CN114703183A - sgRNA and CRISPR/Cas9 lentivirus system for targeted knockout of HIF-1 alpha gene and application - Google Patents
sgRNA and CRISPR/Cas9 lentivirus system for targeted knockout of HIF-1 alpha gene and application Download PDFInfo
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Abstract
The invention discloses a sgRNA for targeted knockout of an HIF-1 alpha gene, a CRISPR/Cas9 lentivirus system and application, and relates to the technical field of biology. The sgRNA is HIF-1 alpha sgRNA1, HIF-1 alpha sgRNA2 or HIF-1 alpha sgRNA 3. The sgRNA for targeted knockout of the HIF-1 alpha gene can effectively target the HIF-1 alpha gene, a CRISPR/Cas9 lentiviral vector system is constructed by adopting the sgRNA, and a cell strain for targeted knockout of the HIF-1 alpha gene is obtained by further transfecting cells. The cell strain with the HIF-1 alpha gene knocked out in a targeted manner, which is prepared by the sgRNA, has the advantages of short preparation time, simplicity in operation, high transfection efficiency of the cell strain, stable expression effect and capability of continuously maintaining the knocking-down effect.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a sgRNA and CRISPR/Cas9 lentivirus system for targeted knockout of HIF-1 alpha gene and application thereof.
Background
HIF-1 alpha is called hypoxia inductive factor-1, and its Chinese name is hypoxia inducible factor-1, which is a transcription factor widely existing in mammals and human bodies under hypoxia condition, and is a key factor responding to hypoxia stress. HIF-1 α is a subunit of hypoxia inducible factor-1 (HIF-1), and is regulated by hypoxia and regulates HIF-1 activity. Under hypoxic conditions, HIF-1 α translocates into the nucleus to bind HIF-1 β to form active HIF-1, which regulates transcription of a variety of genes by binding to hypoxia-responsive elements on target genes. HIF-1 alpha can form different signal channels with a plurality of upstream and downstream proteins, mediate hypoxia signals, regulate and control cells to generate a series of compensatory responses to hypoxia, play an important role in the growth and development of organisms and physiological and pathological processes, and is a focus of biomedical research. Recent studies have shown that HIF-1 α has a close relationship with the oxygen sensing pathway and can be used to regulate gene levels to address oxygen levels at various concentrations.
CRISPR (clustered regularly interspersed short palindromic repeats)/Cas (CRISPR-associated) is a system capable of specifically cutting and degrading exogenous DNA, is a specific acquired immune system in some bacteria, and is guided by specific RNA (ribonucleic acid) and can perform specific cutting. The system is applied to a genome targeted editing tool, and can guide a Cas protein to cut specific DNA by designing specific sgRNA (single guide RNA), and cut the specific DNA under the guidance of the sgRNA to play roles in silencing, cutting or modifying genes and the like. At present, the CRISPR/Cas9 can specifically recognize DNA sequences and can specifically cut the DNA sequences, so that the gene can be regulated.
In order to explore the relationship between HIF-1 alpha and oxygen sensing pathway and study the change mechanism, a cell line for knocking out HIF-1 alpha needs to be constructed. However, the traditional method for constructing the stable cell strain has complex preparation steps and long time consumption, and the prepared cell strain has low transfection efficiency and poor stability. Therefore, by designing sgRNA for targeted knockout of the HIF-1 alpha gene and constructing a cell strain for stable targeted knockout of the HIF-1 alpha gene, the construction method is very important for deeper exploration of the relationship between the HIF-1 alpha and the oxygen sensing pathway.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a sgRNA and CRISPR/Cas9 lentivirus system for targeted knockout of HIF-1 alpha gene and application thereof.
In order to achieve the purpose, the invention adopts the technical scheme that: an sgRNA of an HIF-1 alpha gene targeted knockout, the sgRNA being HIF-1 alpha sgRNA1, HIF-1 alpha sgRNA2 or HIF-1 alpha sgRNA 3;
the sequence of the HIF-1 alpha sgRNA1 is:
HIF-1αsgRNA1oligo1:5’-TAggTTCTTTACTTCGCCGAGATC-3’；
HIF-1αsgRNA1oligo2：5’-AAACGATCTCGGCGAAGTAAAGAA-3’；
the sequence of the HIF-1 alpha sgRNA2 is:
HIF-1αsgRNA2oligo1:5’-TAggCTTTACTTCGCCGAGATC-3’；
HIF-1αsgRNA2oligo2：5’-AAACGATCTCGGCGAAGTAAAG-3’；
the sequence of the HIF-1 alpha sgRNA3 is:
HIF-1αsgRNA3oligo1:5’-TAgGAGCTCCCAATGTCGGAGTT-3’；
HIF-1αsgRNA3oligo2:5’-AAACAACTCCGACATTGGGAGCT-3’。
the CRISPR/Cas9 system is adopted to carry out accurate knockout on a specific gene, and the key for constructing a cell strain of the targeted knockout gene lies in the design of sgRNA. The knockout effect of sgrnas of different sequences on the targeted gene is greatly influenced. The inventor designs three sgRNAs with higher scores on an exon part of HIF-1 alpha through a large amount of screening, constructs a cell strain of targeted knockout of the HIF-1 alpha gene through the three sgRNAs, further more finely researches the change of the cell under the anoxic condition, and researches the phenotype and downstream gene change of the cell by constructing the cell strain of knockout of the HIF-1 alpha, and further researches an oxygen perception pathway by utilizing the cell strain.
The invention also provides a CRISPR/Cas9 plasmid, which contains sgRNA of the targeted knockout HIF-1 alpha gene.
The invention also provides a CRISPR/Cas9 lentiviral vector system, which is constructed by adopting the sgRNA of the targeted knockout HIF-1 alpha gene.
The invention also provides a construction method of the CRISPR/Cas9 lentiviral vector system, which comprises the following steps:
(1) carrying out enzyme digestion on lentivirus vector lenti CRISPRV2 to obtain a CRISPR/Cas9 lentivirus vector;
(2) phosphorylating the DNA sequence of sgRNA of the targeted knockout HIF-1 alpha gene;
(3) and (3) connecting the sgRNA in the step (2) with the CRISPR/Cas9 lentiviral vector in the step (1) to obtain the CRISPR/Cas9 lentiviral vector system.
The invention also provides a kit which comprises the sgRNA of the targeted knockout HIF-1 alpha gene or the CRISPR/Cas9 plasmid.
The invention also provides a cell strain for targeted knockout of HIF-1 alpha gene, which is prepared by transfecting the cell strain by the CRISPR/Cas9 lentiviral vector system.
The invention also provides a preparation method of the cell strain for targeted knockout of the HIF-1 alpha gene, which comprises the following steps:
(1) packaging the CRISPR/Cas9 lentiviral vector system by HEK293T cells to obtain lentiviral particles;
(2) and (2) transfecting the target cell strain by using the lentivirus particles obtained in the step (1) to obtain the cell strain with the HIF-1 alpha gene targeted knockout function.
As a preferred embodiment of the method for preparing a cell line targeted for knocking out the HIF-1 alpha gene, the target cell line in the step (2) is a tumor cell line.
As a preferred embodiment of the preparation method of the cell strain with targeted HIF-1 alpha gene knockout disclosed by the invention, the tumor cell strain is a cervical cancer cell strain.
The invention also provides application of the cell strain with the HIF-1 alpha gene targeted knockout in the research of oxygen perception pathways.
The invention has the beneficial effects that: the sgRNA for targeted knockout of the HIF-1 alpha gene can effectively target the HIF-1 alpha gene, a CRISPR/Cas9 lentiviral vector system is constructed by adopting the sgRNA, and a cell strain for targeted knockout of the HIF-1 alpha gene is obtained by further transfecting cells. The cell strain with the HIF-1 alpha gene knocked out in a targeted manner, which is prepared by the sgRNA, has the advantages of short preparation time, simplicity in operation, high transfection efficiency of the cell strain, stable expression effect and capability of continuously maintaining the knocking-down effect.
Drawings
FIG. 1 is a diagram showing the sequencing results of lentivirus plasmids constructed according to the present invention.
FIG. 2 is a diagram showing the results of detecting HIF-1 alpha protein expression in cell lines of targeted knockout of HIF-1 alpha gene and cell lines of a control group.
FIG. 3 shows the transcriptional level of HIF-1. alpha. mRNA after reverse transcription of a cell line targeted for the knockout of the HIF-1. alpha. gene.
Detailed Description
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Example 1 construction of plasmids targeting knockdown of HIF-1. alpha. Gene Using CRISPR/Cas9 technology
(1) Design Synthesis of sgRNA
An HIF-1 alpha gene exon part is selected by using a CRISPR on-line design tool (website http:// CRISPR. mit. edu /), and is designed, three sgRNA sequences with higher scores (HIF-1 alpha sgRNA1, HIF-1 alpha sgRNA2 or HIF-1 alpha sgRNA3) are obtained according to a scoring system, and no non-specific gene is verified by blast. The sequences of the resulting three pairs of sgRNA oligos are shown in Table 1.
TABLE 1
(2) Vector construction
1) EcoRI and XbaI were selected for digestion of the CD513B plasmid, and the digestion reaction system is shown in Table 2.
TABLE 2
| CD513B | 1μg |
| Quick cut EcoRI | 1μg |
| Quick cut XbaI | 1μg |
| 10×QuickCut Green Buffer | 5ul |
| ddH2O | up to 50μl |
2) The cleaved products were subjected to agarose gel electrophoresis, recovered using a gel recovery kit and run as described.
3) HIF-1. alpha. sgRNA3oligo1, HIF-1. alpha. sgRNA3oligo2 in Table 1 were phosphorylated and annealed using T4PNK to form double strands, and the reaction systems are shown in Table 3. The annealing program of the PCR instrument comprises the following steps: cooling at 95 deg.C for 5min, and at 1.5 min/deg.C for 25 deg.C.
4) The annealed oligo duplex and the digested CD513B plasmid were ligated using T4 ligase and ligated overnight at 16 ℃.
5) The ligated plasmid was amplified in E.coli DH5 α system, plated, single clones were selected for sequencing, and the sequencing results are shown in FIG. 1. The successfully sequenced plasmids were amplified and plasmids were extracted.
(3) Construction of cell line with targeted HIF-1 alpha gene knockout function
1) Virus package
HEK293T cell culture: the HEK293T cell line frozen in the liquid nitrogen tank was taken out and rapidly thawed in a water bath at 37 ℃. Transferring the cell suspension to a 10mL centrifuge tube in a clean bench, adding 5mL DMEM medium, centrifuging at low speed 1000rpm × 5min, discarding the supernatant, adding into 10% FBS-containing DMEM medium in 5% CO2And cultured at 37 ℃. Adherent HEK293T cells were digested for resuspension, plated into 6-well plates, and 5 × 10 added per well5A cell.
Cell transfection: the culture plate is pre-cultured in an incubator for 24 hours, and when the cell density reaches 50-60% confluence, Lipofectamine 3000(Invitrogen) is adopted for transfection. The method mainly comprises the following steps: cas 9-HIF-1. alpha. overexpression plasmid and 1ug sgRNA were diluted 1.5. mu.g with 100. mu.l serum-free DMEM medium, 2ul/ug P3000 was added and mixed well, 1ug:0.75ul Lipofectamine 3000 reagent was diluted with 100ul serum-free DMEM medium and mixed well, the plasmid dilution was mixed well with Lipofectamine 3000 dilution, and incubated at room temperature for 5min and added to wells of the plate containing cells and culture. The cells were placed in CO at 37 deg.C2After culturing for 4-6 h in the incubator, removing the culture medium containing the DNA-lipo3000 mixed solution, replacing a fresh cell culture medium containing 10% FBS, and continuing culturing for 24 h. A control group was set and transfected with the empty plasmid.
2) Virus-infected cells
Culturing conditions of the Hela cell strain: 5% CO2And culturing at 37 ℃ in a DMEM medium.
Viral infection: and (3) inoculating the Hela cells into a 6-well plate, sucking out the original culture medium of the cells after the cell confluence rate reaches 50% overnight, adding a fresh complete culture medium and virus liquid for infection, sucking out the culture medium containing the virus liquid after culturing for 12 hours, and replacing the fresh culture medium for continuous culture.
Screening stable cell strains: and after 48 hours of infection, puromycin is added to screen stable cell strains to obtain the positive cells. And further adopting a limiting dilution method for selection to obtain a stable monoclonal cell strain.
Identification of cell lines targeting knockout of the HIF-1 alpha gene: extracting total protein of the above cells, performing western blot, detecting expression of HIF-1 alpha protein in cells of control group and test group, extracting cell mRNA, performing reverse transcription, and detecting mRNA transcription level of HIF-1 alpha by qPCR. The results are shown in FIGS. 2 and 3. As can be seen in FIG. 2, the accumulation of HIF-1. alpha. protein in the lentivirus-transfected cells was significantly reduced compared to the control, indicating that HIF-1. alpha. protein expression was knocked out. As can be seen from fig. 3, HIF-1 α mRNA levels were significantly reduced (nearly 100%) in cells transfected with sgRNA lentiviral plasmids.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
SEQUENCE LISTING
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<120> sgRNA and CRISPR/Cas9 lentivirus system for targeted knockout of HIF-1 alpha gene and application
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Claims (10)
1. An sgRNA of a targeted knockout HIF-1 alpha gene, wherein the sgRNA is HIF-1 alpha sgRNA1, HIF-1 alpha sgRNA2 or HIF-1 alpha sgRNA 3;
the sequence of the HIF-1 alpha sgRNA1 is:
HIF-1αsgRNA1oligo1:5’-TAggTTCTTTACTTCGCCGAGATC-3’；
HIF-1αsgRNA1oligo2：5’-AAACGATCTCGGCGAAGTAAAGAA-3’；
the sequence of the HIF-1 alpha sgRNA2 is:
HIF-1αsgRNA2oligo1:5’-TAggCTTTACTTCGCCGAGATC-3’；
HIF-1αsgRNA2oligo2：5’-AAACGATCTCGGCGAAGTAAAG-3’；
the sequence of the HIF-1 alpha sgRNA3 is:
HIF-1αsgRNA3oligo1:5’-TAgGAGCTCCCAATGTCGGAGTT-3’；
HIF-1αsgRNA3oligo2:5’-AAACAACTCCGACATTGGGAGCT-3’。
2. a CRISPR/Cas9 plasmid containing a sgRNA targeted to knock-out the HIF-1 a gene of claim 1.
3. A CRISPR/Cas9 lentiviral vector system, wherein the lentiviral vector system is constructed by using sgRNA targeted to knock out HIF-1 alpha gene according to claim 1.
4. The method for constructing the CRISPR/Cas9 lentiviral vector system according to claim 3, comprising the steps of:
(1) carrying out enzyme digestion on lentivirus vector lenti CRISPRV2 to obtain a CRISPR/Cas9 lentivirus vector;
(2) phosphorylating a DNA sequence of sgRNA targeting a knockout HIF-1 α gene of claim 1;
(3) and (3) connecting the sgRNA in the step (2) with the CRISPR/Cas9 lentiviral vector in the step (1) to obtain the CRISPR/Cas9 lentiviral vector system.
5. A kit comprising a sgRNA targeting knockout of an HIF-1 α gene of claim 1 or a CRISPR/Cas9 plasmid of claim 2.
6. A cell strain with targeted HIF-1 alpha gene knockout function, which is prepared by transfecting the cell strain with the CRISPR/Cas9 lentiviral vector system of claim 3.
7. The method for preparing the cell line for targeted knockout of HIF-1 alpha gene according to claim 6, comprising the steps of:
(1) packaging the CRISPR/Cas9 lentiviral vector system by HEK293T cells to obtain lentiviral particles;
(2) and (2) transfecting the target cell strain by using the lentivirus particles obtained in the step (1) to obtain the cell strain with the HIF-1 alpha gene targeted knockout function.
8. The method for preparing the cell line of targeted knockout of HIF-1 α gene according to claim 7, wherein the cell line of interest in step (2) is a tumor cell line.
9. The method for preparing the cell line with targeted knockout of the HIF-1 alpha gene according to claim 8, wherein the tumor cell line is a cervical cancer cell line.
10. The use of the cell line targeted to knock out HIF-1 alpha gene according to claim 6 in the study of oxygen sensing pathway.
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