CN105441451B - A kind of sgRNA targeting sequencing of special target people ABCB1 gene and application - Google Patents
A kind of sgRNA targeting sequencing of special target people ABCB1 gene and application Download PDFInfo
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Abstract
The invention belongs to genetic engineering application fields, and in particular to a kind of sgRNA targeting sequencing of special target people ABCB1 gene and application.The sgRNA targeting sequencing of special target people's ABCB1 gene is Sg1 or Sg2；Its nucleotide sequence is respectively as follows: Sg1:5'-TTGGACTGTCAGCTGCTGTC-3'；Sg2:5'-TGGACTTCCTCTCATGATGC-3'.The present invention designs two single-stranded oligo sequences of synthesis according to sgRNA targeting sequencing, annealing forms double-strand, then it is connect with Cas9 carrier, sgRNA and CRISPR system is introduced into target cell using Cas9 carrier, Cas9 albumen can find matched DNA sequence dna under the guidance of sgRNA, it is sheared, realizes the knockout of Gene A BCB1.
Description
Technical field
The invention belongs to genetic engineering application fields, and in particular to a kind of sgRNA guiding of special target people ABCB1 gene
Sequence and application.
Background technique
CRISPR-Cas9 is bacterium and archeobacteria develop out during evolution it is a kind of for resisting external infringement
Immune intrusion system, virus and exogenous DNA including resisting invasion.In modern genetic engineering application field and TALEN
(transcription activator-like effector nuclease) and ZFN (zinc-finger nuclease)
Technology becomes three big genome edit tools side by side.It is compared to TALEN and ZFN technology, CRISPR-Cas9 technology has special
Property DNA recognition capability, in the second class CRISPR system, Cas9 endonuclease sgRNA guidance under cutting double-stranded DNA, make
At genome double-strand break, non-specific recombination is generated using the unstability of cellular genome reparation and repairs wrong (insert to generate
Enter or lack), the forfeiture of gene function is caused so as to generate frameshift mutation, realizes the purpose of gene knockout.Its
The design and synthesis workload of sgRNA is far smaller than the building process of TALEN and ZFN identification module, and toxicity is well below ZFN
Technology.But CRISPR-Cas9 technology also has the shortcomings that contextual dependency, can be only applied to upstream at present has PAM sequence
Target site.
While multidrug resistance (MDR) is referred to a kind of drug with drug resistance, to other structures difference, action target spot
Different anti-tumor drugs also have drug resistance.Multidrug resistance be cause Medication for Cancer and chemotherapy of tumors failure it is important
One of reason.And one of the main mechanism of multidrug resistance is exactly the mistake of ABC (ATP-binding cassette) transport protein
Amount expression, they can hydrolyze energy supply by ATP and pump out anticancer drug extracellularly.Its principal home's family member includes ABCB1 (P-
GP), ABCC1 (MRP1) and ABCG2 (BCRP) etc..In cell the high expression of ABCB1 will will lead to the generation of multidrug resistance to
The expression for making treatment failure, therefore suppressing or eliminating p-gp can be effectively solved the multidrug resistance occurred in oncotherapy and ask
Topic.
Summary of the invention
In order to overcome in the prior art it is certain it is high expression p-gp albumen cells in occur the disadvantages of multidrug resistance with not
Foot, the primary purpose of the present invention is that a kind of sgRNA targeting sequencing of special target people ABCB1 gene is provided, sgRNA guiding
Sequence can be used for knocking out people's ABCB1 gene, and then suppress or eliminate the expression of p-gp.
Another object of the present invention is to provide it is a kind of using CRISPR-Cas9 system knock out people ABCB1 gene method,
This method edits ABCB1 gene using CRISPR-Cas9 technology, make its normal sequence occur missing or mutation to
Achieve the purpose that knock out the gene.
A further object of the present invention is to provide the applications of the sgRNA targeting sequencing of above-mentioned special target people ABCB1 gene.
A kind of sgRNA targeting sequencing of special target people ABCB1 gene is Sg1 or Sg2；Its nucleotide sequence is respectively as follows:
Sg1:5'-TTGGACTGTCAGCTGCTGTC-3' is located at the 8th exon of Gene A BCB1；
Sg2:5'-TGGACTTCCTCTCATGATGC-3', positioned at the 5th exon of Gene A BCB1；
A method of people ABCB1 gene being knocked out using CRISPR-Cas9 system, is comprised the following steps:
(1) positive oligonucleotides is obtained plus CACCG at the end 5' of above-mentioned targeting sequencing；It is obtained simultaneously according to targeting sequencing
Its corresponding DNA complementary strand, and reverse oligonucleotide is obtained plus AAAC at its 5 ' end；It is respectively synthesized above-mentioned positive few nucleosides
The positive oligonucleotides and reverse oligonucleotide of synthesis are denaturalized by acid and reverse oligonucleotide, and annealing forms double-strand；
(2) double-strand made from step (1) is connect with Cas9 carrier, obtains recombination and knocks out expression vector；
(3) step (2) recombination obtained is knocked out into expression vector and packaging system cotransfection incasing cells, then harvest disease
Poison is purified and is concentrated, and obtains virion；
(4) by infestation with virus particles cell made from step (3), screening surely turns cell, succeeds and knock out ABCB1 gene
Cell；
Cas9 carrier described in step (2) is preferably lentiCRISPRv2 carrier；
Incasing cells described in step (3) is preferably 293T cell；
Package carrier in packaging system described in step (3) is preferably pMD2.G and psPAX2；
Cell described in step (4) is preferably multi-drug resistance of the tumor cell；
Cell described in step (4) is more preferably HCT-8/V or KBv200；
The sgRNA targeting sequencing of special target people's ABCB1 gene is in preparing artitumor multi-medicine-resistant drug
Application；
The tumour is preferably human mouth squamous carcinoma or Human colorectal carcinoma；
The present invention has the following advantages and effects with respect to the prior art:
(1) in order to avoid the generation of miss target phenomenon to greatest extent, selection uses two sgRNA, that is, is located at ABCB1 gene two
A different location.SgRNA and CRISPR system is introduced into target cell using lentiCRISPRv2 carrier, Cas9 albumen
Matched DNA sequence dna can be found under the guidance of sgRNA, sheared.
(2) contain Puromycin resistant gene in carrier, cell is screened using Puromycin, is not transferred to
The cell of lentiCRISPRv2 carrier will be eliminated in screening process.
(3) the sgRNA targeting sequencing of special target people ABCB1 gene provided by the invention, can pass through CRISPR-Cas9 system
System knocks out or editor's ABCB1 gene, and then the expression for suppressing or eliminating p-gp can be effectively solved and occur in oncotherapy
Multidrug resistance problem.
Detailed description of the invention
Fig. 1 is the sequence and its position view of the sgRNA targeting sequencing of special target people's ABCB1 gene.
Fig. 2 is to extract cytogene after the sgRNA designed using the present invention edits the cell strain of high expression p-gp
The sequencing analysis figure that sequencing is compared with wild-type cell is carried out after group.
Fig. 3 is the sequencing peak figure of embodiment 2PCR product sequencing result.
Fig. 4 is the western blot interpretation of result of the p-gp expressing quantity of the cell strain of successful knockout ABCB1 gene
Figure.
Fig. 5 is the cell of successful knockout ABCB1 gene after Doxorubicin, Vincristine and Cisplatin processing
The cell activity result analysis chart of strain.
Fig. 6 is that the edited drug accumulation of cell strain progress that the sgRNA designed using the present invention expresses p-gp to height is real
Test result result analysis chart.
Fig. 7 is the streaming result analysis chart of 3 drug accumulation of embodiment experiment.
Fig. 8 is the quantification treatment analysis chart of 3 drug accumulation of embodiment experiment streaming result.
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
Cell strain used in embodiment is purchased from ATCC, and lentiCRISPRv2 carrier is purchased from Addgene, restriction endonuclease
BsmB I is purchased from Biolabs, and Polyetherimide (PEI) is purchased from purchased from Ploysciences, Puromycin and Polybreen
Sigma；
Embodiment 1
(1) sgRNA is designed
According to the genome sequence (gene ID:5343) of people's ABCB1 gene, 2 targeting people ABCB1 genes are designed
sgRNA.The oligonucleotides sgRNA targeting sequencing of 20nt are as follows: Sg1:5'-TTGGACTGTCAGCTGCTGTC-3'(is located at gene
The 8th exon of ABCB1) and Sg2:5'-TGGACTTCCTCTCATGATGC-3'(be located at it is aobvious outside the 5th of Gene A BCB1
Son) (Fig. 1)；Positive oligonucleotides (Forward oligo) is obtained plus CACCG at its end 5'；It is obtained according to targeting sequencing
Corresponding DNA complementary strand, and reverse oligonucleotide (Reverse oligo) is obtained plus AAAC at its 5 ' end.It is respectively synthesized
Above-mentioned forward direction oligonucleotides and reverse oligonucleotide, by the Forward oligo of the sgRNA oligonucleotide of synthesis and
Reverse oligo is denaturalized in pairs, annealing；The double-strand for being connected into expression vector lentiCRISPRv2 carrier can be formed after annealing,
Designed gRNA target sequence blast is carried out simultaneously to compare to exclude nonspecific target enzyme site, specific oligonucleotides
Sequence is shown in Table 1.
The oligonucleotide sequence of 1 sgRNA targeting sequencing of table
(2) carrier of building expression sgRNA
Viral vectors lentiCRISPRv2 carrier has I restriction enzyme site of BsmB, with I digestion of BsmB, wherein digestion system (20
μ L system) are as follows: I 1 μ L of BsmB；10×NE buffer 2μL；1 μ L of plasmid；ddH2O16μL；Digestion condition are as follows: 37 DEG C of digestion 1h；
By the carrier lentiCRISPRv2 after digestion respectively with step (1) annealing double-strand obtained using T4 ligase into
Row connection, linked system (10 μ L) are as follows: annealing double-strand (Sg1 or Sg2) 2 μ L, lentiCRISPRv2 carrier 2 μ L, 10 × NEB
1 μ L, T4DNA Ligase of T4DNA Ligase Buffer 1 μ L, ddH2O 4μL；Condition of contact are as follows: 16 DEG C of connections are overnight；
By connection product transformed competence colibacillus cell stbl3, specific method for transformation are as follows: -80 DEG C of taking-up competent cells
Stbl3, and dissolves on ice；Then the above-mentioned connection product of 1 μ L, ice bath 30min after mixing are added in 50 μ L competent cells；42
DEG C water-bath 90s, does not shake in the process；1~2min of cooled on ice；Then 800 μ L LB culture mediums, 37 DEG C of shaking table 30min are added；
Apply AMP+Plate (100 μ g/mL) overnight incubation, 37 DEG C of shaking tables expand culture overnight after picking positive colony, and use Hipure
Plasmid Micro Kit C (Magen) extracts plasmid and sequence verification, obtains the carrier of expression sgRNA
(targeting sequencing is by lentiCRISPRv2-hABCB1-Sg1 (targeting sequencing Sg1) and lentiCRISPRv2-hABCB1-Sg2
Sg2) (hABCB1 is expressed as people's gene ABCB1).
Embodiment 2
(1) core plasmid and packaging plasmid pMD2.G and psPAX2 cotransfection are into 293T cell
293T cell is cultivated, the rate of collecting to 293T cell reaches 50%~60%, and 12~18h is best transfection after planting plate
Time；Fresh medium is replaced before transfection, and 3mL culture medium is added in 60mm capsule；The usage amount of plasmid is core matter when transfection
(lentiCRISPRv2-hABCB1-Sg1 made from embodiment 1 or lentiCRISPRv2-hABCB1-Sg 2, separately take grain
LentiCRISPRv2 empty carrier is as control) 4 μ g, 3 psPAX2 μ g, 1 pMD2.G μ g, 24 PEI μ L, supplement DMEM to totality
Product is 200 μ L, and addition sequence is respectively DMEM, PEI and Plasmid DNA (core plasmid, pMD2.G and psPAX2)；Then room temperature is quiet
Only 30min polymerize PEI sufficiently with Plasmid DNA, and above-mentioned culture, which is added dropwise, in rotaring redyeing system after polymerization 293T cell
Capsule in, 37 DEG C of 5%CO are put into after gently shaking up2Continue to cultivate in incubator；
(2) viral harvest and concentration
Collect after transfection for 24 hours, the supernatant of 48h, 72h, 96h 293T cell, collect that be supplemented 3mL after virus new every time
In capsule, the virus stock solution used first harvested can be kept in 4 DEG C of refrigerators fresh culture solution after being closed with sealed membrane；It is all received to virus liquid
1000rpm is centrifuged 5min after the completion of collection, to remove cell fragment, removes cell fragment with 0.45 μm of membrane filtration and other are miscellaneous
Matter；Take filtered virus stock solution used in 100kD ultrafiltration column, 4 DEG C, 4000g is centrifuged 30min, collects about 300 μ L of residue in filter membrane
Viral concentration liquid, the 50 every pipes of μ L are sub-packed in 1.5mL EP pipe, set -80 DEG C can long-term preservation, avoid multigelation；
(3) packaging virus particle infects aim cell strain HCT-8/V and KBv200 respectively
The good aim cell strain HCT-8/V (Human colorectal carcinoma multidrug resistance cell) to be infected of the previous day kind is mentioned in 6 orifice plates
With KBv200 (human mouth squamous carcinoma multidrug resistance cell), cell collects rate and reaches 40~50% and is advisable when cell infection；Before infection
Liquid is changed with 1mL fresh culture, 2 μ are added in the 50 μ L viral concentration liquid made from other 1mL fresh culture dilution step (2)
After L polybrene (Polybreen, 10mg/mL, final working concentration are 10 μ g/mL) mixes, six orifice plate 1 is uniformly instilled dropwise
In hole, gently shake even；According to the property of different cell strains, 6~48h changes fresh medium after infection；The base of lentivirus mediated
Because that can express successively during 48~96h, if efficiency is undesirable can to carry out repeated infection, it is infected expression target gene
Cell is theoretically stable strain；Core carrier has the selection markers of Puro (puromycin), can use Puromycin progress
Stablize the screening of strain；Whole lethasl concentrations of cell strain Puromycin used in measurement experiment are 50 μ g/mL before screening, with complete
Portion's lethasl concentration screens infected cell HCT-8/V or KBv200；
(4) stablize the screening and culture of infection cell strain
Metainfective cell strain HCT-8/V or KBv200 is screened using the Puromicin of 50 μ g/mL, screening is held
Continuous 20d, cultivates the cell after screening；
(5) primer is identified according to two sections of designed sgRNA sequence designs, for reflecting to target fragment after knockout
Fixed, designed primer is as shown in table 2:
Table 2 identifies primer sequence
Using QuickExtract DNA extraction kit to stablize infection cell strain HCT-8/V or KBv200 into
The extracting of row genome carries out PCR identification using identification primer；
Wherein, for the cell strain HCT-8/V after the virion transfection containing lentiCRISPRv2-hABCB1-Sg1
Or KBv200, PCR reaction system (50 μ L) are as follows: 10 × buffer, 5 μ L；dNTP 1μL；1 μ L of template DNA；Detection
1-F(10μM)1μL；Detection 1-R(10μM)1μL；1 μ L of Pfu high fidelity enzyme；ddH2O 40μL；PCR reaction amplification item
Part: 95 DEG C of denaturation 5min；95 DEG C of denaturation 30S, 55 DEG C of annealing 30S, 72 DEG C of extension 2min, 35 recycle；72 DEG C are finally prolonged
Stretch 10min；
For containing lentiCRISPRv2-hABCB1-Sg2 virion transfection after cell strain HCT-8/V or
KBv200, PCR reaction system (50 μ L): 10 × buffer, 5 μ L；dNTP 1μL；1 μ L of template DNA；Detection 2-F
(10μM)1μL；Detection 2-R(10μM)1μL；1 μ L of Pfu high fidelity enzyme；ddH2O 40μL；PCR reaction amplification condition: 95
DEG C denaturation 5min；95 DEG C of denaturation 30S, 46 DEG C of annealing 30S, 72 DEG C of extension 2min, 35 recycle；72 DEG C of last extensions
10min；
Glue recycling is carried out using Gel Extraction Kit (OMEGA) after PCR reaction product race glue, to glue recovery product
Sequencing analysis is carried out, sequencing result is as shown in Figures 2 and 3.Sequencing result shows that two editor's groups are mutated in two ways,
Control group and wild type gene group compare that there is no variations.Present invention hair, which has been successfully established, knocks out the thin of ABCB1 gene
Born of the same parents strain HCT-8/V sg1；HCT-8/V sg2；KBV200sg1；KBV200sg2.
3 In vitro cell experiment of embodiment carries out surveyor's Gene A BCB1 to cell after screening and knocks out effect
(1)western blot
Utilize the cell strain HCT-8/V of successful knockout ABCB1 gene in western blot experimental identification embodiment 2
The p-gp expressing quantity of sg1, HCT-8/V sg2, KBV200sg1 and KBV200sg2, Successful transfection empty carrier
The cell strain HCT-8/V and KBv200 of lentiCRISPRv2 is as control, wherein primary antibody is Anti-ABCB1 (MM SC-
13131, it is purchased from santa cruze), secondary antibody is Anti-mouse IgG, HRP-linked Antibody (article No. 7076, cell
Signaling), specific method is routine western blot operating process.
As a result it as shown in figure 4, in HCT-8/V and KBv200 cell, is transferred to the cell of sg1 and sg2 and is only transferred to carrier
Cell p-gp expression quantity have a notable difference, almost do not express p-gp albumen in sg1 and sg2, can be used as ABCB1 gene and be knocked
One of evidence.
(2) mtt assay detects cell activity
Choose the specific substrate Doxorubicin (be purchased from LC) and Vincristine (purchased from LC) of p-gp with it is non-specific
Property substrate cisplatin (being purchased from LC) with mtt assay detect detection successful knockout ABCB1 gene cell strain HCT-8/V sg1,
The sensibility of HCT-8/V sg2, KBV200sg1 and KBV200sg2 to said medicine.By cell HCT-8/V sg1, HCT-8/V
The cell strain HCT-8/V and KBv200 of sg2, KBV200sg1 and KBV200sg2 and Successful transfection empty carrier lentiCRISPRv2
Control cell is inoculated into 96 orifice plates with the quantity of every 3000~5000 cells in hole, after cell is adherent, is separately added into different dense
Doxorubicin (0.03,0.1,0.3,1,3,10,30 μM), Vincristine (0.03,0.1,0.3,1,3,10,30 μ of degree
) and cisplatin (0.03,0.1,0.3,1,3,10,30,100 μM) M.After cultivating 72h, every hole is added 10 μ L 5mg/mL's
MTT is further cultured for 4h, then abandons the DMSO that 100 μ L are added in the every hole of culture solution, reads the light absorption value in every hole in 570nm with microplate reader.
With Bliss method calculation of half inhibitory concentration value (IC50).
As a result as shown in figure 5, compared with the control cell for being individually transferred to carrier, the cell strain of successful knockout ABCB1 gene
(HCT-8/V sg1, HCT-8/V sg2, KBV200sg1 and KBV200sg2) is to the quick of Doxorubicin and Vincristine
Perception increases, and IC50 value is substantially reduced.And for the non-specific substrate Cisplatin of p-gp, control group with successfully strike
Except the cell strain comparison of ABCB1 gene, IC50 value is not variant.This can be used as using CRISPR-Cas9 system to HCT-8/V
It carries out knocking out one of successful evidence with the ABCB1 gene of KBv200.
(3) drug accumulation is tested
P-gp is by the transmembrane protein of the Gene A BCB1 high glycosylation encoded, it is by consumption ATP by drug from thin
It is intracellular to pump out, to reduce the drug concentration in tumour cell.Select the substrate of two kinds of p-gp: Rh-123 is (purchased from sigma-
Aldrich) and Doxorubicin sees the accumulation of two kinds of molecules in the cell: by cell HCT-8/V sg1, HCT-8/V
The cell strain HCT-8/V and KBv200 of sg2, KBV200sg1 and KBV200sg2 and Successful transfection empty carrier lentiCRISPRv2
Control cell is with every hole 2.5 × 105A quantity is inoculated into 6 orifice plates, after cell is adherent, is separately added into 10 μM of Rh-123
(Rhodamine 123) and Doxorubicin after cultivating 2h, PBS rinse 3 times, shoot fluorescence under fluorescence microscope, so
Cell is digested with pancreatin afterwards, stream type cell analyzer fluorescence intensity is used after being resuspended with PBS, then uses Flow jo software
Carry out quantitative analysis；
As a result as shown in Fig. 6,7,8, two editor's groups (HCT-8/V sg1, HCT-8/V sg2, KBV200sg1 and
KBV200sg2) fluorescence intensity increased significantly compared with the control group, it was demonstrated that the p-gp that editor organizes cell is not functioned two kinds
Drug pumps out cell.And the p-pg of control group can normally play Teat pipette function, therefore fluorescence intensity is lower.Show two editors
Group ABCB1 gene is by successful knockout.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Sequence table
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<213>artificial sequence (Artificial Sequence)
<220>
<223>primer Detection 2-F
<400> 9
tatataccat taaatacttt tacag 25
<210> 10
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223>primer Detection 2-R
<400> 10
ctaattaagt tcctatattt cttct 25
Claims (9)
1. a kind of sgRNA targeting sequencing of special target people ABCB1 gene, it is characterised in that: the special target people ABCB1
The sgRNA targeting sequencing of gene is Sg1 or Sg2；Its nucleotide sequence is respectively as follows:
Sg1:5'-TTGGACTGTCAGCTGCTGTC-3'；
Sg2:5'-TGGACTTCCTCTCATGATGC-3'。
2. a kind of method for knocking out people ABCB1 gene using CRISPR-Cas9 system, the method treat mesh for non-disease
, characterized by comprising the steps of:
(1) end 5' of the sgRNA targeting sequencing obtains positive oligonucleotides plus CACCG in claim 1；Root simultaneously
Its corresponding DNA complementary strand is obtained according to targeting sequencing, and obtains reverse oligonucleotide plus AAAC at its 5 ' end；It is respectively synthesized
Above-mentioned forward direction oligonucleotides and reverse oligonucleotide, the positive oligonucleotides and reverse oligonucleotide of synthesis are denaturalized, annealing, shape
At double-strand；
(2) double-strand made from step (1) is connect with Cas9 carrier, obtains recombination and knocks out expression vector；
(3) step (2) recombination obtained is knocked out into expression vector and packaging system cotransfection incasing cells, then harvest virus is pure
Change and be concentrated, obtains virion；
(4) by the cell of infestation with virus particles in vitro culture made from step (3), screening surely turns cell, knockout of succeeding
The cell of ABCB1 gene.
3. the method according to claim 2 for knocking out people ABCB1 gene using CRISPR-Cas9 system, it is characterised in that:
Cas9 carrier described in step (2) is lentiCRISPRv2 carrier.
4. the method according to claim 2 for knocking out people ABCB1 gene using CRISPR-Cas9 system, it is characterised in that:
Incasing cells described in step (3) is 293T cell.
5. the method according to claim 2 for knocking out people ABCB1 gene using CRISPR-Cas9 system, it is characterised in that:
Package carrier in packaging system described in step (3) is pMD2.G and psPAX2.
6. the method according to claim 2 for knocking out people ABCB1 gene using CRISPR-Cas9 system, it is characterised in that:
The cell of in vitro culture described in step (4) is multi-drug resistance of the tumor cell.
7. the method according to claim 6 for knocking out people ABCB1 gene using CRISPR-Cas9 system, it is characterised in that:
The cell of in vitro culture described in step (4) is HCT-8/V or KBv200.
8. the sgRNA targeting sequencing of special target people ABCB1 gene described in claim 1 is preparing artitumor multi-medicine-resistant
Application in drug.
9. the sgRNA targeting sequencing of special target people ABCB1 gene according to claim 8 is to prepare artitumor multi resistance to
Application in pharmacological property drug, it is characterised in that:
The tumour is people's oral squamous cell carcinomas or Human colorectal carcinoma.
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