CN116790676B - Drug screening method based on doxycycline-induced mediated CRISPR gene knock-in technology - Google Patents
Drug screening method based on doxycycline-induced mediated CRISPR gene knock-in technology Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention discloses a drug screening method based on a doxycycline-induced mediated CRISPR gene knock-in technology, which constructs a doxycycline-induced mediated CRISPR gene knock-in cell model, edits on a genome level, effectively avoids the conditions of possible cell mutation and the like caused by long-term introduction of exogenous genes by adding a doxycycline-induced mediated mode, and increases the stability of a drug screening system; the GFP fluorescent protein detection system is introduced, so that the effectiveness of the gene knock-in model is indicated, the potential off-target effect of the gene editing technology is overcome, the screening of target drugs is facilitated, and the research and development of new drugs are further assisted.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a drug screening method based on a doxycycline-induced mediated CRISPR gene knock-in technology.
Background
Drug discovery programs initiate from the fact that existing drugs have no way to meet new disease needs when a disease or clinical condition occurs, and it is this unmet clinical need that is the underlying driving force for the project. The development of new drugs is from no to no, and the drug is subjected to drug discovery, preclinical research and clinical trial "tribal culture" and finally used for treating diseases. However, in traditional pharmaceutical development, the discovery and testing of new drug candidates typically takes more than ten years, and the total cost associated with the drug discovery process may exceed $ 10 billion. In addition, only a few candidate drugs can be marketed really, and the probability of a new drug being marketed really is only five thousandths. The high cost and lengthy effort of new drug development has also made drug development an adventure for pharmaceutical companies, thereby impeding new drug development progress.
With the continuous development of biotechnology, CRISPR-mediated gene editing has become a powerful tool for new drug development in recent years, which has become a key for releasing potential drug targets, and has profound effects on modern drug discovery and development. New drug development mediated by CRISPR has provided powerful support for diagnosis and treatment of many diseases including genetic diseases and pathogen infection. The systematic knockout of large numbers of candidate genes by CRISPR high-throughput features not only enables the simultaneous use of HTS to examine millions of compounds to accelerate new drug development, but also helps to reveal new drug targets and disease correlations. High throughput compound screening methods include biochemical screening and cell-based screening. Biochemical screening involves primarily assessing the interaction and binding affinity between a compound and a target, whereas cell-based screening exploits knowledge of the functional relationship of such interactions. In recent years, CRISPR improved cell-based screening, enabling rapid, economical model generation by constructing cell lines (immortalized cell lines, primary cells and stem cells) and organoids with disease-specific genetic background by accurate reproduction of precise genome editing of the disease, enabling researchers to perform high throughput screening of compounds to determine the most effective therapeutic drug molecules. The mode can thoroughly change the drug design, save time, reduce cost and accelerate the drug discovery process.
However, CRISPR-mediated gene editing has potential off-target effects, which can affect subsequent screening and research immeasurably, and problems such as change of cell metabolism after gene editing can restrict drug development progress. In addition, since some diseases are caused by gene deletion, and long-term overexpression of the deleted genes can pose a threat to cell and animal survival, designing a high-throughput screening system is of great importance to drug development.
Based on the current situation of a drug screening system, the field needs to research a method capable of inducing a gene editing model to express a specific target point so as to be beneficial to drug screening, and not only can the specific induction expression of a deletion gene in target cells be realized, but also the effectiveness of the model can be indicated, so that the research, development and screening of target point drugs are facilitated.
Disclosure of Invention
In order to solve the technical problems, the invention provides a drug screening method based on a doxycycline induction-mediated CRISPR gene knock-in technology, which comprises the following steps:
step 1, constructing a pLVX-EF1a-IRES-Puro-rtTA3 plasmid, wherein the sequence of the pLVX-EF1a-IRES-Puro-rtTA3 plasmid is shown as SEQ ID NO. 1, and a three-plasmid system is formed by a psPAX2 plasmid, a pMD2.G plasmid and the pLVX-EF1a-IRES-Puro-rtTA3 plasmid for slow virus packaging;
step 2, uniformly mixing the three-plasmid system obtained in the step 1 with the jetPRIME transfection reagent buffer solution, adding the jetPRIME transfection reagent after vortex oscillation, and standing the mixture to obtain a reagent serving as the transfection reagent for standby;
step 3, adding 293FT cells into a 10cm non-resistance culture dish after pretreatment, adding the transfection reagent obtained in the step 2 into the non-resistance culture dish after overnight culture, carrying out transfection, screening 293FT cells by using puromycin after the transfection is finished, and paving the cells after puromycin screening into a 96-well plate for culture by using a continuous gradient dilution method;
step 4, extracting genome DNA (deoxyribonucleic acid) for PCR (polymerase chain reaction) sequencing after the cells obtained in the step 3 form monoclonal amplification, and detecting the expression condition of the target gene rtTA3 to obtain a monoclonal cell line for stably expressing the target gene rtTA 3;
the PCR sequencing was completed using a prepGEM Universal gene extraction kit of MicroGEM, 10 μl of extraction reagent was used per 20000 cells to extract genomic DNA, and PrimeSTAR HS DNA Polymerase was used for the PCR enzyme;
step 5, constructing a Lenti-V2-GAPDH-sgRNA plasmid and a BPK1520-Donor-GAPDH-eGFP-IKZF2 plasmid, wherein the sequence of the Lenti-V2-GAPDH-sgRNA plasmid is shown as SEQ ID NO:2, the sequence of the BPK1520-Donor-GAPDH-eGFP-IKZF2 plasmid is shown in SEQ ID NO:3 is shown in the figure;
step 6, cotransfecting the Lenti-V2-GAPDH-sgRNA plasmid and the BPK1520-Donor-GAPDH-eGFP-IKZF2 plasmid by using the stably expressed rtTA3 monoclonal cell line obtained in the step 4, and after cotransfecting, paving the cells on a 96-well plate and culturing by using a continuous gradient dilution method, wherein the cells are digested and passaged and diluted to have 50 cells in a density of 10 milliliters, and 100 mu L of the cells are paved in the 96-well plate;
after the cells form monoclonal amplification, verifying and screening by using a PCR method to obtain an IKZF2 monoclonal cell line with stable expression;
the PCR sequencing was completed using a prepGEM Universal gene extraction kit of MicroGEM, 10 μl of extraction reagent was used per 20000 cells to extract genomic DNA, and PrimeSTAR HS DNA Polymerase was used for the PCR enzyme;
and 7, adding doxycycline into the stable-expression IKZF2 monoclonal cell line obtained in the step 6 to induce the expression of IKZF2, treating cells by using a target protein specific inhibitor, detecting the degradation trend of the target protein, the expression condition of GFP protein and the activity of the cells, and completing drug screening.
Specifically, in step 2, the amount of the pLVX-EF1a-IRES-Puro-rtTA3 plasmid was 10.65. Mu.g, the amount of the psPAX2 plasmid was 8.598. Mu.g, and the amount of the pMD2.G plasmid was 2.591. Mu.g in the three-plasmid system.
Specifically, in step 2, the amount of the jetPRIME transfection reagent buffer is 1mL, and the amount of the jetPRIME transfection reagent is 2-3 times the sum of the amounts of plasmids in the three plasmid system.
Specifically, in the step 2, the standing condition is that the standing is carried out for 10-15 min at room temperature.
Specifically, in step 3, the pretreatment refers to digestion and resuspension of 293FT cells with pancreatin, and the inoculation amount of the 293FT cells is 10×10 6 。
Specifically, in step 3, the transfection reagent is uniformly injected into the bottom of the non-resistant culture dish.
Specifically, in step 3, the transfection procedure comprises the steps of:
a1, culturing in an incubator for 4-6 hours, and then replacing a fresh antibiotic-free culture medium to continue culturing for 48-72 hours;
a2, collecting supernatant after culturing, placing the supernatant into a 50mL centrifuge tube, centrifuging for 10min at 3000rpm, and sub-packaging and freezing to-80 ℃;
a3, taking 1mL of the virus liquid obtained in the step A2, diluting the virus liquid with a serum-free culture medium until the MOI is 1-5, and uniformly mixing the virus liquid with the polybrene;
a4, taking 5×10 5 293FT cells of (A) were cultured overnight in 6-well plates, and when the cell density reached 70%, the mixture obtained in step A3 was added to the cells and placed in CO at 37 ℃ 2 Culturing in an incubator;
and A5, after culturing for 12-24 hours, changing a fresh antibiotic-free culture medium to continue culturing, and finishing transfection after culturing for 2 days.
Specifically, the concentration of the polybrene is 6-8 mug/mL.
Specifically, in step 6, the operation of co-rotating includes the steps of:
b1, take 5×10 5 Culturing the rtTA3 monoclonal cells obtained in the step 3 in a 6-well plate overnight;
b2, taking 200 mu L of jetPRIME transfection reagent buffer solution, adding 0.5 mu g of Lenti-V2-GAPDH-sgRNA plasmid and 2.5 mu g of BPK1520-Donor-GAPDH-eGFP-IKZF2 plasmid, carrying out vortex oscillation, adding the jetPRIME transfection reagent, and standing for 10-15 min at room temperature, wherein the dosage of the jetPRIME transfection reagent is 2-3 times of the sum of the dosage of the plasmids;
b3, slowly and uniformly pumping the mixed solution obtained in the step B2 into the culture medium of the cells obtained in the step B1, shaking uniformly, and putting into an incubator for culturing;
and B4, after culturing for 4-6 hours, changing a fresh culture medium to continue culturing, and after culturing for 72 hours, completing cotransformation.
Specifically, in the step 7, the target protein specific inhibitor is an NVP-DKY709 inhibitor.
Specifically, in the step 7, the doxycycline-induced procedure includes the following steps:
c1, taking stable expression IKZF2 monoclonal cells, and adding 100 ng/mL doxycycline into a cell culture medium to induce expression for 48h;
c2, digesting the induced cells by pancreatin and re-suspending the cells in a cell culture medium containing 100 ng/mL doxycycline for later use;
c3, cell plating obtained in the step C2 in 96-well cell culture plate, and at 37 ℃,5% CO 2 Culturing overnight in a conditioned incubator;
preparing 4mM storage solution of NVP-DKY709 inhibitor by using dimethyl sulfoxide, and continuously diluting the NVP-DKY709 mixed solution in a 96-well conical plate to obtain 400 times of NVP-DKY709 storage solution;
c5, taking 2 mu L of 400 times NVP-DKY709 obtained in the step C4, storing the liquid into a U-shaped 96-well plate, adding 78 mu L of cells obtained in the step C3, and uniformly mixing to obtain 10 times NVP-DKY709 mother liquor;
adding 10 mu L of 10-fold NVP-DKY709 mother liquor obtained in the step C5 into a 96-well cell culture plate, setting a solvent control group and a blank control group, and adding 90 mu L of the cell suspension obtained in the step C3 into all wells to ensure that the final concentration of dimethyl sulfoxide in all wells is 0.25%;
c7, placing the 96-hole cell plate back into an incubator for culturing for 6-24 hours, and then detecting GFP protein expression results by using a fluorescence microscope;
and C8, placing the cell culture plate cultured for 24 hours in the step C6 at room temperature, detecting the fluorescence intensity by using a fluorescence plate reader, and calculating the degradation trend of the target protein.
The drug screening method based on the doxycycline-induced mediated CRISPR gene knock-in technology has the following beneficial effects:
1. the method provided by the invention combines the doxycycline induction system with the CRISPR gene knock-in technology, edits on a genome level, effectively avoids the conditions of possible cell mutation and the like caused by long-term introduction of exogenous genes by adding the doxycycline induction-mediated mode, and increases the stability of a drug screening system.
2. According to the method provided by the invention, the GFP fluorescent protein detection system is introduced, so that the effectiveness of the gene knock-in model can be indicated, the potential off-target effect of the gene editing technology is overcome, and the stability of the drug screening system is further facilitated.
3. The method provided by the invention realizes the purpose of inducing knockout by constructing BPK1520-Donor-GAPDH-eGFP-IKZF2 plasmid and modifying the Donor plasmid, wherein the fixed-point insertion of the intron TTCTCATCCAAGACTGGCTC sequence between the GAPDH intron, the exon 7 and the exon 8 can not destroy the original structure of cells, can also introduce new editable sequences, is positioned in an intron region by gene editing, does not change the original expression profile of target cells, and is more beneficial to the realization of gene knockout and expression.
4. The drug screening method provided by the invention not only can specifically induce and express the deletion gene in target cells, but also can indicate the effectiveness of the model, thereby being beneficial to screening target drugs and further helping to develop new drugs.
Drawings
FIG. 1 is a flow chart of a doxycycline-induced mediated CRISPR gene knock-in technique of the present invention;
FIG. 2 is a graph showing the results of doxycycline-induced GFP protein expression assay;
FIG. 3 is a graph showing the results of detection of the doxycycline-induced target protein IKZF2 protein degradation assay.
Detailed Description
The following description of the present invention will be made clearly and fully, and it is apparent that the embodiments described are some, but not all, of the embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The reagents used in the following examples are not specifically described as commercial reagents, and table 1 shows the reagent and consumable information used in all the examples, and table 2 shows the instrument and model information used in all the examples.
TABLE 1 reagent and consumable information
| Reagent(s) | Suppliers (suppliers) | Goods number |
| Doxycycline | Sigma | 631311 |
| JetPRIME® | Polyplus | 101000046 |
| Polycurdlan | Sigma | TR-1003-G |
| prepGEM Universal | MicroGEM | PUN0500 |
| Puromycin | Gibco | A1113802 |
| Nano-Glo® HiBiT Lytic Detection System | Promega | N3040 |
Table 2 instrument
| Instrument for measuring and controlling the intensity of light | Suppliers (suppliers) | Goods number |
| Centrifugal machine | Eppendorf | 5810R |
| TS2-FL phase difference inverted microscope imaging system | Nikon | TS2-FL |
| EnVision reader | perkinelmer | 2104 EnVision |
Example 1
As shown in fig. 1, a flowchart of a Doxycycline-induced mediated CRISPR gene knock-in technology of the present invention is shown, in which a Doxycycline-induced expression system-related element is stably expressed in 293FT cells by lentiviral infection, and then GFP fluorescent protein is introduced while inserting a target protein by homologous recombination at a specific site of GAPDH intron by CRISPR Cas 9-mediated gene editing in the constructed cells, and the target protein is induced to be expressed by Doxycycline (Doxycycline).
Firstly, constructing a cell line for stably expressing rtTA3 by using a lentiviral expression system for inducing expression of doxycycline, wherein the specific experimental operation is as follows:
(1) The cultured 293FT cells were digested with pancreatin and resuspended to 10X 10 6 293FT cells of (A) were cultured overnight in 10cm nonreactive dishes.
(2) Construction of pLVX-EF1a-IRES-Puro-rtTA3 plasmid with the sequence shown in SEQ ID NO:1, 1mL of jetPRIME was added to a 1.5 mL EP tube, psPAX2 (8.598. Mu.g), pMD2.G (2.591. Mu.g) and pLVX-EF1a-IRES-Puro rtTA3 (10.65. Mu.g) were added to the buffer, vortexed and mixed with 43.678. Mu.L of jetPRIME (DNA: jetPRIME. Mu.1:2) and then left to stand at room temperature for 10-15 min.
(3) Sucking the prepared transfection reagent, tilting the culture dish, uniformly and slowly pumping into a culture medium, gently shaking, and returning to the incubator (for avoiding floating of cells) for 4-6 h.
(4) After 6h, the culture is continued by changing fresh medium (the cell supernatant containing the mixture of jetPRIME should be discarded into 20% of 84 disinfectant).
(5) After 48h or 72h, collecting virus supernatant, collecting 48 or 72h virus solution into 50mL centrifuge tube, centrifuging at 3000rpm for 10min, packaging into freezing tube, and freezing to-80deg.C at 1 mL/tube.
(6) Taking 5×10 5 293FT cells/well of (E) were cultured overnight in 6-well plates.
(7) And diluting the virus with a serum-free culture medium to MOI of 1-5, and uniformly mixing with polybrene (6-8 mu g/mL). When the cell density reaches 70%, preparing for infection, discarding the culture medium in the 6-hole plate, uniformly mixing 1mL of prepared virus solution with polybrene (6-8 μg/mL), adding into cells, and adding CO at 37deg.C 2 The cells were cultured overnight in an incubator (1000 g of suspension cells were centrifuged at 32℃for 2-4 h).
(8) After 12-24 hours, the cell culture supernatant is replaced by fresh antibiotic-free culture medium for continuous culture (the cell supernatant containing the virus is discarded into 20% 84 disinfectant); if the virus MOI is low at the beginning of infection, the infection can be re-infected before liquid change.
(9) Cells stably expressing rtTA3 were selected by changing fresh medium containing 1. Mu.g/. Mu.L puromycin two days after changing the liquid.
(10) Cells stably expressing rtTA3 were passaged and diluted to a density of 50 cells per 10 ml, 100 μl were plated in 96-well plates and rtTA3 monoclonal cell lines were selected using serial gradient dilution.
(11) The cells forming the monoclonal and amplified were subjected to cell genomic DNA extraction, genomic DNA was extracted using the prepGEM Universal gene extraction kit of MicroGEM, 10 μl of extraction reagent was used per 20000 cells to extract genomic DNA, primeSTAR HS DNA Polymerase was used for PCR amplification to detect the expression of the target gene rtTA3, and then DNA gel was recovered and sequenced by PCR, sequencing primers as shown in table 3:
table 3 PCR primer sequences (shown as sequence listing SEQ NO: 4-SEQ NO: 16)
| Name of the name | Sequence(s) | Length of |
| BPK-MCS-R1 | CGCACCGGTGAATTCGGT | 18-mer |
| EGFP-C-F | CATGGTCCTGCTGGAGTTCGTG | 22-mer |
| eGFP-C_R | ACAGCTCGTCCATGCCGA | 18-mer |
| EGFP-N | CGTCGCCGTCCAGCTCGACCAG | 22-mer |
| eGFP-N_F | AAGGGCGAGGAGCTGTTCA | 19-mer |
| EXFP-R | GTCTTGTAGTTGCCGTCGTC | 20-mer |
| GLA-F1 | TATCGTGGAAGGACTCATGGTA | 22-mer |
| GRA-R | CAGTGATGGCATGGACTGT | 19-mer |
| Hibit-Rev | GCTAATCTTCTTGAACAGCCGCCA | 24-mer |
| Hygro-F | CAGACGTCGCGGTGAGTT | 18-mer |
| IKZF2-F | GGAAACAGAGGCTATTGATGGCT | 23-mer |
| rtTA3-F | ATGTCTAGGCTGGACAAGAGCA | 22-mer |
| rtTA3-R | GGGAGCATGTCAAGGTCAAAAT | 22-mer |
Secondly, constructing a sequence shown as SEQ ID NO:2, which is used for identifying and editing GAPDH genome DNA, and reconstructing a sequence shown as SEQ ID NO:3, wherein the BPK1520-Donor-GAPDH-eGFP-IKZF2 plasmid and the BPK-Donor-GAPDH-eGFP-IKZF 2 plasmid are based on GAPDH homology arms and are used for expressing target genes IKZF2 and GFP, and after construction, the screened rtTA3 monoclonal cell line is used for co-transferring the Lenti-V2-GAPDH-sgRNA plasmid and the BPK1520-Donor-GAPDH-eGFP-IKZF2 plasmid to construct an IKZF2 KI cell line, and the specific experimental steps are as follows:
(1) Taking 5×10 5 rtTA3 monoclonal cells obtained in step 3 were cultured overnight in 6-well plates.
(2) 200. Mu.L of the buffer solution of the jetPRIME transfection reagent is taken, 0.5. Mu.g of the Lenti-V2-GAPDH-sgRNA plasmid and 2. Mu.g of the BPK1520-Donor-GAPDH-eGFP-IKZF2 plasmid are added, vortex oscillation is carried out, the jetPRIME transfection reagent is added, and the mixture is kept stand at room temperature for 10-15 min, wherein the usage amount of the jetPRIME transfection reagent is 2-3 times the sum of the usage amounts of the plasmids.
(3) And (3) slowly and uniformly pumping the mixed solution obtained in the step (2) into the culture medium of the cells obtained in the step (1), shaking uniformly, and putting into an incubator for culturing.
(4) After 4-6h of transfection, the culture was continued by changing fresh medium, and after 72h the cells were passaged by digestion and diluted to a density of 50 cells per 10 ml, 100 μl was plated in 96-well plates.
(5) After the co-transformation was completed, genomic DNA was extracted using 10 μl of extraction reagent per 20000 cells using the prepGEM Universal gene extraction kit of MicroGEM, PCR enzymes were PrimeSTAR HS DNA Polymerase followed by DNA gel recovery and sequencing of PCR, sequencing primers as shown in table 3 above.
(6) According to the PCR detection result, the knocked-in successful cells are plated on a 96-well plate in a serial dilution mode and sorted to obtain an IKZF2 KI cell line.
(7) After the cells formed a single clone, genomic DNA was extracted using 10 μl of extraction reagent per 20000 cells using the prepGEM Universal gene extraction kit of MicroGEM, PCR was performed using PrimeSTAR HS DNA Polymerase, followed by DNA gel recovery and sequencing by PCR, sequencing primers as shown in table 3 above.
Finally, selecting the IKZF2 KI cell line obtained by screening to perform doxycycline induced expression of target IKZF2 gene and GFP, and treating the cells by using an NVP-DKY709 inhibitor, wherein the specific experimental steps are as follows:
(1) The successful IKZF2 cells were knocked in and 100 ng/mL doxycycline was added to the cell culture medium to induce expression for 48h.
(2) The induced cells were adjusted to a concentration of 1X 10 cells per ml by pancreatin digestion 5 Individual cells were resuspended in cell culture medium containing 100 ng/mL doxycycline for later use.
(3) Taking 96-well cell culture plate, adding phosphate buffer solution into A1-A12, B12, C12 and D1-D12, adding 90 μl of suspension into each well of B2-B11 and C2-C11, inoculating 9000 cells per well, adding cell culture medium into B1 and C1 as blank control group, adding B11 and C11 as solvent control group, and culturing 96-well cell culture plate at 37deg.C and 5% CO 2 Culturing in an incubator overnight.
(4) The NVP-DKY709 inhibitor is prepared into 4mM stock solution by dimethyl sulfoxide, and the stock solution is diluted into 10 XNVP-DKY 709 mother solution, wherein the dilution steps are as follows:
firstly, 6 mu L of dimethyl sulfoxide is added to the first row of B3-B10 holes of the 96-hole conical plate, and 10 mu L of 4mM NVP-DKY709 storage solution is added to B2;
secondly, 3 mu L of NVP-DKY709 storage solution is taken from the B2 hole and added into the B3 to be uniformly mixed, 3 mu L of NVP-DKY storage solution is taken from the B3 to be added into the B4 to be uniformly mixed, and the NVP-DKY storage solution is sequentially and continuously diluted until B10 is obtained;
finally, taking a U-shaped 96-well plate, adding 78 mu L of cell culture medium into B2-B11 holes of the U-shaped 96-well plate, taking 2 mu L of diluent in B2-B10 holes of the 96-well conical plate in the last step by using a 12-row discharge gun, adding the diluent into B2-B10 holes of the U-shaped 96-well plate, uniformly mixing, and diluting to obtain 10X NVP-DKY709 mother liquor, wherein the concentration of the 10X NVP-DKY709 mother liquor is 1.4 nM.
(5) 10. Mu.L of 10 XNVP-DKY 709 mother liquor was added to each of the wells B2-B10 and C2-C10 of the 96 well cell culture plates of step (3), and dimethyl sulfoxide was added to the blank control groups (B1 and C1) and the vehicle control groups (B11 and C11) to give a final concentration of dimethyl sulfoxide of 0.25% in all wells.
(6) The 96-well cell plates after the treatment are put back into an incubator for culturing for 24 hours, and then GFP protein expression and cell viability in the cells after 24 hours are detected by a fluorescence microscope, as shown in FIG. 2, wherein the upper left graph represents the result without doxycycline induction, the GFP protein expression rate is extremely low, and the upper right graph represents the result with doxycycline induction, and the GFP protein expression rate is high; the lower panel shows the cell morphology and viability in the bright field, both of which are in good condition.
(7) Target protein degradation trend test: the cell culture plate after 24h of culture is placed at room temperature, 100 mu L of Nano-Glo HiBiT Lytic Detection System detection reagent (LgBiT Protein 1:100 is diluted, nano-Glo HiBiT Lytic Substrate 1:50 is diluted to Nano-Glo HiBiT Lytic buffer solution to prepare detection reagent) is added after balancing, shaking is carried out for 3-10 min on a shaking table at 300-600rpm, then standing is carried out for 10min, fluorescence intensity is detected by 2104 EnVision, and data treatment is carried out, so that the Target Protein Degradation (TPD) trend after NVP-DKY709 inhibitor treatment is determined. Protein degradation ratio (degradation rate%) = (vehicle control-experimental)/(vehicle control-blank) ×100%.
The results of the degradation trend of the target protein after the data processing are shown in fig. 3, the abscissa shows the concentration of NVP-DKY709, and the ordinate shows the protein degradation proportion, and it can be seen that under the condition of 48h of doxycycline induced expression, the protein level of the target protein in the cell is detected through Nano-Glo HiBiT Lytic Detection System after the cell is treated for 24h by using an IKZF2 target protein degradation agent NVP-DKY709, and the protein degradation proportion is intuitively embodied, so that the method provided by the invention can be proved to be used for drug screening.
In summary, the above embodiments are only preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, but any modifications, equivalent substitutions, improvements, etc. within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (11)
1. A drug screening method based on doxycycline-induced mediated CRISPR gene knock-in technology, comprising the steps of:
step 1, constructing a pLVX-EF1a-IRES-Puro-rtTA3 plasmid, wherein the sequence of the pLVX-EF1a-IRES-Puro-rtTA3 plasmid is shown as SEQ ID NO. 1, and a three-plasmid system is formed by a psPAX2 plasmid, a pMD2.G plasmid and the pLVX-EF1a-IRES-Puro-rtTA3 plasmid for slow virus packaging;
step 2, uniformly mixing the three-plasmid system obtained in the step 1 with the jetPRIME transfection reagent buffer solution, adding the jetPRIME transfection reagent after vortex oscillation, and standing the mixture to obtain a reagent serving as the transfection reagent for standby;
step 3, adding 293FT cells into a 10cm non-resistance culture dish after pretreatment, adding the transfection reagent obtained in the step 2 into the non-resistance culture dish after overnight culture, carrying out transfection, screening 293FT cells by using puromycin after the transfection is finished, and paving the cells after puromycin screening into a 96-well plate for culture by using a continuous gradient dilution method;
step 4, extracting genome DNA (deoxyribonucleic acid) for PCR (polymerase chain reaction) sequencing after the cells obtained in the step 3 form monoclonal amplification, and detecting the expression condition of the target gene rtTA3 to obtain a monoclonal cell line for stably expressing the target gene rtTA 3;
step 5, constructing a Lenti-V2-GAPDH-sgRNA plasmid and a BPK1520-Donor-GAPDH-eGFP-IKZF2 plasmid, wherein the sequence of the Lenti-V2-GAPDH-sgRNA plasmid is shown as SEQ ID NO:2, the sequence of the BPK1520-Donor-GAPDH-eGFP-IKZF2 plasmid is shown in SEQ ID NO:3 is shown in the figure;
step 6, cotransformation of the Lenti-V2-GAPDH-sgRNA plasmid and the BPK1520-Donor-GAPDH-eGFP-IKZF2 plasmid by using the stably expressed rtTA3 monoclonal cell line obtained in the step 4, spreading the cells on a 96-well plate by using a continuous gradient dilution method after cotransformation, culturing the cells, verifying the cells by using a PCR method after the cells form monoclonal amplification, and screening the cells to obtain the stably expressed IKZF2 monoclonal cell line;
and 7, adding doxycycline into the stable-expression IKZF2 monoclonal cell line obtained in the step 6 to induce the expression of IKZF2, treating cells by using a target protein specific inhibitor, detecting the degradation trend of the target protein, the expression condition of GFP protein and the activity of the cells, and completing drug screening.
2. The method of claim 1, wherein in step 2, the pLVX-EF1a-IRES-Puro-rtTA3 plasmid is used in an amount of 10.65 μg, the psPAX2 plasmid is used in an amount of 8.598 μg, and the pmd2.G plasmid is used in an amount of 2.591 μg.
3. The method of claim 1, wherein in step 2, the amount of the buffer of the jetPRIME transfection reagent is 1mL and the amount of the jetPRIME transfection reagent is 2-3 times the sum of the amounts of plasmids in the three plasmid system.
4. The method for screening a drug based on the doxycycline-induced mediated CRISPR gene knock-in technology according to claim 1, wherein in step 2, the condition of standing is standing at room temperature for 10-15 min.
5. The method of claim 1, wherein in step 3, the pretreatment is digestion of 293FT cells with pancreatin and resuspension, and the inoculation amount of the 293FT cells is 10×10 6 。
6. The method of claim 1, wherein in step 3, the transfection reagent is uniformly injected into the bottom of the non-resistant culture dish.
7. The method of claim 1, wherein in step 3, the transfection procedure comprises the steps of:
a1, culturing in an incubator for 4-6 hours, and then replacing a fresh antibiotic-free culture medium to continue culturing for 48-72 hours;
a2, collecting supernatant after culturing, placing the supernatant into a 50mL centrifuge tube, centrifuging for 10min at 3000rpm, and sub-packaging and freezing to-80 ℃;
a3, taking 1mL of the virus liquid obtained in the step A2, diluting the virus liquid with a serum-free culture medium until the MOI is 1-5, and uniformly mixing the virus liquid with the polybrene;
a4, taking 5×10 5 293FT cells of (A) were cultured overnight in 6-well plates, and when the cell density reached 70%, the mixture obtained in step A3 was added to the cells and placed in CO at 37 ℃ 2 Culturing in an incubator;
and A5, after culturing for 12-24 hours, changing a fresh antibiotic-free culture medium to continue culturing, and finishing transfection after culturing for 2 days.
8. The method of claim 7, wherein the concentration of the polybrene is 6-8 μg/mL.
9. The method of claim 1, wherein in step 6, the co-transformation comprises the steps of:
b1, take 5×10 5 Culturing the rtTA3 monoclonal cells obtained in the step 3 in a 6-well plate overnight;
b2, taking 200 mu L of jetPRIME transfection reagent buffer solution, adding 0.5 mu g of Lenti-V2-GAPDH-sgRNA plasmid and 2.5 mu g of BPK1520-Donor-GAPDH-eGFP-IKZF2 plasmid, carrying out vortex oscillation, adding the jetPRIME transfection reagent, and standing for 10-15 min at room temperature, wherein the dosage of the jetPRIME transfection reagent is 2-3 times of the sum of the dosage of the plasmids;
b3, slowly and uniformly pumping the mixed solution obtained in the step B2 into the culture medium of the cells obtained in the step B1, shaking uniformly, and putting into an incubator for culturing;
and B4, after culturing for 4-6 hours, changing a fresh culture medium to continue culturing, and after culturing for 72 hours, completing cotransformation.
10. The method of claim 1, wherein in step 7, the inhibitor specific for the target protein is an NVP-DKY709 inhibitor.
11. The method of claim 10, wherein in step 7, the doxycycline-induced procedure comprises the steps of:
c1, taking stable expression IKZF2 monoclonal cells, and adding 100 ng/mL doxycycline into a cell culture medium to induce expression for 48h;
c2, digesting the induced cells by pancreatin and re-suspending the cells in a cell culture medium containing 100 ng/mL doxycycline for later use;
c3, cell plating obtained in the step C2 in 96-well cell culture plate, and at 37 ℃,5% CO 2 Culturing overnight in a conditioned incubator;
preparing 4mM storage solution of NVP-DKY709 inhibitor by using dimethyl sulfoxide, and continuously diluting the NVP-DKY709 mixed solution in a 96-well conical plate to obtain 400 times of NVP-DKY709 storage solution;
c5, taking 2 mu L of 400 times NVP-DKY709 obtained in the step C4, storing the liquid into a U-shaped 96-well plate, adding 78 mu L of cells obtained in the step C3, and uniformly mixing to obtain 10 times NVP-DKY709 mother liquor;
adding 10 mu L of 10-fold NVP-DKY709 mother liquor obtained in the step C5 into a 96-well cell culture plate, setting a solvent control group and a blank control group, and adding 90 mu L of the cell suspension obtained in the step C3 into all wells to ensure that the final concentration of dimethyl sulfoxide in all wells is 0.25%;
c7, placing the 96-hole cell plate back into an incubator for culturing for 6-24 hours, and then detecting GFP protein expression results by using a fluorescence microscope;
and C8, placing the cell culture plate cultured for 24 hours in the step C6 at room temperature, detecting the fluorescence intensity by using a fluorescence plate reader, and calculating the degradation trend of the target protein.
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