CN104178461B - CAS9-carrying recombinant adenovirus and application thereof - Google Patents
CAS9-carrying recombinant adenovirus and application thereof Download PDFInfo
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Abstract
The invention discloses a CAS9-carrying recombinant adenovirus and application thereof. The invention protects a recombinant adenovirus carrying coding gene of CAS9 protein; and the CAS9 protein is disclosed as Sequence 7 in the sequence table. The coding gene of the CAS9 protein is disclosed as 789th-5045th nucleotides from the 5' terminal of Sequence 6 in the sequence table. The invention also protects application of the recombinant adenovirus in preparing a kit for knocking out target gene. The CRISPR (clustered regularly interspaced short palindromic repeat) method has the characteristics of high efficiency, high speed and low cost. The CAS9 recombinant adenovirus disclosed by the invention can be used in a protein function cell platform (1) for quick gene modification and high flux screening, a liver protein function platform (2) for implementing quick and specific gene knock-out at the liver, and a mouse gene modification platform (3) for preparing a gene knock-out/knock-in mouse.
Description
Technical field
The present invention relates to a kind of recombinant adenoviruss carrying cas9 and its application.
Background technology
Continuous development with genome annotation project (genome annotation projects) and perfect, and egg
The enforcement of white matter group plan (proteome projects), scientific research personnel have begun to be eager to have a try it is desirable to group is learned research
Achievement apply to the every field of basic scientific research, and personalized medicine (personalized as soon as possible
Medicine in the middle of).But the group information of magnanimity has also gone out a no small difficult problem to scientific research personnel, that is, this how
These uninteresting data conversions are become significant protein function, or with clinically relevant information？Solve this problem
Core be that and develop a kind of efficient, reliable method as early as possible, to help scientific research personnel's research genotype
(genotype) influence to phenotype (phenotype).Using homologous recombination machinery (homologous
Recombination) gene is oriented with inactivation (targeted gene inactivation) is exactly such a side well
Method, can help the function of the clear and definite gene of scientific research personnel or albumen.But make also can be subject in this way in real work
, such as there is the restriction that efficiency is too low, wastes time and energy and is likely to result in mutation etc. in the restriction of several factors.
Occur in that two kinds of new research meanses nearly ten years, scientific research personnel can be helped to various cells and various organism
Interior almost arbitrarily gene is manually operated.This new technique is exactly the " genome editing technique (genome that we often say
Editing) ", including Zinc finger nuclease (zinc-finger nucleases, zfn) and activating transcription factor sample effector core
Sour enzyme (transcription activator-like effector nucleases, talen).Both nucleases are all
Chimera, is all by through design, sequence-specific dna binding member (programmable, sequence-
Specific dna-binding modules) and nonspecific dna cutting domain be combined into.Zfn and talen is
Dna can be carried out with various genetic modifications, the mechanism of action of both nucleases is all first dna duplex molecule to be cut,
Form dna double-strand break otch (dna double-strand break, dsb), then the nonhomologous end in active cell connects
(this is a kind of non-fidelity, genetic mutation easily to connect (nonhomologous end joining, nhej) repair mechanism
Repair mechanism) or homologous recombination (homology-directed repair, hdr) repair mechanism (this is a kind of high-fidelity
Repair mechanism it is not easy to occur mutation), the repair mechanism using cell itself carries out genetic modification to dna.
This people being combined with nonspecific dna cutting domain by sequence-specific dna binding structural domain
Work nuclease is exactly the important component part of this genome editing technique.The nuclease of these inserted types can be with high effect
Rate, high degree of accuracy carry out manually modified, cutting dna generation dsb to genome, then utilize the dsb such as nhej or hdr to repair
Mechanism complete required for various manually modified.Because scientific research personnel tie to the dna of the albumen such as zinc finger protein and tale albumen
The designed capacity closing domain is more and more stronger, so the range of application of this genome editing technique is also over and over again expanded.This
A little not only simple but also flexible nucleases have played great effect in genetic engineering field, and its importance is also all with day
Increase, the forefront of genetic engineering work has been arrived at station.
Crisprs full name repeats (clustered regularly interspaced for the short palindrome in rule cluster interval
Short palindromic repeats), this is that a class is distributed widely in the repetitive structure in antibacterial and archaeal genome.This
One pattern occur in antibacterial more than 40% and 90% Archimycetess in.Research shows, crispr and a series of associated protein, front
Lead sequence together, the acquired immunity ability to exogenous genes such as antiphagins can be provided for prokaryote.
Originally, a lot of research worker suppose that these strange repetitive sequences are skimble-skamble, but 2005, three lifes
Thing informaticss team reports, dna usual and phage gene order in spacer matches, and shows crispr in microorganism immunity
In may play effect.The eugene koonin of Maryland State American National Biotechnology Information center and his colleague carry
Go out, antibacterial and Archimycetess obtain phage dna, afterwards as one of rna molecule (coupling of external dna can be stoped)
Template saves, just as the system that eukaryotic cell is referred to as the RNA interference (rnai) using destroys rna.
2007, rodolphe barrangou, philippe horvath and other people prove, they can by add
Or delete the spacer dna matching with phage dna, change the resistance to bacteriophage attack for the streptococcus thermophiluss.This
E.I.Du Pont Company is now enable to cultivate stronger bacterial strain for food production.They have also discovered simultaneously wherein hide basic
Principle: when meeting with phage invasion, crispr can react, and now antibacterial is transcribed into spacer dna and dna palindrome
The rna molecule of a string length, tracrrna (an extra rna fragment) and cas9 mono- works generation crrna (from spacer
Rnas).2011, in proposition on " natural " magazine, cas9, tracrrna were together with crrna with certain for charpentier
The external dna that method is attacked and crrna matches.
It is not only Food Science man and microbiologist, the research worker in a lot of fields will appreciate that bacterial immune system
Importance, because it possesses a very valuable characteristic: with certain specific gene order as target.Many Research Teams
It is utilized to delete, add, activate or suppression human body, mouse, Brachydanio rerio, antibacterial, fruit bat, yeast, nematicide and crop plant cells
In target gene, thus demonstrating the broad applicability of this technology.
In the technology of above-mentioned three kinds of accurate manipulation genes, effect of crispr and ease for use are in every respect all more
Victory one is raised.In terms of cutting target dna, crispr system is more more efficient than talens, and can process more bases than talens
Cause.
The experiment of zhang feng and his colleague shows, crispr can lock at once and cut two in human body cell
Gene.With the cooperation of Massachusetts developmental biology man of Alfred North Whitehead biomedical research institute rudolf jaenisch in,
Zhang has divided 5 genes in mouse embryo stem cell.These work are to cultivate transgenic mice to lay the foundation, and this is
One key tool of biomedical research.His team finds, this can be simply by cas9 messenger rna and two guide rnas
In the ovum of injection mouse or germ cell.Additionally, it is different with utilizing a pair of grna introducing by carrying out to cas9 gene changing structure
Cleavage site, the effect problem of missing the target of crispr is also overcome.So far, crispr technology moves towards perfect in itself, and initially enters
Application stage.
Content of the invention
It is an object of the invention to provide a kind of recombinant adenoviruss carrying cas9 and its application.
The recombinant adenoviruss of the claimed encoding gene carrying cas9 albumen；Described cas9 albumen such as sequence table
Sequence 7 shown in.The encoding gene of described cas9 albumen specifically can be if the sequence 6 of sequence table is from 5 ' end 789-5045 position cores
Shown in thuja acid.
The preparation method of described recombinant adenoviruss comprises the steps:
(1) encoding gene of described cas9 albumen is inserted pdonrtmBetween attl1 the and attl2 site of 221 carriers, obtain
To middle interstitial granules；
(2) middle interstitial granules and pad-dest carrier are occurred gateway to react, obtain the coding containing described cas9 albumen
The recombiant plasmid of gene, as recombiant plasmid pad-dest-cas9；
(3) recombiant plasmid pad-dest-cas9 is imported mammalian cell and cultivate, obtain described recombinant adenoviruss.
Described middle interstitial granules concretely uses the double-strand of the encoding gene containing cas9 albumen shown in sequence 6 of sequence table
Dna molecule replaces pdonrtmSmall fragment (containing attl1 and attl2) between attl1 and attl2 of 221 carriers, the restructuring obtaining
Plasmid.
Described mammalian cell concretely 293 cell.
The present invention also protects a kind of recombiant plasmid (recombiant plasmid pad-dest-cas9), and its preparation method is as follows:
(1) encoding gene of described cas9 albumen is inserted pdonrtmBetween attl1 the and attl2 site of 221 carriers, obtain
To middle interstitial granules；
(2) middle interstitial granules and pad-dest carrier are occurred gateway to react, obtain the coding containing described cas9 albumen
The recombiant plasmid of gene.
The present invention also protects the recombinant adenoviruss described in any of the above to be used in the test kit knocking out genes of interest in preparation
Application.The encoding gene of described genes of interest concretely cebpa albumen.The encoding gene of described cebpa albumen specifically may be used
For having the dna molecule of the nucleotide shown in the sequence 3 of sequence table.
The present invention also protects a kind of test kit for knocking out genes of interest, including the recombinant adenovirus described in any of the above
Poison.Described test kit may also include the recombinant slow virus of the encoding gene of the sgrna carrying described genes of interest.Described purpose base
Encoding gene because of concretely cebpa albumen.The encoding gene of described cebpa albumen concretely has the sequence of sequence table
The dna molecule of the nucleotide shown in 3.The encoding gene of the sgrna of described genes of interest specifically can be as the sequence 1 of sequence table or sequence
Shown in row 2.The preparation method of the recombinant slow virus of encoding gene of the described sgrna carrying described genes of interest is specific as follows:
Recombiant plasmid plenti-u6 sgrna is imported mammalian cell and cultivates, obtain carrying the sgrna's of described genes of interest
The recombinant slow virus of encoding gene；The preparation method of described recombiant plasmid plenti-u6sgrna is as follows: 1. composition sequence table
Double-strand dna molecule shown in sequence 1 as template, using the single-stranded dna molecule shown in the sequence 4 of sequence table and sequence table
The molecular primer pair of single-stranded dna shown in sequence 5 carries out pcr amplification, obtains pcr amplified production；2. composition sequence table
Double-strand dna molecule shown in sequence 2 as template, using the single-stranded dna molecule shown in the sequence 4 of sequence table and sequence table
The molecular primer pair of single-stranded dna shown in sequence 5 carries out pcr amplification, obtains pcr amplified production；3. use restriction enzyme
Enzyme sali and xbai enzyme action plko.1 carrier, reclaim carrier framework；4. the pcr amplified production that 1. obtains step, step are 2.
To pcr amplified production and the carrier framework that 3. obtains of step connect, obtain recombiant plasmid plenti-u6 sgrna.Described examination
Agent box may also include described recombiant plasmid plenti-u6 sgrna.Described mammalian cell concretely 293t cell.
Inventor constructs plenti-u6 sgrna slow virus carrier and cas9 adenovirus vector.plenti-u6 sgrna
Slow virus carrier imports mammalian cell and cultivates, and can obtain expressing little guide rna (the small guide of specific gene
Rna, sgrna) recombinant slow virus.Experimental requirement, can design and synthesize for heterogeneic plenti-
U6sgrna carrier.Cas9 adenovirus vector imports mammalian cell and cultivates, and can obtain expressing the restructuring gland of cas9 albumen
Virus.Recombinant adenoviruss co-infection by the recombinant slow virus of the little guide rna of expression specific gene and expression cas9 albumen
Aim cell, it is possible to specific gene in silence aim cell, very easily carries out genome editor to various cells.Also may be used
Little with the recombinant adenoviruss co-infection of the recombinant slow virus of the little guide rna will specific gene expressed and expression cas9 albumen
Mus, can carry out genome editor to albumen in mouse liver (adenoviruss can be enriched with liver), thus need not make
Standby transgenic group mice.The time that prepare transgenosis mice expends is long, about needs 3 months, and the method for application virus infection only needs 1-2
Week.The individual gene expense of prepare transgenosis mice, at about 150,000 yuan, spends in 10,000 yuan needed for the method for application virus infection
Left and right.
Crispr method has efficiency high, and speed is fast, the low feature of expense.The recombinant adenoviruss of the cas9 that the present invention provides
Can be used for: (1) protein function cell platform, rapid gene is modified, high flux screening；(2) liver protein function platform, in liver
Realize quick, specific gene knockout；(3) murine genes transformation platform, prepares gene knockout/knock in mice.
Brief description
Fig. 1 is the result of embodiment 2.
Fig. 2 is the result of the step 3 of embodiment 3.
Fig. 3 is the result of the step 4 of embodiment 3.
Specific embodiment
Below example facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method, if no special instructions, is conventional method.Test material used in following embodiments, if no special instructions, is certainly
Routine biochemistry reagent shop is commercially available.Quantitative test in following examples, is respectively provided with three times and repeats to test, result is made even
Average.
Plko.1 carrier: sigma company, article No.: shc001.293t cell: atcc, numbering is crl-3216.Aml12 is thin
Born of the same parents' (Normal mouse liver cell): atcc, numbering is crl-2254.293 cells: atcc, numbering is crl-1573.Pad-dest carries
Body (pblock-ittm6-dest vector): invitrogen company, catalog number is v48720.C57 mice: Beijing China
Fukang biotech inc.pdonrtm221 carriers (pdonrtm221vector): invitrogen
Company, catalog number (Cat.No.) 12536017.
The preparation of embodiment 1, sgrna virus liquid and cas9 virus liquid
First, the selection of target site
Using ccaat enhancer binding protein-a (ccaat-enhancer binding protein-alpha, cebpa)
As target sequence, verify reliability and the verity of crispr/cas9 system, in order to avoid effect of missing the target, select two grna
The target region of series connection,
The dna sequence of two grna is as follows respectively:
Grna1:5 '-gaaaggacgaaacaccgaaggcggccgggtcgatgtagggttttagagctagaaat-3’；
Grna2:5 '-gaaaggacgaaacaccgcacagccgacagcaggagaagggttttagagctagaaat-3’.
Shown in the sequence 1 of grna1 such as sequence table, (its target region is the sequence 3 of sequence table from 5 ' end 323-345 position cores
Thuja acid).Shown in the sequence 2 of grna2 such as sequence table, (its target region is the sequence 3 of sequence table from 5 ' end 375-397 position nucleoside
Acid).The fragment of the encoding gene of cebpa albumen is as shown in the sequence 3 of sequence table.
2nd, the structure of recombiant plasmid plenti-u6 sgrna
1st, the double-strand dna molecule shown in sequence 1 of composition sequence table.
2nd, with the dna molecule of step 1 synthesis as template, the primer pair using crispr-f and crispr-r composition is carried out
Pcr expands, and obtains pcr amplified production.
Crispr-f (sequence 4 of sequence table): 5 '-gtatttcgatttcttggctttatatatcttgtggaaaggacg
aaacaccg-3’；
Crispr-r (sequence 5 of sequence table): 5 '-gttgataacggactagccttattttaacttgctatttctagc
tctaaaac-3’.
3rd, the double-strand dna molecule shown in sequence 2 of composition sequence table.
4th, with the dna molecule of step 3 synthesis as template, the primer pair using crispr-f and crispr-r composition is carried out
Pcr expands, and obtains pcr amplified production.
5th, use restricted enzyme sali and xbai enzyme action plko.1 carrier, reclaim the carrier framework of about 4kbp.
6th, (nwb, catalog number is e2611, and description is shown in adopt gibson assembly test kithttps:// www.neb.com/protocols/2012/09/25/gibson-assembly-master-mix-assemb ly) and by examination
The operation of agent box description is so that what the pcr amplified production that obtains of the pcr amplified production that obtains of step 2, step 3 and step 5 obtained
Carrier framework connects, and obtains recombiant plasmid plenti-u6 sgrna.
3rd, the preparation of sgrna recombinant slow virus
1st, take the logarithm the 293t cell of trophophase, cultivated with the dmem containing 10% (volume ratio) fbs after trypsinization
Base suspends and obtains cell suspension, by every 20ml cell suspension (containing 1.2 × 107Individual cell) it is seeded to the thin of an a diameter of 15cm
Born of the same parents' culture dish, in 37 DEG C, 5%co2Culture 24h (cell density reaches 70%-80%) in incubator.
2nd, after completing step 1, reject supernatant, add the new dmem culture medium prepared, in 37 DEG C, 5%co2In incubator
Culture 2h.
3rd, after completing step 2, by lipofectamine 2000 reagent, (invitrogen company, catalog number is
12566014) and by specification operation, each Tissue Culture Dish transfect 20 μ g recombiant plasmid plenti-u6sgrna, 37 DEG C,
5%co2Culture 8h in incubator.
4th, after completing step 3, reject supernatant, washs precipitation, Ran Houjia with 20ml pbs buffer (ph7.4,0.01m)
Enter the dmem culture medium containing 10% (volume ratio) fbs of new preparation, in 37 DEG C, 5%co2Culture 48h in incubator.
5th, after completing step 4, take supernatant.
6th, take the supernatant that step 5 obtains, 4 DEG C, 4000g centrifugation 10min, collect supernatant.
7th, by 1 parts by volume lentivirus precipation solution (alstem, catalog number is vc100)
The supernatant obtaining with 4 parts by volume steps 6 is placed in centrifuge tube and mixes, then 4000 × g centrifugation 20min, in centrifuge tube
System is divided into 2 layers, collects the supernatant, as contains the virus liquid of the slow viruss of expression grna1 and grna2, abbreviation sgrna disease
(viral level in sgrna virus liquid is 2 × 10 to venom7pfu/ml).
4th, the structure of recombiant plasmid pad-dest-cas9
1st, the double-strand dna molecule using the encoding gene containing cas9 albumen shown in sequence 6 of sequence table is replaced
pdonrtmSmall fragment (containing attl1 and attl2) between attl1 and attl2 of 221 carriers, obtains middle interstitial granules.Sequence table
In sequence 6, it is the encoding gene of cas9 albumen from 5 ' end 789-5045 position nucleotide.
2nd, middle interstitial granules and pad-dest carrier occur gateway to react (bp restructuring), obtain the volume containing cas9 albumen
The recombiant plasmid of code gene, as recombiant plasmid pad-dest-cas9.
5th, the preparation of cas9 recombinant adenoviruss
1st, take the logarithm 293 cells of trophophase, with the dmem culture medium containing 10% (volume ratio) fbs after trypsinization
Suspension obtains cell suspension, by every 1ml cell suspension (containing 5 × 105Individual cell) be seeded to an a diameter of 60mm cell training
Foster ware, in 37 DEG C, 5%co2Culture 24h (cell confluency rate is 50-70%) in incubator.
2nd, after completing step 1, reject supernatant, add the new dmem culture medium prepared, in 37 DEG C, 5%co2In incubator
Culture 2h.
3rd, use restricted enzyme pac i enzyme action recombiant plasmid pad-dest-cas9 so as to linearisation, obtain linearisation
Plasmid.
4th, after completing step 2, by lipofectamine 2000 reagent (invitrogen company, catalog number:
12566014) and by specification operation, the transfection linearization plasmid (in terms of single culture dish) that obtains of 6 μ g steps 3,37 DEG C,
5%co2Culture 8h in incubator.
5th, after completing step 4, reject supernatant, is washed with 20ml pbs buffer (ph7.4,0.01m), is subsequently adding new
The dmem culture medium containing 10% (volume ratio) calf serum, 1% Pen .- Strep prepared, in 37 DEG C, 5%co2Incubator
Interior culture 7 days.
6th, after completing step 5, scraped with cell and scrape cell from culture dish and move into 50ml conical pipe, be collected by centrifugation thin
Born of the same parents, resuspended with dmem culture medium, obtain cell suspension.
7th, the cell suspension that step 6 obtains is carried out multigelation and (freeze cell in present liquid nitrogen, then in 37 DEG C of water-baths
Middle dissolving, acutely vibrates；Freeze thawing 4 times altogether), collect supernatant, as original viral liquid.
8th, take the logarithm 293 cells of trophophase, with the dmem culture medium containing 10% (volume ratio) fbs after trypsinization
Suspension obtains cell suspension, by every 1ml cell suspension (containing 5 × 105Individual cell) be seeded to an a diameter of 60mm cell training
Foster ware, in 37 DEG C, 5%co2Culture 24h (cell confluency rate is 50-70%) in incubator.
9th, after completing step 8, add the original viral liquid that step 7 obtains, the volume of virus liquid is the body of cell culture fluid
Long-pending 50%, in 37 DEG C, 5%co2In incubator, (culture can be seen that obvious cell cracking or cpe are existing for 2-3 days within 4 days for culture
As), then collect supernatant.
10th, 15%cscl and 40%cscl is added and in beckman centrifuge tube, prepare cscl gradient solution.
11st, the supernatant Deca obtaining step 9 is on the cscl gradient solution that step 10 obtains, 4 DEG C, 30000rpm from
The heart 16 hours, the system in centrifuge tube show two bands (be in upper strata for adenoviruss ghost, be in lower floor for live viruses
Grain), collect underclad portion, dialyse one hour in tbs buffer, then dialyse in the tbs buffer containing 10% glycerol
Twice, obtain the virus liquid containing cas9 recombinant adenoviruss, (viral level in cas9 virus liquid is 10 to abbreviation cas9 virus liquid
×1010pfu/ml).
6th, prepare comparison virus liquid
Replace grna1 and grna2 with two grna of targeting gfp gene, carry out step 2 and three, obtain sgrna virus
The comparison virus liquid of liquid, abbreviation comparison virus liquid.
Embodiment 2, external activity detection
1st, use dmem culture medium suspension aml12 cell, obtaining cell concentration is 5 × 105The cell suspension of individual/ml.
2nd, packet transaction
Experimental group: sgrna virus liquid that cell suspension that 1ml step 1 is obtained, the step 3 of 1ml embodiment 1 obtain,
The cas9 virus liquid mixing that the step 5 of 0.1ml embodiment 1 obtains, in 37 DEG C, 5%co2Culture 48h in incubator；
Matched group: comparison virus liquid that cell suspension that 1ml step 1 is obtained, the step 6 of 1ml embodiment 1 obtain,
The cas9 virus liquid mixing that the step 5 of 0.1ml embodiment 1 obtains, in 37 DEG C, 5%co2Culture 48h in incubator.
3rd, after completing step 2, take cell, extract genome dna, the primer pair using f1 and r1 composition carries out pcr amplification.
f1:5’-attcgcgacccgaagctgcg-3’；
r1:5’-tgttcttgtccaccgacttcttggct-3’.
4th, take the pcr amplified production that step 3 obtains, (setting is not carried out at the comparison of t7e1 enzyme action to carry out t7e1 enzyme action
Reason), then carry out 2% agarose gel electrophoresiies, result is shown in Fig. 1.Matched group carries out the process of t7e1 enzyme action, experimental group and does not carry out
The control treatment of t7e1 enzyme action, band is single.After experimental group carries out t7e1 shearing, smear occurs, that is, in sgrna and cas9
In the presence of albumen, the encoding gene of cebpa albumen there occurs the heteroduplex shearing and creating mispairing, such that it is able to quilt
T7e1 enzyme action.
Embodiment 3, activity in vivo detection
1st, packet transaction
Experimental group: take 4 c57 mices, the sgrna that the step 3 of every tail vein injection 0.1ml embodiment 1 obtains is viral
The cas9 virus liquid that the step 5 of liquid and 0.1ml embodiment 1 obtains；
Matched group: take 1 c57 mice, the comparison virus that the step 6 of every tail vein injection 0.1ml embodiment 1 obtains
The cas9 virus liquid that the step 5 of liquid and 0.1ml embodiment 1 obtains.
2nd, injecting virus liquid, after 7 days, takes the liver of mice.
3rd, take the liver that step 2 obtains, extract genome dna, the primer pair using f1 and r1 composition carries out pcr amplification,
Take pcr amplified production, carry out t7e1 enzyme action (setting does not carry out the control treatment of t7e1 enzyme action), then carry out 2% agarose and coagulate
Gel electrophoresis, result is shown in Fig. 2.Matched group carries out the control treatment that the process of t7e1 enzyme action, experimental group do not carry out t7e1 enzyme action, band
Single.After experimental group carries out t7e1 shearing, smear occurs, that is, in the presence of sgrna and cas9 albumen, cebpa albumen
Encoding gene there occurs the heteroduplex shearing and creating mispairing, such that it is able to by t7e1 enzyme action.Purpose fragment is sequenced,
Result shows, editing ratio up to more than 70%,
4th, take the liver that step 2 obtains, extract total protein, carry out western detection, the one of employing resists for cebp Alpha antibodies
(14aa) (purchased from santa cruz biotechnology, catalog number (Cat.No.): sc-61).Result is shown in Fig. 3, in experimental mice liver
Cebpa protein content be substantially less than control group mice, that is, in the presence of sgrna and cas9 albumen, the coding of cebpa albumen
Gene there occurs shearing, thus cebpa protein content significantly reduces.
Claims (8)
1. a kind of test kit for knocking out genes of interest, including the recombinant adenoviruss of the encoding gene carrying cas9 albumen with take
The recombinant slow virus of the encoding gene of the sgrna with described genes of interest；
Described genes of interest is the gene of coding ccaat enhancer binding protein-a；
Described cas9 albumen is as shown in the sequence 7 of sequence table；
Described " carrying the recombinant slow virus of the encoding gene of the sgrna of described genes of interest " is the encoding gene carrying sgrna1
Recombinant slow virus with the encoding gene of sgrna2；The encoding gene of described sgrna1 is as shown in the sequence 1 of sequence table；Described
The encoding gene of sgrna2 is as shown in the sequence 2 of sequence table.
2. test kit as claimed in claim 1 it is characterised in that: the sequence of the encoding gene such as sequence table of described cas9 albumen
6 from shown in 5 ' end 789-5045 position nucleotide.
3. test kit as claimed in claim 1 or 2 it is characterised in that: " carry the recombinant adenovirus of the encoding gene of cas9 albumen
The preparation method of poison " comprises the steps:
(1) encoding gene of described cas9 albumen is inserted pdonrtmBetween attl1 the and attl2 site of 221 carriers, in obtaining
Interstitial granules；
(2) middle interstitial granules and pad-dest carrier are occurred gateway to react, obtain the encoding gene containing described cas9 albumen
Recombiant plasmid, as recombiant plasmid pad-dest-cas9；
(3) recombiant plasmid pad-dest-cas9 is imported mammalian cell and cultivate, obtain described recombinant adenoviruss.
4. test kit as claimed in claim 3 it is characterised in that: described mammalian cell be 293 cells.
5. a kind of test kit for knocking out genes of interest, including the recombinant adenoviruss of the encoding gene carrying cas9 albumen with take
The recombiant plasmid of the encoding gene of the sgrna with described genes of interest；
Described genes of interest is the gene of coding ccaat enhancer binding protein-a；
Described cas9 albumen is as shown in the sequence 7 of sequence table；
Described " carrying the recombiant plasmid of the encoding gene of the sgrna of described genes of interest " be carry sgrna1 encoding gene and
The recombiant plasmid of the encoding gene of sgrna2；The encoding gene of described sgrna1 is as shown in the sequence 1 of sequence table；Described sgrna2
Encoding gene as shown in the sequence 2 of sequence table.
6. test kit as claimed in claim 5 it is characterised in that: the sequence of the encoding gene such as sequence table of described cas9 albumen
6 from shown in 5 ' end 789-5045 position nucleotide.
7. the test kit as described in claim 5 or 6 it is characterised in that: " carry the recombinant adenovirus of the encoding gene of cas9 albumen
The preparation method of poison " comprises the steps:
(1) encoding gene of described cas9 albumen is inserted pdonrtmBetween attl1 the and attl2 site of 221 carriers, in obtaining
Interstitial granules；
(2) middle interstitial granules and pad-dest carrier are occurred gateway to react, obtain the encoding gene containing described cas9 albumen
Recombiant plasmid, as recombiant plasmid pad-dest-cas9；
(3) recombiant plasmid pad-dest-cas9 is imported mammalian cell and cultivate, obtain described recombinant adenoviruss.
8. test kit as claimed in claim 7 it is characterised in that: described mammalian cell be 293 cells.
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| CN201410400098.XA CN104178461B (en) | 2014-08-14 | 2014-08-14 | CAS9-carrying recombinant adenovirus and application thereof |
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| CN104178461B true CN104178461B (en) | 2017-02-01 |
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| CN105802980A (en) * | 2016-04-08 | 2016-07-27 | 北京大学 | CRISPR/Cas9 system with Gateway compatibility and application of CRISPR/Cas9 system |
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