CN104004778A

CN104004778A - CRISPR/Cas9 system-containing targeted knockout vector and adenovirus and applications thereof

Info

Publication number: CN104004778A
Application number: CN201410249547.5A
Authority: CN
Inventors: 周勇; 温平; 申友锋; 杨晓芳; 李建均; 何久香; 王蜀英; 牟浪; 陈小雨
Original assignee: Chongqing Gao Sheng Biological Medicine LLC
Current assignee: Chongqing Gao Sheng Biological Medicine LLC
Priority date: 2014-06-06
Filing date: 2014-06-06
Publication date: 2014-08-27
Anticipated expiration: 2034-06-06
Also published as: CN104004778B

Abstract

The invention discloses a CRISPR/Cas9 system-containing targeted knockout vector and adenovirus and applications thereof. The targeted knockout vector is prepared through the following steps: after a pX330 U6-Chimeric_BB-CBh-hSpCas9 plasmid is subjected to enzyme digestion and filling-in by using EcoRI and SacII, connecting the pX330 U6-Chimeric_BB-CBh-hSpCas9 plasmid with a pAdTrack-CMV plasmid subjected to enzyme digestion and filling-in by using BstXI; after the obtained product is linearized by using BbsI, connecting the obtained product to a specific target sequence of a desired gene; and after the obtained object is linearized by using PmeI and dephosphorylated by using CIAP alkaline phosphatase, recombining the obtained product with a pAdEasy-1 plasmid. The targeted knockout vector can mutate gene sequences in target sequence areas, and the mutation rate is high, up to 30.6-45.8%, therefore, the targeted knockout vector can be used for gene site-directed mutation, and lays a foundation for gene therapy.

Description

The target knockout carrier that contains CRISPR/Cas9 system and adenovirus and application

Technical field

The invention belongs to expression system field, be specifically related to the target knockout carrier and adenovirus and the application that contain CRISPR/Cas9 system.

Background technology

CRISPR/Cas9 system is a kind of a kind of Acquired immunity system being present in bacterium and archeobacteria; to eliminate external nucleic acid or phage; and CRISPR site stays and can express the microRNA matching with intrusive viruses genome sequence in autogene group, in order to protect bacterium and archeobacteria not to be subject to viral infringement.When after the bacterium that contains CRISPR/Cas9 system and archeobacteria infection virus, CRISPR RNA just can be by complementary sequence in conjunction with viral genome, and expresses CRISPR relevant enzyme, because CRISPR relevant enzyme belongs to nuclease, can cut viral DNA molecule, thereby stop virus replication.After 2013, investigators deliver many sections of articles and introduce CRISPR/Cas9 system on the magazine such as " Science ", " Nature Biotechnology ", and success realizes accurate genetic modification on the species such as the mankind, mouse, zebra fish.But the relevant experimental subjects of CRISPR/Cas9 is all confined to the operation of cell levels at present, for example, clone or unicellular embryo etc., be not directly used in living animal by CRISPR/Cas9 gene target operative technique.

Adenovirus carrier method is one of the most promising gene transfer method in gene therapy.Carrier easily builds and operation, and host range is wide, infectious strong, and adenovirus carrier can be transferred to foreign gene in various target cells or tissue effectively.Can enter different tissues through different approaches, can infect the non-division cells after differentiation, and adenoviral gene unconformability be in host cell, without the danger of insertion mutation activated oncogene, foreign gene can be expressed freely.In recent years, adenovirus carrier has caused scientific research personnel's concern, is widely used in research and the application of the gene therapy of the diseases such as inherited disease, transmissible disease and tumour.But do not modify accurately for some gene.As: the sickleshaped anemia of sickleshaped human inheritance's disease is extremely causing due to protoheme S.A base T (thymus pyrimidine) of normal expression protoheme S protein gene sports A (guanine), causes a L-glutamic acid in protoheme S albumen to sport α-amino-isovaleric acid.Red blood corpuscle is become a kind of heredopathia of crescent cell by normal discoid cytometaplasia.Patient can cause poor circulation and have an intense pain because of red blood corpuscle dysfunction and corrupted.We can very 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 of bringing due to transgenation as the mankind, such as tumour, sickleshaped anemia, albinism etc., will obtain breakthrough progress.

Summary of the invention

In view of this, one of object of the present invention is to provide the target that contains CRISPR/Cas9 system knockout carrier; Two of object of the present invention is to provide the adenovirus containing target knockout carrier; Three of object of the present invention be to provide target knockout carrier in the application of preparing in induced gene sudden change reagent; Four of object of the present invention is to provide target knockout carrier in the application of preparing in tumor inhibitor.

1, the target knockout carrier that contains CRISPR/Cas9 system, described target knockout carrier cuts, fills with EcoRI and SacII enzyme the pAdTrack-CMV plasmid that rear connection is cut, filled through BstXI enzyme by pX330-U6-Chimeric_BB-CBh-hSpCas9 plasmid, then by the specific target sequence that is connected into goal gene after BbsI linearizing, then with obtaining with the restructuring of pAdEasy-1 plasmid after the linearizing of Pme I and CIAP alkaline phosphatase dephosphorylation.

Preferably, described goal gene is GFP or c-myc gene.

Preferably, the specific target sequence that goal gene is GFP is as shown in SEQ ID NO.1, SEQ ID NO.4 or SEQ ID NO.7.

Preferably, goal gene is that the specific target sequence of c-myc gene is as shown in SEQ ID NO.12, SEQ ID NO.15 or SEQ ID NO.18.The Nucleotide that wherein acts on specific target sequence shown in SEQ ID NO.12 is the two strands that SEQ ID NO.13 and SEQ ID NO.14 annealing form, the nucleotides sequence that acts on target sequence shown in SEQ ID NO.15 is classified the two strands of SEQ ID NO.16 and the de-fire formation of SEQ ID NO.17 as, and the nucleotides sequence that acts on target sequence shown in SEQ ID NO.18 is classified the two strands of SEQ ID NO.19 and SEQ ID NO.20 annealing formation as.

Preferred, described specific target sequence is as shown in SEQ ID NO.12.

2, the adenovirus that contains described target knockout carrier.

3, the application of described target knockout carrier in the reagent of preparation sudden change specific target sequence.

4, the application of described target knockout carrier in the reagent of preparation inhibition tumour.

Beneficial effect of the present invention is: the invention discloses a kind of target knockout carrier and adenovirus and application of the CRISPR/Cas9 of containing system, this target knockout carrier can be in target sequence region mutator gene sequence, and it is high to knock out rate, reach 30.6%-45.8%, therefore this target knockout carrier can be used in site-directed point mutation, can be for gene therapy, for gene therapy disease is clinically laid a good foundation.

Brief description of the drawings

In order to make object of the present invention, technical scheme and beneficial effect clearer, the invention provides following accompanying drawing:

Fig. 1 is for turning GFP mouse PCR product through T7Endonuclease I restriction enzyme digestion and electrophoresis detected result figure.

Fig. 2 is the cell result figure (A: infect Ad-pSpCas9-T2 virus liquid mouse of the each tissue of flow cytometry analysis mouse; B: infect Ad-pSpCas9 (BB) virus liquid mouse; C: infect physiological saline mouse).

Fig. 3 is the GFP gene amplification product sequencing result that turns GFP mouse (base is lost in "-" representative, and base is inserted in "+" representative) that infects Ad-pSpCas9-T2 virus liquid.

Fig. 4 is for turning c-myc DNA murine PCR product through T7Endonuclease I restriction enzyme digestion and electrophoresis detected result figure.

Fig. 5 is the c-myc gene amplification product sequencing result that turns c-myc mouse (base is lost in "-" representative, and base is inserted in "+" representative) that infects Ad-pSpCas9-T4 virus liquid.

Fig. 6 is for turning c-myc DNA murine tail vein injection adenovirus survival statistics.

Embodiment

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, conventionally according to normal condition, for example, condition described in molecular cloning experiment guide (third edition, the work such as J. Pehanorm Brooker), or the condition of advising according to manufacturer.

Embodiment 1

1, build Crispr/Cas9 adenovirus carrier

(1) pX330-U6-Chimeric_BB-CBh-hSpCas9 plasmid (Addgene plasmid ID:42230) is cut with EcoRI and SacII enzyme, enzyme is cut after product through 1% agarose electrophoresis, the fragment that recovery contains hSpCas9, and called after U6-Chimeric_BB-CBh-hSpCas9.

(2) pAdTrack-CMV adenovirus carrier is carried out to enzyme with BstXI and cut except CMV promotor and GFP fragment, enzyme is cut after product through 1% agarose electrophoresis, reclaims carrier framework, and called after pAdTrack carrier framework.

(3) step (1) enzyme being cut to gained recovery product and step (2) gained carrier framework klenow enzyme fills, then the connection of spending the night under 16 DEG C of conditions with T4 ligase enzyme, obtain recombinant vectors, called after pAdTrack-U6-Chimeric_BB-CBh-hSpCas9.

The recombinant vectors pAdTrack-U6-Chimeric_BB-CBh-hSpCas9 obtaining is transformed to bacillus coli DH 5 alpha competence, and coat in the solid LB flat board that Kan concentration is 100 μ g/mL, be inverted overnight incubation for 37 DEG C.The well-grown mono-clonal of picking, is in the LB liquid nutrient medium of 100 μ g/mL in 15mL Kan concentration, and 37 DEG C are shaken bacterium and spend the night, and plasmid extraction in a small amount extracts plasmid by agarose gel electrophoresis, the carrier that detected magnitude is correct.Then recombinant vectors pAdTrack-U6-Chimeric_BB-CBh-hSpCas9 correct size is served to the order-checking of Hai Shenggong biotechnology company limited.Crispr/cas9 adenovirus carrier called after pAdTrack-pSpCas9 (BB) correct order-checking.

2, build and knock out GFP gene C rispr/Cas9 adenovirus carrier

(1) adenovirus carrier pAdTrack-pSpCas9 (BB) is cut with BbsI enzyme, reclaim linear fragment.

(2) utilize the oligonucleotide of online tool ZiFiT Targeter version4.0 design for 3 target spots of GFP gene, be specially:

First target spot is T1:5 '-ccgcgccgaggtgaagttcg-3 ' (SEQ ID NO.1); The oligonucleotide of design is to being: 5 '-caccgccgcgccgaggtgaagttcg-3 ' (SEQ ID NO.2); 5 '-aaaccgaacttcacctcggcgcggc-3 ' (SEQ ID NO.3);

Second target spot is T2,5 '-gtgaaccgcatcgagctgaa-3 ' (SEQ ID NO.4); The oligonucleotide of design is to being: 5 '-caccgtgaaccgcatcgagctgaa-3 ' (SEQ ID NO.5); 5 '-aaacttcagctcgatgcggttcac-3 ' (SEQ ID NO.6);

The 3rd target spot is T3,5 '-aggaggacggcaacatcctg-3 ' (SEQ ID NO.7); The oligonucleotide of design is to being: 5 '-caccgaggaggacggcaacatcctg-3 ' (SEQ ID NO.8); 5 '-aaaccaggatgttgccgtcctcctc-3 ' (SEQ ID NO.9).

(3) after being heated to respectively to 95 DEG C, 3 pairs of oligonucleotide are slowly down to annealing at room temperature, form double-stranded, then pAdTrack-pSpCas9 (BB) linear fragment after being connected into respectively step (1) enzyme and cutting, to connect product, transform bacillus coli DH 5 alpha competent cell, and coat overnight incubation on the LB solid plate that Kan concentration is 100 μ g/mL, the well-grown mono-clonal of picking, be in the LB liquid nutrient medium of 100 μ g/mL in 15mL Kan concentration, 37 DEG C are shaken bacterium and spend the night, extract plasmid, must knock out respectively GFP gene C rispr/Cas9 adenovirus carrier, 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.coli BJ5183 competence preparation: by-80 DEG C of frozen E.coli BJ5183 bacterial strains (being called for short BJ5183), line the LB flat board containing Streptomycin sulphate (30～50 μ g/mL), be inverted for 37 DEG C and cultivate 12～16h, then picking mono-clonal shakes bacterium cultivation, uses CaCl ₂legal system is as competent cell.

(2) pAdEasy-1 plasmid transforms BJ5183 competence: the E.coli BJ5183 competence that adopts thermal shock method step of converting (1) to prepare pAdEasy-1 plasmid, then be applied to containing Amp concentration is the LB flat board of 100 μ g/mL, picking mono-clonal is in the liquid LB substratum 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) by pAdTrack-pSpCas9-T1, pAdTrack-pSpCas9-T2 and pAdTrack-pSpCas9-T3 use respectively Pme I linearization for enzyme restriction, then use the plasmid of CIAP alkaline phosphatase dephosphorylation Pme I single endonuclease digestion, prevent self-cyclisation.

(4) linear plasmid after step (3) dephosphorylation is adopted respectively thermal shock method transform the BJ5183 competence containing pAdEasy-1 plasmid, linear plasmid proceeds to containing recombinating with plasmid pAdEasy-1 after the BJ5183 competence of pAdEasy-1 plasmid, obtains respectively pAd-pSpCas9-T1, pAd-pSpCas9-T2 and pAd-pSpCas9-T3 recombinant plasmid.

PAdTrack-pSpCas9 is used to Pme I linearization for enzyme restriction simultaneously, then use CIAP alkaline 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 intracellular toxin plasmid DNA

A, the pAd-pSpCas9-T1 that gets respectively extraction, pAd-pSpCas9-T2, pAd-pSpCas9-T3 and pAd-pSpCas9 (BB) recombinant plasmid 1 μ L add in 100 μ L DH5 α competent cells, blow even, place standing 20min in ice, put into again 42 DEG C of water-bath 90s, be placed in rapidly ice bath 3min, add 500 μ L LB liquid nutrient mediums, place 180rpm37 DEG C of 1h of shaking table, get bacterium liquid 100 μ L and evenly coat 37 DEG C of overnight incubation of LB solid medium that Amp concentration is 100 μ g/mL.

B, to get single bacterium colony be in the LB liquid nutrient medium of 100 μ g/mL in 3mL Amp concentration, 250rpm, 37 DEG C of shaking culture 8 hours; Therefrom get 300 μ L bacterium liquid and be inoculated in the LB liquid nutrient medium that 300mL Amp concentration is 100 μ g/mL, and in 250rpm, 37 DEG C of shaking culture 12～16 hours;

C, collection bacterium liquid, then centrifugal 15min under 4 DEG C, 4000rpm condition, abandon supernatant, collect thalline, then extract plasmid according to QIAGEN EndoFree Plasmid Maxi Kit test kit specification sheets operation steps, obtain the plasmid without endotoxic pAd-pSpCas9-T1, pAd-pSpCas9-T2, pAd-pSpCas9-T3 and pAd-pSpCas9 (BB).

(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, constantly rock during this time solution in centrifuge tube is heated evenly as far as possible; After thawing, adding rapidly 7mL volume fraction is (DMEM nutrient solution need to be preheated to 37 DEG C in advance) in the DMEM nutrient solution of 10% foetal calf serum, and rifle head is blown and beaten gently to acellular group and existed, then centrifugal 6min under 1300rpm condition, supernatant discarded; Be preheated in advance to adding 2mL in centrifuge tube the DMEM nutrient solution that 37 DEG C of volume fractions are 10% foetal calf serum again, piping and druming cell suspends it, according to 5 × 10 ⁴cell is inoculated in culture dish by individual cell, and in 37 DEG C, containing 5% CO ₂in incubator, cultivate 24h, then change liquid, changed liquid every 2 days later and go down to posterity in the time that cell density reaches 90%.

(3) packaging of pAd-pSpCas9-T1, pAd-pSpCas9-T2 and pAd-pSpCas9-T3

Transfection the day before yesterday, according to every hole 4 × 10 ⁵individual HEK293 cell is inoculated into 6 orifice plates, and after cultivation 24h, cell approximately has 70% fusion, and 4h before transfection, with cleaning 2 cells without dual anti-PBS, is changed to the optimization nutrient solution Opti-MEMI (2mL/ hole) without dual anti-serum-free by the nutrient solution containing serum; 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 specification sheets:

A. optimize nutrient solution with Opti-MEMI 4 μ g are diluted to 250 μ L, incubated at room without endotoxic pAd-pSpCas9-T1 plasmid;

B. optimize nutrient solution by 10 μ L Lipofectamine with Opti-MEMI equally ^tM2000 are diluted to 250 μ L, incubated at room 5min; First two steps must not operate and exceeded 25min;

C. by the plasmid of hatching and Lipofectamine ^tM2000 mix, and cumulative volume is 500 μ L, and whole process is as far as possible soft, mixing solutions incubated at room 20min;

D. 500 μ L mixed solutions are added in a hole of culture plate, rock gently culture plate and mix nutrient solution;

E. be 5%CO by nutrient solution at 37 DEG C, volume fraction ₂incubator cultivate, after 6h, nutrient solution is changed to the DMEM nutrient solution that volume fraction is 10% foetal calf serum; Wherein pack by above-mentioned steps in 4 of 6 orifice plates holes, and one, two other hole is liposome contrast, and one is blank.

F. transfection 24h observation of cell pathology (Cytopathic effect, CPE) situation, after transfection 8～10d, form 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 liquor of collecting is the virus liquid after packaging, called after Ad-pSpCas9-T1, and this virus liquid can be directly used in follow-up cell infection or in-80 DEG C of preservations.

According to method packaging pAd-pSpCas9-T2 plasmid, pAd-pSpCas9-T3 plasmid and empty carrier plasmid pAd-pSpCas9 (BB) same as described above, obtain respectively virus liquid Ad-pSpCas9-T2, Ad-pSpCas9-T3 and Ad-pSpCas9 (BB).

(4) virus titer is measured

By every hole 2 × 10 ⁴the density inoculation HEK293 of individual cell, in 96 orifice plates, adopts TCID ₅₀the virus titer of Ad-pSpCas9-T1 is measured and calculated to method and Karbers method.Will be with multiple proportions (10 ^-1～10 ^-10) the virus liquid cells infected of dilution, every hole adds 100 μ L viral dilution liquid, cultivates the CEP pathology of 9～10d statistics cell.According to Karbers formula: T=10 × 10 ^{1+d (s – 0.5)}tCID ₅₀/ mL calculates the viral titre that obtains; Wherein s is positive ratio sum, namely used extent of dilution sum.D=log10 extent of dilution=1 (this is for the extent of dilution of 10 times); And according to formula: T=1 × 10 ^xtCID ₅₀/ mL=1 × 10 ^{x – 0.7}pFU/mL, can be changed to PFU/mL unit by virus titer.

The purity of virus liquid and titre are as follows as calculated:

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, virus infection turns GFP mouse cell

Get and turn GFP mouse and (turn the preparation of GFP mouse by Okabe M, Ikawa M, Kominami K, Nakanishi T, Nishimune Y. " Green mice " as asource of ubiquitous green cells.FEBS Lett1997; The method preparation of 407:313-9 report) tail point sets up fibroblast, then bed board: after resuspended the cell dissociation of logarithmic 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 nutrient solution, renews fresh nutrient solution, adds respectively the virus liquid of Ad-pSpCas9-T1, Ad-pSpCas9-T2 and Ad-pSpCas9-T3, infection multiplicity is 400, simultaneously with PBS and Ad-pSpCas9 (BB) virus liquid in contrast, after mixing, put into incubator and cultivate, about 24 hours, change liquid, collecting cell after 48 hours, extracts cell DNA.Pcr amplification GFP gene target spot gene order, pcr amplification upstream primer is Cas9-GFP-F:5 '-gtgagcaagggcgaggag-3 ' (SEQ ID NO.10); Downstream primer is Cas9-GFP-R:5 '-tggtagtggtcggcgagc-3 ' (SEQ ID NO.11), PCR product is cut with T7Endonuclease I enzyme, nucleic acid detected through gel electrophoresis, then use BIO-RAD imaging system (ChemiDocXRS) the software analysis statistics non-homogeneous recombinational repair of target spot (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 at random 3 groups, 3 every group (1 group # is that 1,2,3,2 group # are that 4,5,6,3 group # are 7,8,9), first group of tail vein injection Ad-pSpCas9-T2 virus liquid (viral dosage 1 × 10 ¹¹pFU/kg) injection, second group of isodose adenovirus Ad-pSpCas9 of tail vein injection (BB), the physiological saline of the 3rd group of injection equivalent, once virus of injection in every 7 days, injects 3 times continuously, raises altogether 21 days.Get the heart, liver, spleen, lung, the nephridial tissue that turn GFP mouse, and set up clone, and the cell of respectively organizing with flow cytometry analysis mouse, and sub-electing green fluorescence negative cells and positive cell, result is as shown in Figure 2.Result shows, the each tissue of injection Ad-pSpCas9-T2 virus liquid all can sub-elect the negative cells (Fig. 2 A) of different ratios, and the mouse 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 two groups of cells of injection Ad-pSpCas9 (BB) virus liquid and physiological 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, the artificial adenovirus carrier that knocks out GFP gene C RISPR/Cas9 element of integrating, Ad-pSpCas9-T2 virus liquid is at acceptor (turning GFP mouse) animal model, effectively targeting modification target gene GFP, has confirmed that adenovirus mediated CRISPR/Cas9 system can carry out the modification of gene target in living animal.

Green fluorescence negative cells and the positive cell extraction DNA that the each tissue of GFP mouse sub-elects that turn of Ad-pSpCas9-T2 virus liquid will be infected, then pcr amplification GFP gene target site sequence, amplified fragments is cloned in pMD18-T carrier, recombinant vectors is delivered to the order-checking of Shanghai Sheng Gong biotechnology company limited, and result as shown in Figure 3.Result demonstration, 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 target knock out and turn c-myc DNA murine

1, build adenovirus expression carrier

Utilize online tool ZiFiT Targeter version4.0, at 3 CRISPR/Cas9 target spots of the upper design of c-myc gene Second Exon (GenBank:AH005318.1), and design corresponding oligonucleotide, be specially:

First target spot is T4, and nucleotides sequence is classified 5 '-tcgctacgtccttctcccca-3 ' (SEQ ID NO.12) as; The oligonucleotide of its design is to being 5 '-caccgtcgctacgtccttctcccca-3 ' (SEQ ID NO.13); 5 '-aaactggggagaaggacgtagcgac-3 ' (SEQ ID NO.14);

Second target spot is T5, and nucleotides sequence is classified 5 '-gcagccgcccgcgcccagtg-3 ' (SEQ ID NO.15) as; The oligonucleotide of its design is to being: 5 '-caccgcagccgcccgcgcccagtgg-3 ' (SEQ ID NO.16); 5 '-aaaccactgggcgcgggcggctgcg-3 ' (SEQ ID NO.17);

The 3rd target spot is T6, and nucleotides sequence is classified 5 '-cagatgatgaccgagttact-3 ' (SEQ ID NO.18) as; The oligonucleotide of its design is to being: 5 '-caccgcagatgatgaccgagttact-3 ' (SEQ ID NO.19); 5 '-aaacagtaactcggtcatcatctgc-3 ' (SEQ ID NO.20).

Then obtain respectively plasmid pAdTrack-pSpCas9-T4, pAdTrack-pSpCas9-T5 and pAdTrack-pSpCas9-T6 according to the method for embodiment 1.And prepare respectively Ad-pSpCas9-T4, Ad-pSpCas9-T5 and Ad-pSpCas9-T6 virus liquid according to the method for embodiment 1, and then measure virus liquid titre, its measurement result is as follows:

Ad-pSpCas9-T4 virus liquid, purity is A260/A280=1.47, titre is 2.5 × 10 ¹¹;

Ad-pSpCas9-T5 virus liquid, purity is A260/A280=1.51, titre is 1.7 × 10 ¹¹;

Ad-pSpCas9-T6 virus liquid, purity is A260/A280=1.48, titre is 3.1 × 10 ¹¹.

2, virus liquid infects acceptor mouse

Get and turn c-myc DNA murine tail point and set up fibroblast and (turn the preparation of c-myc DNA murine by Harris A W, Harris A W, Pinkert C A, Crawford M, et al.The E mu-myc transgenic mouse.A model for high-incidence spontaneous lymphoma and leukemia of early B cells[J] .The Journal of experimental medicine, 1988, 167 (2): the method preparation of 353-371. report), then bed board, after resuspended the cell dissociation of logarithmic 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 nutrient solution, renew fresh nutrient solution, according to infection multiplicity 400particle/cell, cell is divided into 5 groups, infects respectively virus liquid Ad-pSpCas9-T4, Ad-pSpCas9-T5, Ad-pSpCas9-T6, Ad-pSpCas9 (BB) and PBS, after mixing, can put into incubator cultivation and within 24 hours, change liquid, peptic cell after 72 hours, extracts cell DNA.

Detect primer according to the shot design of c-myc gene, be specially, upstream primer is M1-F:5 '-gagcgagctgcagccgcccgcg-3 ' (SEQ ID NO.21), and downstream primer is M1-R5 '-gcccgcgggcggggctcagg-3 ' (SEQ ID NO.22).Then taking extract cell DNA as template, increase taking sequence shown in SEQ ID NO.21 and SEQ ID NO.22 as primer respectively.The PCR product T7 endonuclease (T7E1) that amplification is obtained carries out enzyme to be cut, enzyme cut after under 80-120V condition, detect with agarose gel electrophoresis 40min, result as shown in Figure 4.Result shows, the sudden change that virus liquid Ad-pSpCas9-T4 causes target spot most effective.

By the cell extraction DNA of virus liquid Ad-pSpCas9-T4 infection, utilize the upstream and downstream primer amplification c-myc gene of c-myc gene, extension amplification outcome, to pMD18-T carrier, is delivered to the order-checking of Shanghai Sheng Gong biotechnology company limited, and result is as shown in Figure 5.Result shows can form sudden change in target area after virus liquid Ad-pSpCas9-T4 cells infected.

3, turn c-myc DNA murine tail vein injection adenovirus

Get 30 mouse (10 week age) that turn c-myc gene, be divided at random 3 groups, every group 10, first group of injecting virus liquid Ad-pSpCas9-T4, second group and the 3rd group of mouse respectively injecting virus liquid Ad-pSpCas9 (BB) and PBS in contrast, are injected once weekly, in SPF level animal rearing room, raise mouse 30 weeks, observe mouse survival rate and record result, result as shown in Figure 6.Result shows, the mouse of tail vein injection PBS group suffers from 9 of tumor mortality, the mouse of tail vein injection Ad-pSpCas9 (BB) virus liquid is all dead because suffering from tumour, and tail vein injection Ad-pSpCas9-T4 virus liquid mouse is because suffering from 5 of tumor mortality, and the death time.Injection adenovirus Ad-pSpCas9-T4 knock-out mice c-myc gene is described, thereby can suppresses the morbidity of mouse tumor, extend the survival time of mouse.

4, turn the order-checking of c-myc DNA murine gene target site

3 dead mouse of group and survival mice are dissected, core, liver, spleen, lung and nephridial tissue, the DNA that extracts each tissue is template, taking the sequence of SEQ ID NO.21 and SEQ ID NO.22 as primer carries out pcr amplification c-myc gene, PCR product is reclaimed and is cloned in PMD18-T carrier, after conversion, every mouse organized 20 single bacterium colonies of picking, serve Hai Shenggong order-checking, then add up non-homogeneous restructuring amendment (NHEJ) according to sequencing result, result is as shown in table 1.

The non-homogeneous restructuring amendment statistics of the each tissue of table 1, dead mouse and survival mice

As shown in Table 1, all there is non-homogeneous restructuring amendment (NHEJ) in various degree in the c-myc gene in each histoorgan of the mouse of experimental group.Show that adenovirus mediated CRISPR/Cas9 system can be successfully carries out target and knock out turning in c-myc DNA murine gene c-myc gene, suppress thereby reach the object that mouse tumor occurs.

Finally explanation is, above preferred embodiment is only unrestricted in order to technical scheme of the present invention to be described, although the present invention is described in detail by above preferred embodiment, but those skilled in the art are to be understood that, can make various changes to it in the form and details, and not depart from the claims in the present invention book limited range.

Claims

1. contain the target knockout carrier of CRISPR/Cas9 system, it is characterized in that: described target knockout carrier cuts, fills by pX330-U6-Chimeric_BB-CBh-hSpCas9 plasmid EcoRI and SacII enzyme the pAdTrack-CMV plasmid that rear connection is cut, filled through BstXI enzyme, then by the specific target sequence that is connected into goal gene after BbsI linearizing, then with obtaining with the restructuring of pAdEasy-1 plasmid after the linearizing of Pme I and CIAP alkaline phosphatase dephosphorylation.

2. the target knockout carrier that contains according to claim 1 CRISPR/Cas9 system, is characterized in that: described goal gene is GFP or c-myc gene.

3. the target knockout carrier that contains according to claim 2 CRISPR/Cas9 system, is characterized in that: the specific target sequence that goal gene is GFP is as shown in SEQ ID NO.1, SEQ ID NO.4 or SEQ ID NO.7.

4. the target knockout carrier that contains according to claim 2 CRISPR/Cas9 system, is characterized in that: goal gene is that the specific target sequence of c-myc gene is as shown in SEQ ID NO.12, SEQ ID NO.15 or SEQ ID NO.18.

5. the target knockout carrier that contains according to claim 4 CRISPR/Cas9 system, is characterized in that: described specific target sequence is as shown in SEQ ID NO.12.

6. contain the adenovirus of target knockout carrier described in claim 1-5 any one.

7. the application of target knockout carrier in the reagent of preparation sudden change specific target sequence described in claim 1-5 any one.

8. the application of target knockout carrier in the reagent of preparation inhibition tumour described in claim 5.