invention will be further described with reference to examples, and it is apparent that the described examples are only some of the examples of the present application, not all the examples. All other embodiments, which can be made by one of ordinary skill in the art based on the embodiments herein without making any inventive effort, shall fall within the scope of the present application.
Experimental material
Raji-Luc cells were purchased from Beijing Vietnam Biotechnology Co., ltd, and the original plasmids pCAG-PBase, PB-CRISPR-cas9 and PB-CRISPR-sgnna used for electrotransformation were all commercial plasmids.
RPMI1640 medium, AIM-V medium, PBS solution, penicillin-streptomycin solution, restriction enzymes, T4 ligase, agar, yeast extract, tryptone, sodium chloride, ampicillin, DH5 a competent cells, 50 x TAE solution, DNA marker DNA ladder, 10 x DNA Loading Buffer and agarose were all purchased from beijing orchid bordetella commercial limited. The gel recovery kit, the plasmid miniprep kit, the plasmid macropprep kit and the DNA purification kit were all purchased from Nanjinouzan biotechnology Co.Ltd. FBS fetal bovine serum was purchased from beijing bernovo biotechnology limited. 7-AAD antibodies, mouse anti-human MYC antibodies, APC mouse anti-human CD19 antibodies, FITC mouse anti-human CD38 antibodies were purchased from Shenzhen Daidae Biotechnology Co., ltd.
Second, experimental method
A specific experimental flow chart of the present invention is shown in figure 1.
1. Construction of PB-CRISPR-CD19sgRNA plasmid
sgRNA (small guide RNA) is a guide RNA (gRNA) that directs insertion or deletion of uridine residues into the plastids (kinetoplastid) during RNA editing, a small non-coding RNA that can be paired with pre-mRNA. Off-target and on-target were designed and verified at the IDT website, and appropriate sgRNA sequences were selected for primer synthesis by the qinghaos. According to the invention, various factors are comprehensively considered from a plurality of sgRNA sequences, and 4 sgRNA sequences are selected for synthesis. Annealing the synthesized primer fragments to form required target fragments, carrying out PCR amplification, purifying and recovering amplified products, connecting the amplified products with a linearization vector CRISPR-sgRNA-vector by adopting T4 at a constant temperature of 16 ℃ for 12 hours, converting the amplified products into competent escherichia coli, and carrying out amplification culture, extracting plasmids and concentrating, wherein the amplified products are shown in figure 2.
Table targets the sgRNA sequence of CD19
Culture of Raji-Luc cells
Resuscitates Raji-Luc cells at 37deg.C, 5% CO 2 Sterile culturing in cell incubator, and passaging with RPMI medium containing 10% FBS every 48 hrInoculation density of 5X 10 5 And (3) culturing to logarithmic growth phase for subsequent experiments.
3. Screening for optimal sgRNA sequences
Raji-Luc cells were divided into 4 groups of 1X 10 each 6 Each of the plasmids designated as sgRNA1, sgRNA2, sgRNA3 and sgRNA4, was centrifuged for 5 minutes at 300g, the supernatant was discarded, and the supernatant was resuspended in serum-free RPMI medium, 4. Mu.g each of the pCAG-PBase transposase, PB-CRISPR-cas9 and PB-CD19 sgRNA plasmids was added to each group, and the total was 12. Mu.g, and the mixture was transferred to an electric rotating cup after mixing. The K562 mode was selected for electrotransfection using a Berle electrotransport. After the electric shock is finished, the cells are placed in an incubator for 30min, and then double serum is slowly added dropwise. Subculturing with RPMI1640 medium containing 10% FBS every 48 hr to give inoculation density of 5×10 5 Per ml, and survival and proliferation were recorded. After electrotransfection for 48 hours, 4 groups of cells after electrotransformation and original Raji-Luc cells without electrotransformation are taken, 400g are centrifuged for 5min, the supernatant is removed, the cells are washed by a proper amount of PBS, the supernatant is centrifuged, the supernatant is removed, the cells are resuspended by adding a staining buffer solution respectively, and the cells are stained for 1h in a dark place by using a APC anti human CD flow antibody. After staining, cells were washed with PBS, centrifuged, resuspended in PBS and the CD19 knockout efficiency of cells was measured by flow cytometry. The electrotransfection results for the different sgRNA sequences are shown in fig. 3, and the cell viability and proliferation are shown in fig. 4.
4. Preparation of Raji-Luc CD19KO cells by electrotransfection of PB-CRISPR-CD19 sgRNA1 plasmid and screening of stable knockout monoclonal cell lines
Taking 1×10 7 The Raji-Luc cells were centrifuged at 300g for 5 minutes, the supernatant was discarded, and the suspension was resuspended in serum-free RPMI medium containing the sgRNA1 electrotransfer plasmid and transferred to an electrotransfer cup for shock manipulation. After the electric shock is finished, placing the cells in an incubator for 30min, slowly dripping double serum, and placing at 37 ℃ and 5% CO 2 The cells were cultured aseptically in a cell incubator. After electrotransfection for 48 hours, appropriate amounts of the electrotransformed Raji-Luc CD19KO cells and the non-electrotransformed original Raji-Luc cells were taken, centrifuged for 5min at 400g, the supernatant was removed, the cells were washed with an appropriate amount of PBS, centrifuged, the supernatant was removed, the cells were resuspended in 2% FBS-PBS staining buffer, and FITC anti human CD, APC anti human CD19 and 7-AAD flow-type anti-tumor were added thereto, respectivelyThe body was stained in dark for 1h. Washing cells with PBS after staining, centrifuging, re-suspending with PBS, detecting transfection efficiency of cells with flow cytometry, and collecting 7-AAD - CD38 + CD19 - The target cells were subjected to flow sorting, the sorted cells were diluted to a limit of 2.5 cells/ml, and 200. Mu.l of the selected monoclonal cells were added to each well of a 96-well plate. The monoclonal cell strain in the 96-well plate is subjected to expansion culture, a proper amount of monoclonal cells are again dyed by antibodies FITC anti human CD and APC anti human CD, untransfected Raji-Luc cells are used as positive control, and the expression conditions of CD19 and CD38 on the surface of Raji-Luc CD19KO cells are compared and detected to determine whether the screened cells successfully knock out the surface CD19 antigen.
5. Detection of cell line surface luciferase expression
Taking original Raji-Luc cell, raji-Luc CD19KO cell No. 2 monoclonal, and Raji-Luc CD19KO cell No. 20 monoclonal each 1×10 5 Sequentially and gradually diluting to 1×10 4 ，1×10 3 ，1×10 2 10/100. Mu.L of the cells were inoculated into 96 Kong Baiban, and 100. Mu.L of Bright-Lumi was added to each well TM The firefly luciferase detection reagent was reacted for 5min, and then a chemiluminescent mode was selected to detect the relative luminescence (relative light unit, RLU) of each well cell of each group.
Raji-Luc CD19KO cell genome sequencing
The original Raji-Luc cell, raji-Luc CD19KO cell No. 2 and No. 20 monoclonal extract genome are respectively taken, PCR amplification is carried out, the product is sequenced, and the sequencing result is compared with the original gene.
7. Preparation of CD19CAR-T and CD38 CAR-T cells
Healthy volunteers harvest 10ml of venous blood, extract PBMC cells, add IL-2 and OK-T3 activated T cells, culture for 48 hours, divide into CD19CAR-T group and CD38 CAR-T group, respectively transduce with retrovirus prepared in the earlier stage of the laboratory, the CAR structure is classical second generation CAR structure and carries detected MYC tag, and obtain CD19CAR-T and CD38 CAR-T cells. After 48h transduction, appropriate amounts of CD19CAR-T and CD38 CAR-T cells were taken, respectively, and after staining with PE-MYC antibody, the flow was fineThe cytometer detects the transduction efficiency. CAR-T cells after transduction were placed at 37℃in 5% CO 2 Sterile culturing in cell incubator, and passaging once every 48h with AIM-V complete medium containing 100U/mL IL-2 at an inoculation density of 1×10 6 And each ml.
8. Luciferase chemiluminescence method for verifying killing effect of CAR-T on Raji-Luc and Raji-Luc CD19KO cells
Taking Raji-Luc and Raji-Luc CD19KO cells in logarithmic growth phase at 4×10 4 A density of 50. Mu.L per well was seeded in 96-well whites. Each divided into three experimental groups: target cell+Pan-T cell group, target cell+CD1CAR-T cell group, target cell+CD38CAR-T cell group, each group is set with an effective target ratio of 1:1, 1: 2. 1: 4. 1:8, three complex holes each. Taking proper amounts of CAR-T and Pan-T cells, processing to proper density, adding 50 μl of CAR-T cells or Pan-T cells into each well of the 96-well plate, placing at 37deg.C, 5% CO 2 Co-culturing in a cell culture box. After 8h incubation, 100. Mu.L Bright-Lumi was added to each well TM After reacting for 5min, the firefly luciferase detection reagent selects a chemiluminescence mode, detects relative luminescence luminosity (relative light unit, RLU) of each group of cells, calculates killing efficiency, and calculates the formula: killing efficiency = 1- (experimental well-blank well)/(maximum lysis well-blank space) ×100%.
9. Statistical method
Experimental data were analyzed using GraphPad Prism 8 statistical software. Metering data toThe comparison between groups is shown by t-test. P (P)<A difference of 0.05 is statistically significant, P<0.05，**P<0.01。
Third, experimental results
1. Construction of PB-CRISPR-CD19sgRNA plasmid
Sequencing results show that the sequence of the 4 constructed PB-CRISPR-CD19sgRNA plasmids is consistent with the expected sequence, and PB-CRISPR-CD19 sgRNA1, PB-CRISPR-CD19 sgRNA2, PB-CRISPR-CD19 sgRNA3 and PB-CRISPR-CD19 sgRNA4 plasmids are successfully constructed.
2. Transfection effects of different sgRNA sequences
After 48 hours of electrotransfection, the expression rate of CD19 was lower for four groups of cells, sgRNA1, sgRNA2, sgRNA3 and sgRNA4, than for the original Raji-Luc, 41.04%, 34.19%, 29.17% and 32.22%, respectively, see FIG. 3, thus demonstrating a stronger sequence targeting of sgRNA 1. In the subsequent culture, the cell status of the sgRNA1 group recovered best, the proliferation rate was also fastest, and the cells of the sgRNA4 group had basically died by day 8, while the survival rate of the sgRNA2.3 group exceeded seventy percent, the proliferation fold factor was lower than that of the sgRNA1 group, 5.18 and 6.23, respectively, and the proliferation fold factor of the sgRNA1 group was 13.26, see fig. 4. It was thus seen that the cell status was optimal after electrotransfection of the sgRNA group cells CD19, whereby the sgRNA1 was selected as plasmid sequence for the subsequent construction of Raji-Luc CD19KO cells.
3. Obtaining stably expressed monoclonal cells
The method comprises the steps of obtaining No. 2 monoclonal and No. 20 monoclonal Raji-Luc CD19KO cells through flow cytometry sorting and limiting dilution, and the results show that the electric transduction efficiency of the two groups of cells obtained after sorting reaches more than 99% (namely CD19 negative and CD38 positive cells), so that the Raji-Luc CD19KO cells are successfully prepared.
No significant difference in luciferase expression between Raji-Luc CD19KO cells and original Raji-Luc cells
As shown by the detection result of the enzyme labeling instrument (see FIG. 6), the luciferase expression of the No. 2 monoclonal Raji-Luc CD19KO cell and the No. 20 monoclonal Raji-Luc CD19KO cell is not significantly different from that of the original Raji-Luc cell, so that the subsequent experiment can be continued.
Raji-Luc CD19KO cells were mutated at the corresponding gene locus
Sequencing results showed that the Raji-Luc CD19 KO2 monoclonal had a large fragment of base loss at the CD19 gene, while the 20 monoclonal had a mutation of one base, see FIG. 7.
6. Preparation of CD19CAR-T and CD38 CAR-T cells
As shown in fig. 8, after 48 hours of transduction, significant cell clustering occurred in both CD19CAR-T and CD38 CAR-T groups (see fig. 8 a), transduction efficiencies of 72.4% and 72.9%, respectively, indicating successful expression of CD19CAR and CD38 CAR molecules on the T lymphocyte surface, successful preparation of CD19CAR-T and CD38 CAR-T cells (see fig. 8 b).
Raji-Luc CD19KO cells were unable to activate the corresponding CAR-T cells for killing
Taking Raji-Luc cells and the Raji-Luc CD19KO cells obtained by construction as target cells respectively, taking CD19CAR-T and CD38 CAR-T cells successfully constructed before a laboratory as effector cells, and determining the killing efficiency experimental results of luciferase as shown in FIG. 9, wherein when the target cells are Raji-Luc cells, the killing capacity of the CD19CAR-T and the CD38 CAR-T cells is equivalent and obviously stronger than that of Pan-T cells (FIG. 9 a); when the target cells were Raji-Luc CD19KO cells, the killing capacity of CD19CAR-T cells was comparable to Pan-T cells, and the killing capacity of CD38 CAR-T cells was significantly greater than Pan-T cells and CD19CAR-T cells (fig. 9 b). This result indicates that the CD19 knockout Raji-Luc cell line is unable to activate the corresponding CAR-T cells for killing.
It is to be understood that this invention is not limited to the particular methodology, protocols, and materials described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are also encompassed by the appended claims.
Claims (9)
- A method for constructing a Raji-Luc cell line with a cd19 gene knocked out, comprising the steps of:step 1: designing a plurality of sgRNA sequences targeting CD 19;step 2: constructing a plurality of PB-CRISPR-CD19sgRNA plasmids by using the plurality of sgRNA sequences in the step 1;step 3: electrotransfection of Raji-Luc cells with the multiple PB-CRISPR-CD19sgRNA plasmids of step 2;step 4: detecting the CD19 knockout efficiency of the Raji-Luc cells obtained in the step 3 after electrotransfection by using a flow cytometer, determining the growth state of the cells by using cell culture, and screening out the optimal sgRNA sequence;step 5: the PB-CRISPR-CD19sgRNA plasmid constructed by the optimal sgRNA sequence is used for carrying out batch electrotransfection on Raji-Luc cells;step 6: screening the stable knocked-out monoclonal Raji-Luc cell strain.
- 2. The method of claim 1, wherein the optimal sgRNA sequence is SEQ id No.1: GCTAGGTCCGAAACATTCCAC.
- 3. The method according to claim 1, wherein in step 6, the electroporated Raji-Luc CD19 cells are resuspended in staining buffer and stained in the dark with a flow antibody; after the dyeing is finished, detecting the transfection efficiency of the cells by using a flow cytometer, carrying out flow sorting, and screening the stably knocked-out monoclonal Raji-Luc cell strain by using a cell limit dilution method after sorting.
- 4. A Raji-Luc cell line with CD19 gene knockout obtained by constructing according to the method of any one of claims 1-3.
- 5. An sgRNA specifically targeting a CD19 gene, wherein the targeting sequence of the sgRNA is SEQ ID No.1: GCTAGGTCCGAAACATTCCAC.
- 6. The CRISPR-specific targeting CD19 gene-based sgRNA is characterized in that the targeting sequence of the sgRNA is SEQ ID No.1: GCTAGGTCCGAAACATTCCAC.
- 7. Use of the sgRNA of claim 5 for knocking out the CD19 gene or for preparing a CD19 gene knockout cell line.
- 8. A kit for knocking out an sgRNA gene, comprising the sgRNA of claim 5, or a targeting vector for targeting the knocking out of the sgRNA gene; the targeting vector for targeted knocking out of the sgRNA gene comprises the coding sequences of the sgRNA and CRISPR protein genes as claimed in claim 5.
- 9. A knockout vector comprising the sgRNA sequence of claim 5.
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