CN114790463B - Construction method and application of monoclonal cell strain for stably transfecting CRISPR/dCAS9 system - Google Patents
Construction method and application of monoclonal cell strain for stably transfecting CRISPR/dCAS9 system Download PDFInfo
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Abstract
The invention discloses a construction method of a monoclonal cell strain for stably transfecting a CRISPR/dCAS9 system, which comprises the following steps: 1) Lentivirus transfection; 2) Ultracentrifugation; 3) Cell infection and first-time flow fluorescence sorting; 4) And (5) secondary flow type fluorescence sorting. The invention belongs to the technical field of genetic engineering, and provides a construction method and application of a monoclonal cell strain for stably transfecting a CRISPR/dCAS9 system by combining a lentivirus transfection method, an ultracentrifugation method and a flow type fluorescence sorting method, so that the defects that the CRISPR/dCAS9 system is difficult to transfect and cannot be stably expressed for a long time are overcome, the stable expression of the CRISPR/dCAS9 system is realized, and the targeted regulation and control of DNA methylation can be realized.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a construction method and application of a monoclonal cell strain for stably transfecting a CRISPR/dmas 9 system.
Background
DNA methylation was the earliest epigenetic regulatory mode studied and refers to the addition or removal of a methyl group at the 5-position carbon atom of cytosine by DNA methylation modifying enzymes (methyltransferases and demethylases). DNA methylation is widely involved in gene transcription regulation, cell differentiation, embryo development, X chromosome inactivation, gene imprinting, tumorigenesis and development and other cellular processes. However, due to the limitation of the technical level, the existing DNA methylation regulation mainly regulates the change of DNA methylation at the whole genome level by changing the activity of DNA methylation modification enzyme, but the requirement of targeted regulation of DNA methylation of specific genes cannot be met. Methylation modification is a highly refined and continuous process, so that a specific DNA methylation editing technology is needed, and under the condition that other genes are not affected, the function and action mechanism of DNA methylation of a single gene and even specific CpG sites are researched, so that an effective tool is provided for deeply researching the function of DNA methylation modification and research and development of related medicines.
Bacteria and archaebacteria evolved CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR associated proteins) systems are small RNA mediated adaptive immune systems that recognize and destroy or silence foreign DNA (viruses and plasmids) that enter the host, and after engineering, can be used for gene editing and epigenetic regulation. Researchers have mutated the nuclease domain of Cas9 in streptococcus on this basis to produce Cas9 with a deletion of endonuclease activity, known as read Cas9 (dCas 9). dCas9 is no longer able to cleave DNA, but it can still target and bind DNA with the same precision under the guidance of sgrnas. By designing specific sgrnas complementary to target DNA and fusing dCas9 with DNA methyl modification enzymes, CRISPR/dCas technology is expected to achieve specific DNA methylation regulation. Patent application CN 111073902A discloses a CRISPR/dCAS9 vector for improving the expression level of a gliotoxin biosynthesis gene, a construction method and application thereof, and establishes a CRISPR/dCAS9 specific transcription regulation system suitable for the Edodes, thereby promoting the transcription regulation of the Edodes FS110 gliotoxin biosynthesis.
The slow virus vector is modified from HIV virus, and can introduce target gene into target cell for long-time stable expression. Compared with partial adenovirus vector, slow virus transfection can greatly improve the mediating efficiency of target genes. However, for some plasmids with larger molecular weight, transfection using lentiviral vectors also has the problem of low transfection efficiency. The CRISPR/dCas9 system can be used for methylation modification, but has limitations in that plasmids are large, transfection efficiency is low, or stable transfection is difficult. Therefore, it is of great importance to provide a monoclonal cell line which stably expresses the CRISPR/dCas9-TET1CD system and which is capable of long-term stable in vitro culture.
Disclosure of Invention
In order to solve the problems (the defects of low transfection efficiency or difficult stable transfection) existing in the prior art, the invention firstly provides a construction method and application of a monoclonal cell strain for stably transfecting the CRISPR/dCAS9 system by combining a slow virus transfection method, an ultracentrifugation method and a flow type fluorescence sorting method, overcomes the defects of difficult transfection and incapability of long-time stable expression of the CRISPR/dCAS9 system, realizes stable expression of the CRISPR/dCAS9 system, and can realize targeted regulation and control of DNA methylation.
The purpose of the present invention will be further explained by the following detailed description.
The invention provides a construction method of a monoclonal cell strain for stably transfecting a CRISPR/dCAS9 system, which comprises the following steps:
1) Lentiviral transfection: inoculating and culturing 293FT cells, transfecting a core plasmid and a lentivirus packaging plasmid system by using a transfection reagent when the cell fusion degree reaches 70% -90%, and collecting virus liquid at 48h and 96 h;
2) Ultracentrifugation: filtering the collected virus liquid with a filter membrane, uniformly mixing and ultracentrifugating for 2-2.5h at 4 ℃ and 22000-26000rpm; centrifuging, removing supernatant, adding PBS solution, and re-suspending to obtain virus protein re-suspension, and preserving at 4deg.C;
3) Cell infection and first-pass fluorescence sorting: inoculating and culturing target cells A549 in a 6-hole plate, culturing for 20-28h, then dripping the virus protein heavy suspension to infect twice, carrying out passage amplification culture on the target cells for 2-4 generations, carrying out first-time flow fluorescence sorting, and recovering cells by using a flow tube to obtain a cell strain expressing a CRISPR/dCAS9 system;
4) Second-time flow fluorescence sorting: carrying out secondary flow fluorescence sorting on the cell strain expressing the CRISPR/dCAS9 system, wherein the cell recovery mode is 96-hole plate recovery, and 1 cell is recovered in each hole; observing the cell adherence condition by using a microscope 4-8h after the second flow fluorescence sorting, primarily screening monoclonal cells, digesting the cells by using trypsin and transferring when the cells are grown in a 96-well plate, carrying out passage amplification for 2-3 generations, and carrying out flow fluorescence analysis verification to obtain the monoclonal cell strain for stably transfecting the CRISPR/dCAS9 system.
The invention uses CRISPR/dCAS9-TET1CD as a core plasmid, uses psPAX2 and pMD2.G with specific mass ratio as a slow virus packaging plasmid system for transfection, collects virus liquid at 48h and 96h, and carries out ultracentrifugation under specific conditions to obtain concentrated virus particles, thereby improving transfection efficiency; because the molecular weight of the transfected plasmid is large, the transfection efficiency is relatively low, and after target cells are subjected to passage expansion culture for 2-4 generations, enough cells (the number of the cells is more than 250 ten thousand) are used for sorting, so that enough positive cells are obtained under the condition of low positive rate, and the proliferation culture of the positive cells is facilitated. Meanwhile, a small amount of positive cells are sorted out by using a fluorescent marker of the target gene and through first-time flow fluorescent sorting, and cells are recovered by using a flow tube, so that the defect that positive cell strains cannot be obtained through drug screening under low transfection efficiency is overcome. Because the fluorescent cells obtained by the first flow fluorescent sorting are not hundred percent positive cells by using the flow tube for recovery, namely the non-positive cells can be mistaken as positive cells, and the proportion of the positive cells gradually decreases due to the cell growth advantages of the non-positive cells in the passage process. Based on the finding, the invention further develops secondary flow fluorescence sorting, adopts different cell recovery modes, overcomes the defect that the proportion of positive cells is gradually reduced due to the advantage of cell growth in the passage process of non-positive cells obtained by the primary flow sorting, selects positive cells for independent culture, and finally obtains a stably expressed monoclonal cell strain after the passage culture is verified by flow fluorescence analysis (only positive rate is analyzed and cells are not recovered).
Preferably, the transfection reagent is Polyethylenimine (PEI) MAX; the core plasmid is CRISPR/dCAS9-TET1CD, the lentiviral packaging plasmid system comprises psPAX2 and pMD2.G, the mass ratio of the CRISPR/dCAS9-TET1CD, the psPAX2 and the pMD2.G is 6:6:5, and the dosage ratio of the transfection plasmid to the transfection reagent is 1 mug to 3 mug. The transfected plasmids include core plasmids and lentiviral packaging plasmid systems.
Preferably, the viral titer of the viral protein suspension is 1X 10 8 -1.68×10 8 TU/mL。
Preferably, the filter is a Millipore filter with a pore size of 0.45 μm.
Preferably, the channels of the first-time flow fluorescence sorting and the second-time flow fluorescence sorting are tagBFP, the excitation wavelength is 400nm, and the emission wavelength is 450nm.
Correspondingly, the invention also provides a monoclonal cell strain for stably transfecting the CRISPR/dCAS9 system, which is constructed by adopting the construction method.
In addition, the invention also provides application of the monoclonal cell strain stably transfected with the CRISPR/dCAS9 system in preparing medicaments for treating DNA methylation related diseases.
Compared with the prior art, the invention has the beneficial effects that: the invention provides a construction method and application of a monoclonal cell strain for stably transfecting a CRISPR/dCAS9 system by combining a lentiviral transfection method, an ultracentrifugation method and a flow type fluorescence sorting method and optimizing the operation conditions of a lentiviral packaging plasmid system, such as composition and mass ratio of the lentiviral packaging plasmid system, virus liquid collection, ultracentrifugation, virus titer and the like, and overcomes the defects that the CRISPR/dCAS9 system is difficult to transfect and cannot be stably expressed for a long time, realizes the stable expression of the CRISPR/dCAS9 system, and can realize targeted regulation and control on DNA methylation. The invention provides a stable expression CRISPR/dCAS9-TET1CD monoclonal cell strain, which can utilize the monoclonal cell strain to carry out DNA methylation modification change on specific genes or loci, provides a favorable tool for researching the DNA methylation function and action mechanism of the specific genes or loci, and is expected to play an important role in preparing medicaments for treating DNA methylation related diseases.
Drawings
FIG. 1 is a schematic flow chart of the construction method provided by the invention.
FIG. 2 is a graph of the CRISPR/dCAs9-TET1CD plasmid used in the present invention.
FIG. 3 is a graph showing the results of the first-time flow fluorescence sorting of the example of the present invention.
FIG. 4 is a graph showing the detection results of cells subjected to first-time flow fluorescence sorting in the embodiment of the present invention after 7 generations of amplification culture.
FIG. 5 is a graph showing the results of the verification of a monoclonal cell line obtained in the example of the present invention.
FIG. 6 is a graph showing the results of flow-through fluorescence sorting of comparative example 2 according to the present invention.
FIG. 7 is a graph showing the results of flow-through fluorescence sorting of comparative example 3 according to the present invention.
FIG. 8 is a graph showing the results of measuring the mRNA expression levels of the monoclonal cell lines dCAs9 and TET1CD obtained in the example of the present invention.
FIG. 9 is a graph showing the methylation detection result of the Line 1DNA of the monoclonal cell Line provided by the embodiment of the invention.
Detailed Description
The invention will be described in further detail with reference to the drawings and examples.
In the present invention, the materials and reagents involved are all conventional commercial products or can be obtained by conventional technical means in the art, such as CRISPR/dCAS9-TET1CD from Addgene, psPAX2 from Addgene, pMD2.G from Addgene, polyethylenimine MAX from Polysciences.
Culture conditions of 293FT and a549 cells: DMEM medium (containing 10% by volume of fetal calf serum), 5% CO 2 Culturing at 37 ℃.
EXAMPLE one construction of monoclonal cell lines stably transfected with the CRISPR/dCAS9 System
A schematic flow chart of a construction method of a monoclonal cell strain for stably transfecting a CRISPR/dCAS9 system is shown in figure 1, and the construction method specifically comprises the following steps:
1) Lentiviral transfection: 293FT cells were seeded in 10-cm cell culture dishes at a cell seeding number of 5X 10 6 Transfecting CRISPR/dCAs9-TET1CD core plasmid and a lentiviral packaging plasmid system (comprising psPAX2 and pMD2. G) by using Polyethylenimine MAX transfection reagent, wherein the concentration of PEI application solution is 1mg/ml, the mass ratio of CRISPR/dCAs9-TET1CD, psPAX2 and pMD2.G is 6:6:5, the dosage of CRISPR/dCAs9-TET1CD is 6 mug, and the dosage ratio of transfection reagent is 51 mu L; fresh medium was changed 8h after transfection, and virus solution was collected 48h and 96 h.
2) Ultracentrifugation: the collected virus solution was applied to a 0.45 μm wellFiltering with Millipore membrane, mixing, evenly distributing to 6 centrifuge tubes, and ultracentrifugation for 2h at 4deg.C and 25000rpm; centrifuging, removing supernatant, and adding 200 μl1×PBS to each centrifuge tube to obtain virus protein suspension with virus titer of 1.05X10 8 TU/mL,4 ℃.
3) Cell infection and first-pass fluorescence sorting: target cells A549 were seeded and cultured in 6-well plates with a cell seeding number of 2X 10 5 Dropwise adding 100 mu L of the virus protein heavy suspension for each infection after culturing for 24 hours, carrying out infection twice, changing liquid after 12 hours of infection, carrying out first-time flow fluorescence sorting after carrying out passage amplification culture on target cells for 3 generations, and recovering the cells by using a flow tube, wherein the channel is tagBFP (excitation wavelength is 400nm and emission wavelength is 450 nm), thus obtaining a cell strain expressing a CRISPR/dCS 9 system; a small amount of positive cells are separated through first-time flow fluorescence separation, a relatively high primary screening positive rate is obtained, the defect that positive cell strains cannot be obtained through drug screening under low transfection efficiency is overcome, and a cell detection result diagram is shown in figure 3. The results of the first flow fluorescent sorting of cells after passage 7 were examined and shown in FIG. 4. 53.0% is due to the technical defect caused by the recovery of cells using a flow tube in the first flow sorting, and the positive rate decreases with increasing passage number after sorting, so that it is necessary to sort out the monoclonal to ensure 100% positive cells.
4) Second-time flow fluorescence sorting: carrying out secondary flow fluorescence sorting on the cell strain expressing the CRISPR/dCAS9 system, wherein the cell recovery mode is 96-hole plate recovery, and 1 cell is recovered in each hole; and 6h after the second flow fluorescence sorting, observing the cell adherence condition by using a microscope, primarily screening monoclonal cells, digesting the cells by using trypsin and transferring when the cells are grown in a 96-well plate, carrying out passage amplification for 2 generations, and carrying out flow fluorescence analysis verification to obtain the monoclonal cell strain of the stably transfected CRISPR/dCAS9 system. The verification result is shown in fig. 5, the defect that positive cells are difficult to sort out by 100% in the flow sorting technology is overcome, the positive cells are singly sorted, and then the positive cells are singly selected for culture, and finally the monoclonal cell strain with stable expression is obtained.
Comparative example 1
The inventor uses the existing calcium phosphate coprecipitation method to transfect 293FT cells according to the mass ratio of VSVG (delta 8.9=8:1:6), viruses produced by each 293 cell infect target cells A549, virus liquid is collected for 48 hours and 72 hours after transfection, and the screening method is purine screening, and the target cells stably transfected with the CRISPR/dCAS9 system cannot be obtained due to large plasmids and low transfection efficiency.
Comparative example 2
The inventor inoculates 293FT cells in 2 cell culture dishes of 10cm, uses Polyethylenimine (PEI) -MAX transfection reagent, PEI application liquid concentration is 1mg/ml, and transfects CRISPR/dCAS9-TET1CD core plasmid and lentiviral packaging plasmid system (comprising psPAX2 and pMD2. G), wherein the mass ratio of CRISPR/dCAS9-TET1CD, psPAX2 and pMD2.G is 4:3:1, the dosage ratio of transfection plasmid to transfection reagent is 1 mug to 3 mug, and virus liquid is collected in 48h and 96 h; directly filtering the collected virus liquid into an Amicon Ultra-15ml ultrafiltration tube, and concentrating the virus liquid by centrifugation at 4 ℃ for 30min in an Allegra X-15R table-type refrigerated centrifuge at 4000 Xg; to the concentrated virus solution, 100. Mu.l of complete medium containing 10% by volume of FBS was added to prepare a virus protein suspension, which was stored at 4 ℃. 100. Mu.L of the viral protein resuspension was added dropwise for each infection, and the infection was twice.
Comparative example 2 differs from example one mainly in that: the mass ratios of CRISPR/dCAS9-TET1CD, psPAX2 and pMD2.G were varied, concentrated using ultrafiltration tubes, without ultracentrifugation. Comparative example 2 cell lines expressing the CRISPR/dCas9 system were obtained by first flow fluorescent sorting, and the cell detection results are shown in fig. 6 with a sorting positive rate of only 0.071%.
Comparative example 3
The inventors inoculated 293FT cells in 5 10cm cell culture dishes, transfected CRISPR/dCAS9-TET1CD core plasmids with lentiviral packaging plasmid systems (comprising psPAX2 and pMD2. G) using Polyethylenimine (PEI) -MAX transfection reagent at a concentration of 1mg/ml, the mass ratio of CRISPR/dCAS9-TET1CD, psPAX2 and pMD2.G was 6:6:5, the dose ratio of transfected plasmid to transfection reagent was 1 μg to 3 μl, the transfected 293FT cells were expanded to 5 dishes, and virus solution was collected at 48h and 96 h; filtering the collected virus liquid with a Millipore filter membrane with the aperture of 0.45 μm, uniformly mixing, evenly distributing the virus liquid into 4 centrifuge tubes, and ultracentrifugating for 1.5h under the conditions of 4 ℃ and 25000rpm; after centrifugation, the supernatant was removed, 200. Mu.L of 1 XPBS was added to each centrifuge tube to resuspend the viral proteins, and the resulting viral protein suspensions were stored at 4 ℃. 100. Mu.L of the viral protein resuspension was added dropwise for each infection, and the infection was twice.
Comparative example 3 differs from example one mainly in that: less viral load and shorter centrifugation time results in lower viral titer of the viral protein resuspension. Comparative example 3 cell lines expressing the CRISPR/dCas9 system were obtained by first flow fluorescent sorting, and the cell detection results are shown in fig. 7 with a sorting positive rate of only 0.337%.
Comparative example 4
The inventor inoculates 293FT cells in 10 cell culture dishes with the concentration of PEI application solution of 1mg/ml by using a Polyethylenimine (PEI) -MAX transfection reagent, transfects CRISPR/dCAS9-TET1CD core plasmid and a lentiviral packaging plasmid system (comprising psPAX2 and pMD2. G), the mass ratio of CRISPR/dCAS9-TET1CD, psPAX2 and pMD2.G is 6:6:5, the dosage ratio of the transfection plasmid to the transfection reagent is 1 mug to 3 mug, and the virus solution is collected in 48h and 96 h; filtering the collected virus liquid with a Millipore filter membrane with the aperture of 0.45 μm, uniformly mixing, evenly distributing the virus liquid into 6 centrifuge tubes, and ultracentrifugating for 2 hours under the conditions of 4 ℃ and 25000rpm; after centrifugation, the supernatant was removed, 200. Mu.L of 1 XPBS was added to each centrifuge tube to resuspend the viral proteins, and the resulting viral protein suspensions were stored at 4 ℃. 200. Mu.L of the viral protein resuspension was added dropwise for each infection, and the infection was twice.
Comparative example 4 differs from example one mainly in that: the viral protein resuspension per infection was added in 200 μl drops. Results: viral titers are too high, leading to target cell death, and positive cells are not sorted.
Example two detection of mRNA expression level of A549 monoclonal cell strain stably expressing CRISPR/dCAS9 System
Extracting total RNA of A549 monoclonal cell strain obtained in example I and stably expressing CRISPR/dCAS9 system by TRIzol method, and using TAKARA company reverseReverse transcription is carried out by the transcription kit to generate cDNA according to TOYOBO companyqRT-PCR was performed using Green qPCR kit, and the primer sequences used for qRT-PCR are shown in Table 1.
TABLE 1 primers used in the present invention
As shown in FIG. 8, the A549 monoclonal cell strain which stably expresses the CRISPR/dCAS9 system was successfully constructed, and the mRNA expression amounts of dCAS9 and TET1CD were obviously higher than those of the control group.
EXAMPLE three stable expression CRISPR/dCAS9 System A549 monoclonal cell Line 1DNA methylation detection
Extracting the genome DNA detection condition of the A549 monoclonal cell strain stably expressing the CRISPR/dCAS9 system obtained in the embodiment to examine the influence on the methylation of the whole genome DNA, and specifically comprising the following steps:
(1) digesting the constructed cell strain, and extracting cell genome DNA by using a TIANGEN blood/cell/tissue genome DNA extraction kit;
(2) bisulphite modification:
bisulphite modification of genomic DNA using QIAGEN EpiTect Bisulfite Kit (cat# 59104);
(3) pyrosequencing:
pyrophosphate sequencing was performed on bisulfite modified DNA using QIAGEN PyroMarkPCRKit (cat# 978703) and the primers required for sequencing are shown in Table 1.
As shown in FIG. 9, the stable expression CRISPR/dCAS9-TET1CD monoclonal cell line did not alter the DNA methylation level of the non-targeted gene.
The foregoing is a further detailed description of the invention in connection with the preferred embodiments, and it is not intended that the invention be limited to the specific embodiments described. It will be apparent to those skilled in the art that several simple deductions or substitutions may be made without departing from the spirit of the invention, and these should be considered to be within the scope of the invention.
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Claims (3)
1. The construction method of the monoclonal cell strain for stably transfecting the CRISPR/dCAS9 system is characterized by comprising the following steps: the method comprises the following steps:
1) Lentiviral transfection: inoculating and culturing 293FT cells, transfecting a core plasmid and a lentivirus packaging plasmid system by using a transfection reagent when the cell fusion degree reaches 70% -90%, and collecting virus liquid at 48h and 96 h;
2) Ultracentrifugation: filtering the collected virus liquid with a filter membrane, uniformly mixing and ultracentrifugating for 2-2.5h at 4 ℃ and 22000-26000rpm; centrifuging, removing supernatant, adding PBS solution, and re-suspending to obtain virus protein re-suspension, and preserving at 4deg.C;
3) Cell infection and first-pass fluorescence sorting: inoculating and culturing target cells A549 in a 6-hole plate, culturing for 20-28h, then dripping the virus protein heavy suspension to infect twice, carrying out passage amplification culture on the target cells for 2-4 generations, carrying out first-time flow fluorescence sorting, and recovering cells by using a flow tube to obtain a cell strain expressing a CRISPR/dCAS9 system;
4) Second-time flow fluorescence sorting: carrying out secondary flow fluorescence sorting on the cell strain expressing the CRISPR/dCAS9 system, wherein the cell recovery mode is 96-hole plate recovery, and 1 cell is recovered in each hole; 4-8h after the second flow fluorescent sorting, observing the cell adherence condition by using a microscope, primarily screening monoclonal cells, digesting the cells by using trypsin and transferring when the cells are grown in a 96-well plate, carrying out passage amplification for 2-3 generations, and carrying out flow fluorescent analysis verification to obtain a monoclonal cell strain for stably transfecting the CRISPR/dCAS9 system;
the transfection reagent is Polyethylenimine MAX; the core plasmid is CRISPR/dCAS9-TET1CD, the lentiviral packaging plasmid system comprises psPAX2 and pMD2.G, the mass ratio of the CRISPR/dCAS9-TET1CD, the psPAX2 and the pMD2.G is 6:6:5, and the dosage ratio of the transfection plasmid to the transfection reagent is 1 mug to 3 mug;
the viral titer of the viral protein resuspension was 1.05X10 8 TU/mL。
2. The method for constructing a monoclonal cell line stably transfected with the CRISPR/dCas9 system according to claim 1, wherein: the filter membrane is a Millipore filter membrane, and the pore size is 0.45 μm.
3. The method for constructing a monoclonal cell line stably transfected with the CRISPR/dCas9 system according to claim 1, wherein: the channels of the first-time flow type fluorescence sorting and the second-time flow type fluorescence sorting are tagBFP respectively, the excitation wavelength is 400nm, and the emission wavelength is 450nm.
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