CN113025613B - ADORA2A gene knockout cell and construction method and application thereof - Google Patents

ADORA2A gene knockout cell and construction method and application thereof Download PDF

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CN113025613B
CN113025613B CN202110266853.XA CN202110266853A CN113025613B CN 113025613 B CN113025613 B CN 113025613B CN 202110266853 A CN202110266853 A CN 202110266853A CN 113025613 B CN113025613 B CN 113025613B
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陈春
陆铭浩
黄姚彪
杨连阳
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Abstract

The invention discloses an ADORA2A gene knockout cell, a construction method and application thereof, and belongs to the technical field of biological engineering. The ADORA2A gene knockout cell is constructed by using the CRISPR/Cas9 technology, has stable heredity and no obvious difference with a control cell in aspects of morphology, proliferation and the like, is an ideal ADORA2A gene knockout cell model, and can be used for researching the specific functions of ADORA2A protein in the aspects of tumor resistance, cell inflammation, virus infection and the like.

Description

ADORA2A gene knockout cell and construction method and application thereof
Technical Field
The invention relates to the technical field of biological engineering, in particular to an ADORA2A gene knockout cell and a construction method and application thereof.
Background
Natural killer cells (NK) are important immune cells in the body that are able to kill tumor cells directly without relying on antibodies and complement. In the tumor microenvironment in solid tumors, NK cells are often in a "functionally depleted" state, and adenosine in the tumor microenvironment impairs the survival, proliferation, cytotoxic functions of NK cells, thereby promoting tumor progression; therefore, the research on the immunosuppressive effect of adenosine in solid tumors is of great value.
In the traditional adenosine receptor research, a mouse is taken as a research object, and an inhibitor is used for inhibiting the adenosine receptor; however, the immune system of mice still differs from that of humans, and still has great limitations for revealing human biology, especially in terms of bioimmunity. Therefore, how to build a new research platform for adenosine immunosuppression is a problem to be solved urgently by the technicians in the field.
Disclosure of Invention
In view of the above, the invention provides an ADORA2A gene knockout cell, a construction method and an application thereof, and the ADORA2A gene knockout cell has a good application potential for exploring the effect of an adenosine A2A receptor (A2AR) in immune cells.
In order to achieve the purpose, the invention adopts the following technical scheme:
a gRNA of a targeted knockout human ADORA2A gene, the gRNA is two, and the nucleotide sequence is as follows:
SEQ ID NO.2：5’-CTCCTCGGTGTACATCACGG-3’，
SEQ ID NO.3：5’-CTGAACCCTAGGAGAGTCTA-3’。
a CRISPR/Cas9 electric transfer system for targeted knockout of human ADORA2A gene comprises gRNA of the targeted knockout human ADORA2A gene.
A method for constructing an ADORA2A gene knockout cell, comprising the following steps:
(1) according to the gRNA of the targeted knockout human ADORA2A gene, a double-stranded target fragment of the targeted human ADORA2A gene containing the gRNA is prepared.
(2) And (2) connecting the double-chain target fragment prepared in the step (1) with a vector to obtain a gene knockout vector for targeted knockout of the human ADORA2A gene.
(3) And (3) electrically transferring the gene knockout vector constructed in the step (2) into a host cell to obtain a monoclonal cell with ADORA2A gene function deletion.
Preferably, the nucleotide sequence of the forward fragment of the double-stranded target fragment is shown in SEQ ID NO. 4:
5’-GGCGCGCCGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGCTCCTCGGTGTACATCACGGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTATTGGGGATCCATTGGGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGCTGAACCCTAGGAGAGTCTAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTGGTACC-3’。
preferably, the host cell is a human cell.
An ADORA2A gene knockout cell constructed according to the above method.
The ADORA2A gene knockout cell is applied to the construction of a solid tumor immunity research cell model.
In conclusion, the ADORA2A gene is knocked out on the human cell genome for the first time by using the CRISPR/Cas9 technology, and the method is simple to operate and short in period.
The ADORA2A function of the ADORA2A gene knockout cell is completely lost, the morphology of the knockout cell is not obviously different from that of a control cell, the proliferation is stable, and the ADORA2A gene knockout cell model is an ideal ADORA2A gene knockout cell model and can be used for researching the specific functions of ADORA2A protein on cells in the aspects of tumor resistance, cell inflammation, virus infection and the like.
Compared with cells without ADORA2A gene knockout, ADORA2A gene knockout cells can remarkably enhance the killing capacity of tumor cells under adenosine environment.
Drawings
Figure 1 shows a map of the position of insertion of a double-stranded fragment of interest into a CRISPR vector.
FIG. 2 is a graph showing the sequencing results of knockout cells NK92-2AK, wherein:
F：GGCCATGCCCATCATGGGCTCCTCGGTGTACATCA-del 8459bp-ACTCTCCTAGGGTTCAGGAGCTGCTGGGCCCAGAG；
R：CTGTGGCCATGCCCATCATGGGCTCCTCGGTGTAC-del 8522bp-in 30bp-AAAATGTAAGTGTGAGGAAACCCTTTTTATTTTAT。
FIG. 3 is a diagram showing the result of verifying the ADORA2A loss of function in ADORA2A knockout cell NK92-2 AK.
FIG. 4 shows the comparison of NK92-2AK with control cells at passage 15.
Figure 5 shows the results of a proliferation rate study in an adenosine microenvironment.
FIG. 6 shows the results of the tumor cell killing ability study in adenosine microenvironment.
FIG. 7 shows the results of IFN- γ secretion capacity studies in adenosine microenvironment.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Definition of
The term "gene knockout" refers to inactivation of a particular target gene by homologous recombination. The invention leads the ADORA2A gene in a host cell to be knocked out by a gene knocking-out technology. The obtained ADORA2A gene knockout NK92 cells can exert normal anti-tumor functions in an adenosine-rich microenvironment.
The term "CRISPR/Cas 9" is an adaptive immune defense developed by bacteria and archaea during long-term evolution, and can be used to fight invading viruses and foreign DNA. Crispr (clustered regulated short palindromic repeats) is a genetic system used by bacteria to defend against viral attack/evade mammalian immune responses. Scientists use grnas to guide Cas9 nuclease to cut and modify at specific genomic sites of various cells.
The term gRNA is guide RNA (guide RNA), which refers to a small non-coding RNA that directs the insertion or deletion of uridine residues into the kinetoplast (kinetoplastid) during RNA editing; can be paired with pre-mRNA.
Example 1
Construction of ADORA2A Gene knockout cell NK92-2AKO
(1) The following 2 gRNAs were designed based on the human ADROA2A gene sequence (25764 bp in total length, wherein the CDS region encoding amino acids is shown in SEQ ID NO. 1):
gRNA-A1：5’-CTCCTCGGTGTACATCACGG-3’，SEQ ID NO.2；
gRNA-A2：5’-CTGAACCCTAGGAGAGTCTA-3’，SEQ ID NO.3。
the above gRNA was synthesized artificially.
Synthesizing a double-stranded target fragment hADORA2A gene hADORA2A-gRNA containing the gRNA by a primer bridging method according to the gRNA, wherein the nucleotide sequence of a forward fragment of the gRNA is shown in SEQ ID NO. 4:
5’-GGCGCGCCGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGCTCCTCGGTGTACATCACGGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTATTGGGGATCCATTGGGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGCTGAACCCTAGGAGAGTCTAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTGGTACC-3’。
(2) the CRISPR vector TKO138 (purchased from Starfish organisms) is subjected to enzyme digestion at 37 ℃ for 2h by using AscI and KpnI according to an enzyme digestion system in the table 1; the enzyme digestion product is connected with hADORA2A-gRNA at 16 ℃ overnight according to a connection system shown in the table 2, and a gene knockout carrier TKO138-1 (shown in figure 1) of the targeted knockout human ADORA2A gene is obtained.
TABLE 1
Components Dosage of
CRISPR vectors 1μg
AscI 1μL
KpnI 1μL
10×NEBuffer 5μL
H 2 O to50μL
TABLE 2
Figure BDA0002972358480000051
Figure BDA0002972358480000061
The TKO138-1 is transformed into DH5 alpha competent cells, after being cultured at 37 ℃ overnight, the selected clonic bacteria are sequenced to obtain correct positive clonic bacteria containing TKO 138-1.
The correct positive clones were cloned as follows 1: inoculating 500 proportion into LB liquid culture medium with Amp + resistance, placing in a shaking table at 37 ℃, shaking and culturing for 16h at 180r/min, and then extracting endotoxin-free plasmid by using an endotoxin-free plasmid miniprep kit to obtain TKO138-1 plasmid.
(3) Culturing wild NK92 cells (purchased from China center for type culture Collection, number GDC052), and electrically transferring TKO138-1 into NK92 cells according to the operating instruction of a Neon electrotransformation apparatus when the cells are in good state and most of the cells are grown, wherein the electrotransformation system is shown in Table 3, and the electrotransformation program is shown in Table 4; after culturing for 48h, obtaining knockout cell heterozygote NK92-2AK mix 。
TABLE 3
Components Dosage of
NK92 cells 2×10 5 An
TKO138-1 plasmid 1μg
Buffer R to 10μL
TABLE 4
Pulse voltage (v) Pulse width (ms) Pulse number (times)
1300 10 3
Subsequently, the KO corresponding to ADORA2A gene was obtained by limiting dilution culture:
mixing NK92-2AK mix Cell dilutionTo 10/mL, transferred to 96-well plates at 100. mu.L per well; half of the fresh medium was replaced at two days intervals and continued to culture for a sufficient number of cells.
Extracting a KO genome, carrying out PCR amplification on a sequence near a target point by taking the KO genome as a template, and carrying out amplification primers:
ADORA2AF：5’-GTAAGGGGTGGCACTTTCCG-3’，SEQ ID NO.5；
ADORA2AR：5’-TAGCTCTGCGCTACTCAGCA-3’，SEQ ID NO.6；
and sequencing the PCR product to determine the sequence information near the target point, and comparing the sequence information with the sequence information of the corresponding position in the genome of the wild type NK92 cell to obtain the monoclonal cell line NK92-2AKs with the ADORA2A gene function deletion.
A monoclonal cell NK92-2AK is selected from a monoclonal cell line NK92-2AKs, and the genome knockout result shows that a target point has large-fragment base deletion (figure 2).
qPCR validation of ADORA2A Gene knockout cells
To further confirm the deletion of ADORA2A gene in ADORA2A knock-out cells, total RNA of wild type NK92(WT) and NK92-2ak (ko) were extracted, reverse-transcribed into cDNA, and ADORA2A expression was detected using cDNA as a template, and primers were quantified:
qADORA2AF：5’-ATCGCCATTGACCGCTACAT-3’，SEQ ID NO.7；
qADORA2AR：5’-TGGTTCTTGCCCTCCTTTGG-3’，SEQ ID NO.8。
the results are shown in figure 3, the KO group qPCR can not detect the mRNA expression of ADORA2A, and the result shows that the deletion of mRNA caused by the deletion of the coding region gene of the ADORA2A gene in NK92 cells leads to the deletion of the function of the ADORA2A gene in cells, and ADORA2A gene knockout cells NK92-2AK are obtained.
Although the method takes an NK cell line NK92 as an example, for other cell lines and primary cells of other origins, a corresponding host cell with the ADORA2A gene function deletion can be obtained by the method.
Cellular morphology and genetic stability of ADORA2A Gene knockout cells
As shown in FIG. 4, no obvious morphological difference was observed between NK92-2AK and NK92 normal cells during 15 consecutive passages. Also, no mRNA expression of ADORA2A was detected in NK92-2AK group after serial passage, indicating that the constructed cells were very stable.
Example 2 proliferation Rate Studies of NK92-2AK under adenosine microenvironment
The CCK8 method is adopted to detect the relative proliferation rate of NK92-2AK cells after the cells are treated with 50 mu M adenosine for 12 hours:
after counting, the same amount (20000) of NK92-2AK cells (KO) and wild type NK92 cells (WT) were divided into two groups, inoculated in a 96-well plate, and then 100. mu.L of Adenosine (ADO) -free medium and 50. mu.M adenosine-containing medium were added, respectively. After 12h of incubation, 10. mu.L of CCK8 solution was added, and after incubation in a carbon dioxide incubator for 2h, absorbance was measured at a wavelength of 450nm using a microplate reader.
Each cell was treated for 4 replicates each, and the results are shown in FIG. 5, where there was a significant decrease in proliferation rate after 50 μ M adenosine treatment of wild type NK92 cells compared to the control, whereas proliferation rate was unaffected for NK92-2AK cells treated with 50 μ M adenosine. The results showed that ADORA2A knock-out cell NK92-2AK was able to proliferate normally in the presence of 50. mu.M adenosine, with an adenosine-resistant phenotype.
Example 3 tumor cell killing and IFN-gamma secretion Capacity Studies of NK92-2AK in adenosine microenvironment
1. Research on killing capacity of NK92-2AK tumor cells under adenosine microenvironment
When the A549 cells (purchased from Shanghai department of sciences cell bank, catalog number SCSP-503) are in good state and the fusion degree is 90%, digesting the cells by pancreatin, counting the cells, inoculating the cells into a 96-well plate, adding 10000 cells per well, and culturing for 12h by adding 100 mu L of A549 cell culture medium to adhere to the wall.
After counting, the same amount (20000) of NK92-2AK cells (KO) and wild type NK92 cells (WT) were divided into two groups, inoculated into 96-well plates containing A549 cells, and then 100. mu.L of Adenosine (ADO) -free medium and 50. mu.M adenosine-containing medium were added, respectively. After 12h of incubation, 10. mu.L of CCK8 solution was added, and after incubation in a carbon dioxide incubator for 2h, absorbance was measured at a wavelength of 450nm using a microplate reader.
Each cell was treated 4 replicates each, and the results are shown in figure 6, with NK92-2AK having significantly enhanced tumor killing compared to wild-type NK92 cells in the presence of 50 μ M adenosine; the ADORA2A gene knockout cell NK92-2AK is shown to have stronger anti-tumor capability in an adenosine microenvironment.
2. Research on IFN-gamma secretion capability of NK92-2AK under adenosine microenvironment
When the A375 cells (purchased from Shanghai department of sciences cell bank, catalog number SCSP-533) are in good state and the fusion degree is 90%, digesting with pancreatin, counting, inoculating into a 96-well plate, 10000 cells per well, adding 100 μ L of A375 cell culture medium, culturing for 12h, and allowing them to adhere to the wall. After counting, the same amount (20000) of NK92-2AK cells (KO) and wild type NK92 cells (WT) were divided into two groups, inoculated into a 96-well plate containing A375 cells, and then added with 100. mu.L of Adenosine (ADO) -free medium and 50. mu.M adenosine-containing medium, respectively. After 12h of culture, centrifuging and taking the supernatant, and detecting the IFN-gamma secretion condition by flow.
Each cell was treated 3 replicates each, and the results are shown in figure 7, with significant reduction in NK92 cell IFN- γ secretion in the presence of 50 μ M adenosine, compared to the control, while adenosine had no significant effect on NK92-2AK cell IFN- γ secretion; the ADORA2A gene knockout cell NK92-2AK is shown to be capable of normally secreting IFN-gamma in an adenosine microenvironment and has normal anti-tumor capability.
The general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Sequence listing
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<212> DNA
<213> Artificial
<400> 3
ctgaacccta ggagagtcta 20
<210> 4
<211> 734
<212> DNA
<213> Artificial
<400> 4
ggcgcgccga gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg 60
ttagagagat aattggaatt aatttgactg taaacacaaa gatattagta caaaatacgt 120
gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg 180
actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt 240
ggaaaggacg aaacaccgct cctcggtgta catcacgggt tttagagcta gaaatagcaa 300
gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt 360
attggggatc cattgggagg gcctatttcc catgattcct tcatatttgc atatacgata 420
caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca 480
aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt 540
taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata 600
tatcttgtgg aaaggacgaa acaccgctga accctaggag agtctagttt tagagctaga 660
aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt 720
gcttttttgg tacc 734
<210> 5
<211> 20
<212> DNA
<213> Artificial
<400> 5
gtaaggggtg gcactttccg 20
<210> 6
<211> 20
<212> DNA
<213> Artificial
<400> 6
tagctctgcg ctactcagca 20
<210> 7
<211> 20
<212> DNA
<213> Artificial
<400> 7
atcgccattg accgctacat 20
<210> 8
<211> 20
<212> DNA
<213> Artificial
<400> 8
tggttcttgc cctcctttgg 20

Claims (7)

1. gRNA targeted for knock-out of the human ADORA2A gene, characterized in that,
the gRNA is two, and the nucleotide sequence is as follows:
SEQ ID NO.2：5’-CTCCTCGGTGTACATCACGG-3’，
SEQ ID NO.3：5’-CTGAACCCTAGGAGAGTCTA-3’。
2.a CRISPR/Cas9 electrotransfer system for targeted knockout of human ADORA2A gene, comprising a gRNA targeted for knockout of human ADORA2A gene of claim 1.
3.A method for constructing an ADORA2A gene knockout cell, comprising the steps of:
(1) the gRNA targeted for knockout of the human ADORA2A gene according to claim 1, a double-stranded fragment of interest targeted to the human ADORA2A gene containing the gRNA;
(2) connecting the double-chain target fragment prepared in the step (1) with a vector to obtain a gene knockout vector for targeted knockout of the human ADORA2A gene;
(3) and (3) electrically transferring the gene knockout vector constructed in the step (2) into a host cell to obtain a monoclonal cell with ADORA2A gene function deletion.
4. The method of claim 3, wherein the ADORA2A knockout cell is constructed,
the nucleotide sequence of the forward fragment of the double-stranded target fragment is shown as SEQ ID NO. 4:
5’-GGCGCGCCGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGCTCCTCGGTGTACATCACGGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTATTGGGGATCCATTGGGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGCTGAACCCTAGGAGAGTCTAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTGGTACC-3’。
5. the method of claim 3, wherein the ADORA2A knockout cell is constructed,
the host cell is a human cell.
6. An ADORA2A knockout cell constructed according to the method of any one of claims 3 to 5.
7. Use of an ADORA2A gene knockout cell of claim 6 for constructing a cell model for solid tumor immune research.
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Title
A2AR Adenosine Signaling Suppresses Natural Killer Cell Maturation in the Tumor Microenvironment;Arabella Young等;《Cancer Research》;20171211;第78卷(第4期);第1003-1016页 *
Blockade of A(2A) receptors potently suppresses the metastasis of CD73(+) tumors;Paul A. Beavis等;《PNAS》;20130903;第110卷(第36期);第14711-14716页 *
小鼠脑组织腺苷A2A受体缺失模型的建立与评价;戴双双 等;《生物工程学报》;20080630;第24卷(第4期);第700-706页 *

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