CN104004778B - Targeting knockout carrier containing CRISPR/Cas9 system and adenovirus thereof and application - Google Patents
Targeting knockout carrier containing CRISPR/Cas9 system and adenovirus thereof and application Download PDFInfo
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Abstract
The invention discloses a kind of targeting knockout carrier containing CRISPR/Cas9 system and adenovirus thereof and application, this targeting knockout carrier is by pX330-U6-Chimeric_BB-CBh-hSpCas9 plasmid EcoRI and SacII enzyme action, fill rear connection through BstXI enzyme action, the pAdTrack-CMV plasmid filled, then with the specific target sequence being connected into genes of interest after BbsI linearisation, obtain with recombinating with pAdEasy-1 plasmid after Pme I linearisation and CIAP alkali phosphatase dephosphorylation again, the targeting knockout carrier obtained can in target sequence region mutagenesis gene order, and mutation rate is high, reach 30.6%-45.8%, may be used for site-directed point mutation, for gene therapy is laid a good foundation.
Description
Technical field
The invention belongs to expression system field, be specifically related to targeting knockout carrier containing CRISPR/Cas9 system and adenovirus thereof and application.
Background technology
CRISPR/Cas9 system is a kind of a kind of acquired immune system be present in antibacterial and archeobacteria; to eliminate external nucleic acid or phage; and CRISPR site leaves the microRNA can expressed and match with intrusive viruses genome sequence in autogene group, in order to protect antibacterial and archeobacteria not by the infringement of virus.After the antibacterial containing CRISPR/Cas9 system and archeobacteria infect virus, CRISPRRNA just by complementary series in conjunction with viral genome, and express CRISPR relevant enzyme, because CRISPR relevant enzyme belongs to nuclease, can viral DNA molecules be cut, thus stop virus replication.After 2013, researcheres are delivered many sections of articles and are introduced CRISPR/Cas9 system on the magazine such as " Science ", " NatureBiotechnology ", and success realizes accurate genetic modification on the species such as the mankind, mice, Brachydanio rerio.But the experimental subject that current CRISPR/Cas9 is correlated with all is confined to the operation of cellular level, such as, cell line or one cell embryos etc., be not directly used in living animal by CRISPR/Cas9 gene target operating technology.
Adenovirus vector method is one of the most promising gene transfer method in gene therapy.Carrier easily builds and operates, and host range is wide, infectious strong, and exogenous gene can be transferred in various target cell or tissue by adenovirus vector effectively.Can enter different tissues through different approaches, can infect the non-division cells after differentiation, and adenoviral gene unconformity is in host cell, without the danger of insertion mutation activated oncogene, exogenous gene can be expressed freely.In recent years, adenovirus vector causes the concern of scientific research personnel, is widely used in the investigation and application of the gene therapy of the diseases such as heredopathia, infectious disease and tumor.But do not modify accurately for some gene.As: the sickleshaped anemia of sickleshaped human genetic diseases is because the exception of haemachrome S causes.A base T (thymus pyrimidine) of normal expression haemachrome S protein gene sports A (guanine), causes a glutamic acid mutation in haemachrome S protein to be valine.Erythrocyte becomes a kind of hereditary of crescent cell by normal discoid cytometaplasia.Patient can cause poor circulation because of erythrocyte dysfunction and corrupted and have an intense pain.We very can carry out targeting modification operation to the genome of animal body at the adenovirus mediated CRISPR/Cas9 gene target operating system of invention, so for the treatment of the disease brought due to gene mutation as the mankind, such as tumor, sickleshaped anemia, albinism etc., will obtain breakthrough progress.
Summary of the invention
In view of this, an object of the present invention is to provide the knockout carrier of the targeting containing CRISPR/Cas9 system; Two of object of the present invention is to provide the adenovirus containing targeting knockout carrier; What three of object of the present invention was to provide targeting knockout carrier is preparing the application in induced gene sudden change reagent; Four of object of the present invention is to provide targeting knockout carrier preparing the application in tumor inhibitor.
1, the targeting knockout carrier containing CRISPR/Cas9 system, described targeting knockout carrier by pX330-U6-Chimeric_BB-CBh-hSpCas9 plasmid EcoRI with SacII enzyme action, fill and be connected through BstXI enzyme action, the pAdTrack-CMV plasmid that fills afterwards, then with the specific target sequence being connected into genes of interest after BbsI linearisation, then obtain with recombinating with pAdEasy-1 plasmid after Pme I linearisation and CIAP alkali phosphatase dephosphorylation.
Preferably, described genes of interest is GFP or c-myc gene.
Preferably, the genes of interest specific target sequence that is GFP is as shown in SEQIDNO.1, SEQIDNO.4 or SEQIDNO.7.
Preferably, genes of interest is for the specific target sequence of c-myc gene is as shown in SEQIDNO.12, SEQIDNO.15 or SEQIDNO.18.The nucleotide wherein acting on specific target sequence shown in SEQIDNO.12 is the double-strand that SEQIDNO.13 and SEQIDNO.14 annealing is formed, the nucleotides sequence acting on target sequence shown in SEQIDNO.15 is classified as the double-strand that SEQIDNO.16 and SEQIDNO.17 takes off fire formation, and the nucleotides sequence acting on target sequence shown in SEQIDNO.18 is classified as the double-strand of SEQIDNO.19 and SEQIDNO.20 annealing formation.
Preferred, described specific target sequence is as shown in SEQIDNO.12.
2, the adenovirus containing described targeting knockout carrier.
3, the application of described targeting knockout carrier in the reagent of preparation sudden change specific target sequence.
4, the application of described targeting knockout carrier in the reagent preparing Tumor suppression.
Beneficial effect of the present invention is: the invention discloses a kind of targeting knockout carrier containing CRISPR/Cas9 system and adenovirus thereof and application, this targeting knockout carrier can in target sequence region mutagenesis gene order, and it is high to knock out rate, reach 30.6%-45.8%, therefore this targeting knockout carrier can be used in site-directed point mutation, may be used for gene therapy, for gene therapy disease is clinically laid a good foundation.
Accompanying drawing explanation
In order to make object of the present invention, technical scheme and beneficial effect clearly, the invention provides following accompanying drawing:
Fig. 1 is for turning GFP mice PCR primer through T7EndonucleaseI restriction enzyme digestion and electrophoresis testing result figure.
Fig. 2 is the cell results figure (A: infect Ad-pSpCas9-T2 virus liquid mice that flow cytometry analysis mice is respectively organized; B: infect Ad-pSpCas9 (BB) virus liquid mice; C: infect normal saline mice).
Fig. 3 is the GFP gene amplification product sequencing result (base is lost in "-" representative, and base is inserted in "+" representative) turning GFP mice infecting Ad-pSpCas9-T2 virus liquid.
Fig. 4 is for turning c-myc DNA murine PCR primer through T7EndonucleaseI restriction enzyme digestion and electrophoresis testing result figure.
Fig. 5 is the c-myc gene amplification product sequencing result (base is lost in "-" representative, and base is inserted in "+" representative) turning c-myc mice infecting Ad-pSpCas9-T4 virus liquid.
Fig. 6 is for turning c-myc DNA murine tail vein injection adenovirus survival statistical result.
Detailed description of the invention
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.The experimental technique of unreceipted actual conditions in embodiment, usually conveniently condition, the such as condition described in Molecular Cloning: A Laboratory guide (third edition, the work such as J. Pehanorm Brooker), or according to the condition that manufacturer advises.
Embodiment 1
1, Crispr/Cas9 adenovirus vector is built
(1) by pX330-U6-Chimeric_BB-CBh-hSpCas9 plasmid (AddgeneplasmidID:42230) EcoRI and SacII enzyme action, enzyme action afterproduct through 1% sepharose electrophoresis, reclaim the fragment containing hSpCas9, and called after U6-Chimeric_BB-CBh-hSpCas9.
(2) pAdTrack-CMV adenovirus vector BstXI is carried out enzyme action and remove CMV promoter and GFP fragment, enzyme action afterproduct, through the sepharose electrophoresis of 1%, reclaims carrier framework, and called after pAdTrack carrier framework.
(3) step (1) enzyme action gained recovery product and step (2) gained carrier framework klenow enzyme are filled, then spend the night under 16 DEG C of conditions with T4 ligase and connect, obtain recombinant vector, called after pAdTrack-U6-Chimeric_BB-CBh-hSpCas9.
By the recombinant vector pAdTrack-U6-Chimeric_BB-CBh-hSpCas9 transformation of E. coli DH5 α competence obtained, and to coat Kan concentration be in the solid LB flat board of 100 μ g/mL, is inverted overnight incubation for 37 DEG C.The well-grown monoclonal of picking, be in the LB fluid medium of 100 μ g/mL in 15mLKan concentration, 37 DEG C are shaken bacterium and spend the night, and mini-scale plasmid extracts, and extract plasmid by agarose gel electrophoresis, the carrier that detected magnitude is correct.Then recombinant vector pAdTrack-U6-Chimeric_BB-CBh-hSpCas9 correct for size is served the order-checking of Hai Shenggong biological engineering company limited.Crispr/cas9 adenovirus vector called after pAdTrack-pSpCas9 (BB) correct for order-checking.
2, structure knocks out GFP gene C rispr/Cas9 adenovirus vector
(1) adenovirus vector pAdTrack-pSpCas9 (BB) is used BbsI enzyme action, reclaim linear fragment.
(2) utilize online tool ZiFiTTargeterversion4.0 design for the oligonucleotide of 3 target spots of GFP gene, be specially:
First target spot is T1:5 '-ccgcgccgaggtgaagttcg-3 ' (SEQIDNO.1); The oligonucleotide of design is to being 5 '-caccgccgcgccgaggtgaagttcg-3 ' (SEQIDNO.2); 5 '-aaaccgaacttcacctcggcgcggc-3 ' (SEQIDNO.3);
Second target spot is T2,5 '-gtgaaccgcatcgagctgaa-3 ' (SEQIDNO.4); The oligonucleotide of design is to being 5 '-caccgtgaaccgcatcgagctgaa-3 ' (SEQIDNO.5); 5 '-aaacttcagctcgatgcggttcac-3 ' (SEQIDNO.6);
3rd target spot is T3,5 '-aggaggacggcaacatcctg-3 ' (SEQIDNO.7); The oligonucleotide of design is to being 5 '-caccgaggaggacggcaacatcctg-3 ' (SEQIDNO.8); 5 '-aaaccaggatgttgccgtcctcctc-3 ' (SEQIDNO.9).
(3) slowly annealing at room temperature is down to after 3 pairs of oligonucleotide being heated to 95 DEG C respectively, form double-strand, then pAdTrack-pSpCas9 (BB) linear fragment after step (1) enzyme action is connected into respectively, product will be connected, transformation of E. coli DH5 α competent cell, and coat overnight incubation on LB solid plate that Kan concentration is 100 μ g/mL, the well-grown monoclonal of picking, be in the LB fluid medium of 100 μ g/mL in 15mLKan concentration, 37 DEG C are shaken bacterium and spend the night, extract plasmid, GFP gene C rispr/Cas9 adenovirus vector must be knocked out respectively, and difference called after pAdTrack-pSpCas9-T1, pAdTrack-pSpCas9-T2 and pAdTrack-pSpCas9-T3.
3, the structure of pAd-pSpCas9-T1, pAd-pSpCas9-T2 and pAd-pSpCas9-T3
(1) E.coliBJ5183 competence preparation: by-80 DEG C of frozen E.coliBJ5183 bacterial strains (being called for short BJ5183), the LB lined containing streptomycin (30 ~ 50 μ g/mL) is dull and stereotyped, be inverted cultivation 12 ~ 16h for 37 DEG C, then picking monoclonal shakes bacterium cultivation, uses CaCl
2legal system is as competent cell.
(2) pAdEasy-1 Plastid transformation BJ5183 competence: the E.coliBJ5183 competence adopting thermal shock method step of converting (1) to prepare pAdEasy-1 plasmid, then being applied to containing Amp concentration is that the LB of 100 μ g/mL is dull and stereotyped, picking monoclonal is in the LB liquid medium of 100 μ g/mL in containing Amp concentration, after bacterium liquid muddiness, then use CaCl
2the standby BJ5183 competence containing pAdEasy-1 plasmid of legal system.
(3) pAdTrack-pSpCas9-T1, pAdTrack-pSpCas9-T2 and pAdTrack-pSpCas9-T3 are used respectively Pme I linearization for enzyme restriction, then use the plasmid of CIAP alkali phosphatase dephosphorylation Pme I single endonuclease digestion, prevent self-cyclisation.
(4) thermal shock method is adopted by the linear plasmid after step (3) dephosphorylation to transform the BJ5183 competence containing pAdEasy-1 plasmid respectively, recombinate with plasmid pAdEasy-1 after linear plasmid proceeds to the BJ5183 competence containing pAdEasy-1 plasmid, obtain pAd-pSpCas9-T1, pAd-pSpCas9-T2 and pAd-pSpCas9-T3 recombiant plasmid respectively.
Simultaneously by pAdTrack-pSpCas9 Pme I linearization for enzyme restriction, then use CIAP alkali phosphatase dephosphorylation, then transform the BJ5183 competence containing pAdEasy-1 plasmid, obtain empty carrier pAd-pSpCas9 (BB) plasmid.
4, adenovirus packaging
(1) without the preparation of endotoxin plasmid DNA
A, pAd-pSpCas9-T1, pAd-pSpCas9-T2, pAd-pSpCas9-T3 and pAd-pSpCas9 (BB) the recombiant plasmid 1 μ L getting extraction respectively add blow in 100 μ LDH5 α competent cells even, place in ice and leave standstill 20min, put into 42 DEG C of water-bath 90s again, be placed in rapidly ice bath 3min, add 500 μ LLB fluid mediums, place shaking table 180rpm37 DEG C of 1h, get bacterium liquid 100 μ L and be spread evenly across LB solid medium 37 DEG C of overnight incubation that Amp concentration is 100 μ g/mL.
B, to get single bacterium colony be in the LB fluid medium of 100 μ g/mL in 3mLAmp concentration, 250rpm, 37 DEG C of shaken cultivation 8 hours; Therefrom getting 300 μ L bacterium liquid, to be inoculated in 300mLAmp concentration be in the LB fluid medium of 100 μ g/mL, and in 250rpm, 37 DEG C of shaken cultivation 12 ~ 16 hours;
C, collection bacterium liquid, then at 4 DEG C, centrifugal 15min under 4000rpm condition, abandon supernatant, collect thalline, then extract plasmid according to QIAGENEndoFreePlasmidMaxiKit test kit description operating procedure, obtain without endotoxic pAd-pSpCas9-T1, pAd-pSpCas9-T2, pAd-pSpCas9-T3 and pAd-pSpCas9 (BB) plasmid.
(2) recovery of cell strain HEK293 and cultivation
Get the HEK293 cell of liquid nitrogen cryopreservation, be put in rapidly in 37 DEG C of water-baths and thaw, period constantly rocks and solution in centrifuge tube is heated evenly as far as possible; Adding rapidly 7mL volume fraction after thawing is (DMEM culture fluid needs to be preheated to 37 DEG C in advance) in the DMEM culture fluid of 10% hyclone, and rifle head is blown and beaten to the existence of acellular group gently, then centrifugal 6min under 1300rpm condition, supernatant discarded; In centrifuge tube, add 2mL be again preheated to the DMEM culture fluid that 37 DEG C of volume fractions are 10% hyclone in advance, piping and druming cell makes it suspend, according to 5 × 10
4cell is inoculated in culture dish by individual cell, and in 37 DEG C, CO containing 5%
2cultivate 24h in incubator, then change liquid, changed when liquid to cell density reaches 90% every 2 days later and go down to posterity.
(3) packaging of pAd-pSpCas9-T1, pAd-pSpCas9-T2 and pAd-pSpCas9-T3
Day before transfection, according to every hole 4 × 10
5individual HEK293 cell is inoculated into 6 orifice plates, and after cultivating 24h, cell about has 70% fusion, 4h before transfection, cleans 2 cells with without dual anti-PBS, and the culture fluid containing serum is changed to the optimization culture liquid Opti-MEMI (2mL/ hole) without dual anti-serum-free; Lipofectamine
tM2000 (purchased from Invitrogen companies) as transfection reagent by pAd-pSpCas9-T1 transfection to HEK293 cell, concrete steps are carried out (reagent dosage that all reagent is a hole) according to description:
A. 4 μ g are diluted to 250 μ L without endotoxic pAd-pSpCas9-T1 plasmid, incubated at room with Opti-MEMI optimization culture liquid;
B. use Opti-MEMI optimization culture liquid by 10 μ LLipofectamine equally
tM2000 are diluted to 250 μ L, incubated at room 5min; First two steps must not operate more than 25min;
C. by the plasmid of hatching and Lipofectamine
tM2000 mixings, cumulative volume is 500 μ L, and whole process is as far as possible soft, mixed solution incubated at room 20min;
D. 500 μ L mixed liquors are added in a hole of culture plate, rock culture plate mixing culture fluid gently;
E. by culture fluid 37 DEG C, volume fraction is 5%CO
2incubator cultivate, after 6h, culture fluid is changed to the DMEM culture fluid that volume fraction is 10% hyclone; Wherein 4 holes of 6 orifice plates are packed by above-mentioned steps, and one, two other hole is control liposome, and one is blank.
F. transfection 24h observation of cell pathological changes (Cytopathiceffect, CPE) situation, formed after transfection 8 ~ 10d and generally have plaque appearance, now cell can be scraped, by cell harvesting in cryopreservation tube, in-196 DEG C (in liquid nitrogen) and 37 DEG C of multigelations 5 times, the centrifugal 5min of 12000rpm, the supernatant collected is the virus liquid after packaging, called after Ad-pSpCas9-T1, and this virus liquid can be directly used in subsequent cell and to infect or in-80 DEG C of preservations.
According to method same as described above packaging pAd-pSpCas9-T2 plasmid, pAd-pSpCas9-T3 plasmid and empty carrier plasmid pAd-pSpCas9 (BB), obtain virus liquid Ad-pSpCas9-T2, Ad-pSpCas9-T3 and Ad-pSpCas9 (BB) respectively.
(4) virus titer measures
By every hole 2 × 10
4the density inoculation HEK293 of individual cell, in 96 orifice plates, adopts TCID
50method and Karbers method measure and calculate the virus titer of Ad-pSpCas9-T1.Will with multiple proportions (10
-1~ 10
-10) the virus liquid infection cell that dilutes, every hole adds 100 μ L viral dilution liquid, cultivates the CEP pathological changes that 9 ~ 10d adds up cell.According to Karbers formula namely: T=10 × 10
1+d (s – 0.5)tCID
50/ mL calculating is viral obtains titre; Wherein s is positive rate's sum, namely used dilution factor sum.D=log10 dilution factor=1 (this is for the dilution factor of 10 times); And according to formula: T=1 × 10
xtCID
50/ mL=1 × 10
x – 0.7pFU/mL, can be changed to PFU/mL unit by virus titer.
As calculated the purity of virus liquid and titre as follows:
Virus liquid Ad-pSpCas9-T1, purity A260/A280=1.47, titre 2.5 × 10
11;
Virus liquid Ad-pSpCas9-T2, purity A260/A280=1.51, titre 1.7 × 10
11;
Virus liquid Ad-pSpCas9-T3, purity A260/A280=1.48, titre 3.1 × 10
11;
Virus liquid Ad-pSpCas9 (BB), purity A260/A280=1.50, titre 1.6 × 10
11.
5, viral infection turns GFP mouse cell
Get and turn GFP mice and (turn the preparation of GFP mice by OkabeM, IkawaM, KominamiK, NakanishiT, NishimuneY. " Greenmice " asasourceofubiquitousgreencells.FEBSLett1997; The method preparation of 407:313-9 report) tail point sets up fibroblast, then bed board: after resuspended for the cell dissociation of exponential phase, by 1 × 10
5/ L density is inoculated in 12 orifice plates, grow overnight to 70 ~ 80% is paved with 12 orifice plates, then sucking-off culture fluid, renews fresh culture fluid, adds the virus liquid of Ad-pSpCas9-T1, Ad-pSpCas9-T2 and Ad-pSpCas9-T3 respectively, infection multiplicity is 400, in contrast with PBS and Ad-pSpCas9 (BB) virus liquid, put into incubator after mix homogeneously and cultivate, 24 hours change liquid simultaneously, 48 h before harvest cells, extract cell DNA.Pcr amplification GFP gene target gene order, pcr amplification forward primer is Cas9-GFP-F:5 '-gtgagcaagggcgaggag-3 ' (SEQIDNO.10); Downstream primer is Cas9-GFP-R:5 '-tggtagtggtcggcgagc-3 ' (SEQIDNO.11), PCR primer T7EndonucleaseI enzyme action, nucleic acid gel electrophoresis detection, then use BIO-RAD imaging system (ChemiDocXRS) software analysis statistics target spot non-homogeneous recombination repair (NHEJ) efficiency, result as shown in Figure 1.As shown in Figure 1, the cell GFP gene target site NHEJ of infection adenovirus Ad-pSpCas9-T2 virus liquid is most effective is 48%, filters out this virus liquid for turning GFP mouse tail vein injection.
6, tail vein injection turns GFP DNA murine
Turn GFP DNA murine by 9, be divided into 3 groups at random, often organize 3 (1 group # is 1,2,3, and 2 group # are 4,5,6, and 3 group # are 7,8,9), first group of tail vein injection Ad-pSpCas9-T2 virus liquid (viral dosage 1 × 10
11pFU/kg) inject, second group of isodose adenovirus Ad-pSpCas9 (BB) of tail vein injection, the normal saline of the 3rd group of injection equivalent, injection in every 7 days once virus, injection 3 times, amounts to raising 21 days continuously.Get turn GFP mice the heart, liver, spleen, lung, nephridial tissue, and set up cell line, and with the cell that flow cytometry analysis mice is respectively organized, and sub-elect green fluorescence negative cells and positive cell, result is as shown in Figure 2.Result shows, injection Ad-pSpCas9-T2 virus liquid respectively organizes the negative cells (Fig. 2 A) that all can sub-elect different proportion, and the mice major organs (heart, liver, spleen, lung, kidney) of injection Ad-pSpCas9-T2 virus liquid, in genome, GFP gene knocks out efficiency and can reach 30.6%-45.8%; And the two groups of cells injecting Ad-pSpCas9 (BB) virus liquid and normal saline are all with green fluorescence, do not sub-elect green fluorescence negative cells (Fig. 2 B and Fig. 2 C).The above results shows, artificial integration knocks out the adenovirus vector of GFP gene C RISPR/Cas9 element, Ad-pSpCas9-T2 virus liquid is at receptor (turning GFP mice) animal model, can effective targeting modification target gene GFP, confirm that adenovirus mediated CRISPR/Cas9 system can carry out the modification of gene target in living animal.
The GFP mice that turns infecting Ad-pSpCas9-T2 virus liquid is respectively organized the green fluorescence negative cells and positive cell extraction DNA that sub-elect, then pcr amplification GFP gene target site sequence, amplified fragments is cloned in pMD18-T carrier, recombinant vector is delivered to the order-checking of Shanghai Sheng Gong biological engineering company limited, result as shown in Figure 3.Result shows, and in gfp positive cell, GFP gene order has part to undergo mutation, and the GFP gene order of green fluorescence negative cells all has sudden change.
Embodiment 2, adenovirus mediated CRISPR/Cas9 targeting knock out and turn c-myc DNA murine
1, adenovirus expression carrier is built
Utilize online tool ZiFiTTargeterversion4.0, at upper design 3 the CRISPR/Cas9 target spots of c-myc gene Second Exon (GenBank:AH005318.1), and the oligonucleotide that design is corresponding, be specially:
First target spot is T4, and nucleotides sequence is classified as 5 '-tcgctacgtccttctcccca-3 ' (SEQIDNO.12); The oligonucleotide of its design is to being 5 '-caccgtcgctacgtccttctcccca-3 ' (SEQIDNO.13); 5 '-aaactggggagaaggacgtagcgac-3 ' (SEQIDNO.14);
Second target spot is T5, and nucleotides sequence is classified as 5 '-gcagccgcccgcgcccagtg-3 ' (SEQIDNO.15); Oligonucleotide of its design is to being 5 '-caccgcagccgcccgcgcccagtgg-3 ' (SEQIDNO.16); 5 '-aaaccactgggcgcgggcggctgcg-3 ' (SEQIDNO.17);
3rd target spot is T6, and nucleotides sequence is classified as 5 '-cagatgatgaccgagttact-3 ' (SEQIDNO.18); Oligonucleotide of its design is to being 5 '-caccgcagatgatgaccgagttact-3 ' (SEQIDNO.19); 5 '-aaacagtaactcggtcatcatctgc-3 ' (SEQIDNO.20).
Then plasmid pAdTrack-pSpCas9-T4, pAdTrack-pSpCas9-T5 and pAdTrack-pSpCas9-T6 is obtained respectively according to the method for embodiment 1.And prepare Ad-pSpCas9-T4, Ad-pSpCas9-T5 and Ad-pSpCas9-T6 virus liquid respectively according to the method for embodiment 1, then measure virus liquid titre, its measurement result is as follows:
Ad-pSpCas9-T4 virus liquid, purity is A260/A280=1.47, and titre is 2.5 × 10
11;
Ad-pSpCas9-T5 virus liquid, purity is A260/A280=1.51, and titre is 1.7 × 10
11;
Ad-pSpCas9-T6 virus liquid, purity is A260/A280=1.48, and titre is 3.1 × 10
11.
2, virus liquid infects Recipient mice
Get and turn c-myc DNA murine tail point and set up fibroblast and (turn the preparation of c-myc DNA murine by HarrisAW, HarrisAW, PinkertCA, CrawfordM, etal.TheEmu-myctransgenicmouse.Amodelforhigh-incidencesp ontaneouslymphomaandleukemiaofearlyBcells [J] .TheJournalofexperimentalmedicine, 1988, the method preparation of 167 (2): 353-371. reports), then bed board, after resuspended for the cell dissociation of exponential phase, by 1 × 10
5/ L density is inoculated in 12 orifice plates, grow overnight to 70 ~ 80% is paved with 12 orifice plates, then sucking-off culture fluid, renew fresh culture fluid, according to infection multiplicity 400particle/cell, cell is divided into 5 groups, infects virus liquid Ad-pSpCas9-T4, Ad-pSpCas9-T5, Ad-pSpCas9-T6, Ad-pSpCas9 (BB) and PBS respectively, incubator can be put into after mix homogeneously and cultivate and change liquid in 24 hours, peptic cell after 72 hours, extracts cell DNA.
Shot design according to c-myc gene detects primer, be specially, forward primer is M1-F:5 '-gagcgagctgcagccgcccgcg-3 ' (SEQIDNO.21), and downstream primer is M1-R5 '-gcccgcgggcggggctcagg-3 ' (SEQIDNO.22).Then with extract cell DNA for template, respectively with sequence shown in SEQIDNO.21 and SEQIDNO.22 for primer increases.By increasing, the PCR primer T7 Cobra venom endonuclease (T7E1) obtained carries out enzyme action, and after enzyme action under 80-120V condition, detect with agarose gel electrophoresis 40min, result as shown in Figure 4.Result shows, virus liquid Ad-pSpCas9-T4 is most effective to the sudden change that target spot causes.
By the cell extraction DNA that virus liquid Ad-pSpCas9-T4 infects, utilize the upstream and downstream primer amplification c-myc gene of c-myc gene, extension amplification outcome is in pMD18-T carrier, and deliver to the order-checking of Shanghai Sheng Gong biological engineering company limited, result as shown in Figure 5.Sudden change can be formed in target area after result shows virus liquid Ad-pSpCas9-T4 infection cell.
3, c-myc DNA murine tail vein injection adenovirus is turned
Get the mice (10 week age) that 30 turn c-myc gene, be divided into 3 groups at random, often organize 10, first group of injecting virus liquid Ad-pSpCas9-T4, injecting virus liquid Ad-pSpCas9 (BB) and PBS in contrast, inject weekly once, raise mice 30 weeks in SPF level animal feeding room respectively for second group and the 3rd group of mice, observe mouse survival rate and record result, result as shown in Figure 6.Result shows, the mice of tail vein injection PBS group suffers from tumor mortality 9, the mice of tail vein injection Ad-pSpCas9 (BB) virus liquid is all dead because suffering from tumor, and tail vein injection Ad-pSpCas9-T4 virus liquid mice is because suffering from tumor mortality 5, and the death time comparatively.Injection adenovirus Ad-pSpCas9-T4 knock-out mice c-myc gene is described, thus the morbidity of mouse tumor can be suppressed, extend the time-to-live of mice.
4, the order-checking of c-myc DNA murine gene target site is turned
3 dead mices of group and survival mice are dissected, core, liver, spleen, lung and nephridial tissue, the DNA extracting each tissue is template, with the sequence of SEQIDNO.21 and SEQIDNO.22 for primer carries out pcr amplification c-myc gene, PCR primer is reclaimed and is cloned in PMD18-T carrier, after conversion, every mice organizes picking 20 single bacterium colonies, serve Hai Shenggong order-checking, then according to non-homogeneous restructuring amendment (NHEJ) of sequencing result statistics, result is as shown in table 1.
The non-homogeneous restructuring amendment statistical result that table 1, dead mice and survival mice are respectively organized
As shown in Table 1, the c-myc gene in each histoorgan of mice of experimental group all there occurs non-homogeneous restructuring amendment (NHEJ) in various degree.Show that adenovirus mediated CRISPR/Cas9 system can successfully be carried out targeting knock out turning c-myc gene in c-myc DNA murine gene, thus reach the object suppressing mouse tumor to occur.
What finally illustrate is, above preferred embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although by above preferred embodiment to invention has been detailed description, but those skilled in the art are to be understood that, various change can be made to it in the form and details, and not depart from claims of the present invention limited range.
Claims (5)
1. the application of targeting knockout carrier in the reagent preparing Tumor suppression containing CRISPR/Cas9 system, is characterized in that: described targeting knockout carrier is used by pX330-U6-Chimeric_BB-CBh-hSpCas9 plasmid
ecoRi and
saciI enzyme action, fill rear connection through BstXI enzyme action, the pAdTrack-CMV plasmid that fills, then use
bbsbe connected into the specific target sequence of genes of interest after I linearisation, then obtain with recombinating with pAdEasy-1 plasmid after Pme I linearisation and CIAP alkali phosphatase dephosphorylation.
2. application according to claim 1, is characterized in that: described genes of interest is GFP or c-myc gene.
3. application according to claim 2, is characterized in that: the specific target sequence that genes of interest is GFP is as shown in SEQIDNO.1, SEQIDNO.4 or SEQIDNO.7.
4. application according to claim 2, is characterized in that: genes of interest is for the specific target sequence of c-myc gene is as shown in SEQIDNO.12, SEQIDNO.15 or SEQIDNO.18.
5. application according to claim 4, is characterized in that: described specific target sequence is as shown in SEQIDNO.12.
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