CN110004181A - A kind of additive type CRISPR/Cas9 expression vector and its construction method and application - Google Patents
A kind of additive type CRISPR/Cas9 expression vector and its construction method and application Download PDFInfo
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- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
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Abstract
A kind of additive type CRISPR/Cas9 expression vector and its construction method and application, belong to cell engineering field.Host cell gene group is easily integrated into order to solve existing CRISPR/Cas expression system, the problem of destroying sequence in genome, the present invention provides a kind of additive type CRISPR/Cas9 expression vectors, the carrier is the CRISPR/Cas9 expression vector containing S/MAR sequence, refer to the CRISPR/Cas9 expression vector containing S/MAR sequence, the S/MAR sequence, nucleotide is as shown in SEQ ID NO:1；The S/MAR sequence is located in the carrier between the coding region sequence that can encode albumen and bGH poly (A) sequence.Additive type CRISPR/Cas9 expression vector provided by the invention can be free in dyeing vivoexpression Cas9 albumen and single-stranded guidance RNA, can be used for gene knockout research.
Description
Technical field
The invention belongs to cell engineering fields, and in particular to a kind of additive type CRISPR/Cas9 expression vector and its
Construction method and application.
Background technique
CRISPR/Cas9 system is compiled as emerging strong gene editing and genetic screening tool by gene
Volume, there is extensive application prospect in terms of the fields of biomedicine such as the foundation of disease model and gene therapy.CRISPR/
Cas9 system mainly includes two members, Cas9 albumen and single-stranded guidance RNA during exercising gene editing function
(sigle-guide RNA, sgRNA).Wherein Cas9 albumen and single-stranded guidance RNA are mainly by expression vector in histocyte
Transcriptional expression obtains, and common expression vector is generally retrovirus expression vector or plasmid-type expression vector.However,
CRISPR/Cas9 system still suffers from some technical problems in actual application, and one of problem is that expression
The carrier of CRISPR/Cas9 system is easily integrated into host cell gene group, after entering host cell to destroy gene
Sequence in group, there are potential security risks.
Summary of the invention
It is easily integrated into host cell gene group in order to solve existing CRISPR/Cas expression system, destroys sequence in genome
The problem of column, provides a kind of additive type CRISPR/Cas9 expression vector, refers to the CRISPR/Cas9 table containing S/MAR sequence
Up to carrier, the S/MAR sequence, nucleotide is as shown in SEQ ID NO:1；The S/MAR sequence, which is located in the carrier, to be compiled
Between the coding region sequence and bGH poly (A) sequence of code albumen.
It further limits, the coding region sequence that can encode albumen is EGFP coding region sequence.
It further limits, the sgRNA sequence of the CRISPR/Cas9 expression vector, is one in SEQ ID NO:2-4
Kind.
It further limits, additive type CRISPR/Cas9 expression vector also contains G418 resistant gene, the G418 resistance
The nucleotide sequence of gene is as shown in SEQ ID NO:5.
The present invention also provides the construction methods of above-mentioned additive type CRISPR/Cas9 expression vector, are connected by digestion
It connects or the method for homologous recombination is by S/MAR sequence construct can encode the code area sequence of albumen into CRISPR/Cas9 expression vector
Between column and bGH poly (A) sequence.
It further limits, the construction method of the additive type CRISPR/Cas9 expression vector includes the following steps:
1) G418 resistant gene is building up in px330-EGFP carrier, obtains carrier framework px330-EGFP-G418；Institute
The nucleotide sequence of G418 resistant gene is stated as shown in SEQ ID NO:5；
2) on the carrier framework px330-EGFP-G418 for obtaining S/MAR sequence construct to step 1), carrier framework is obtained
px330-EGFP-G418-S/MAR；
3) by sgRNA sequence be inserted into carrier px330-EGFP-G418-S/MAR expression skeleton U6 promoter after BbsI enzyme
In enzyme site, additive type CRISPR/Cas9 expression vector px330-EGFP-G418-S/MAR-sgRNA, the sgRNA sequence are obtained
Column one of such as SEQ ID NO:2-4.
Further limiting, G418 resistant gene and px330-EGFP carrier described in step 1) use BsmBI digestion respectively,
Px330-EGFP carrier framework digestion large fragment and G418 resistant gene digestion products are recycled, through T4DNA ligase obtains carrier
Skeleton px330-EGFP-G418.
It further limits, S/MAR sequence described in step 2) and px330-EGFP-G418 carrier are used respectively
Bsp1407 digestion, recycling px330-EGFP-G418 carrier framework digestion large fragment and S/MAR sequence digestion products, through T4DNA
Enzyme connection, obtains carrier framework px330-EGFP-G418-S/MAR.
It further limits, sgRNA sequence described in step 3) and carrier framework px330-EGFP-G418-S/MAR distinguish
After BbsI digestion, recycling carrier framework px330-EGFP-G418-S/MAR digestion large fragment and sgRNA sequence digestion products,
Through T4DNA enzymatic connection, obtains additive type CRISPR/Cas9 expression vector px330-EGFP-G418-S/MAR-sgRNA.
Additive type CRISPR/Cas9 expression vector of the present invention can be used for gene knockout research.
Beneficial effect
Matrix attachment region (matrix attachment region, MAR) refers in eucaryote chromatin can be with
Paralinin (nuclear matrix) or nuclear skeleton generate the DNA sequence dna of specific binding, also known as nuclear skeleton attachment region
(scaffold attachment region,SAR).It is denoted as S/MAR (or SMAR) sequence in the present invention, S/MAR sequence is
Exposed DNA sequence dna in chromosome stablizes the space bit of chromosome mainly in conjunction with paralinin in nucleus or nuclear skeleton
It sets.After S/MAR sequence is connect with CRISPR/Cas9 pUC pUC in the present invention, the matter of CRISPR/Cas9 and S/MAR building
Grain carrier, which cyclic annular can be stablized to dissociate, to be present in outside chromosome, is carried with existing frequently-used CRISPR/Cas9 viral vectors and plasmid
Body is compared, and does not need to be integrated into host cell gene group, will not destroy sequence in genome, this vector safety is high, potential
Security risk is small.
S/MAR can be such that CRISPR/Cas9 expression vector is formed independent in extrachromosomal cyclic structure simultaneously, to avoid
Gene silencing caused by gene position effect in chromosome.In view of this relationship between S/MAR sequence and gene expression, especially
It overcomes position effect, avoids transgene silencing, has been applied to genetically modified organism engineering currently as a kind of cis-regulating element
In.By constructing suitable expression vector, zooblast is imported after the side of target gene connects S/MAR, target gene
Expression can be stablized, foreign gene expression levels difference declines between trans genie individual.
Additive type CRISPR/Cas9 expression vector provided by the invention can be free in dyeing vivoexpression Cas9 albumen
With single-stranded guidance RNA, exogenous origin gene integrator is avoided into host cell gene group, to destroy sequence in genome, is had
Effect evades potential security risk.
Detailed description of the invention
Fig. 1 PCR amplification G418 sequence electrophoretogram, wherein M is DNA Marker8000, and 1-3 is G418 sequence；
Fig. 2 PCR amplification S/MAR sequence electrophoretogram, wherein M is DNA Marker8000, and 1-2 is SMAR sequence；
Fig. 3 is connected into G418 and S/MAR sequence carrier restriction enzyme digestion and electrophoresis figure, and wherein M is DNA Marker15000, and 1 is carrier
Px330-EGFP-G418 is through Bsp1407 restriction enzyme digestion and electrophoresis as a result, 2 be carrier px330-EGFP-G418-S/MAR through BglII digestion
Electrophoresis result figure；
Fig. 4 sgRNA annealing electrophoresis result figure, wherein M is DNA Marker15000, and 1 is CYP20A1 gene sgRNA sequence
Column, 2 be SIRT3 gene sgRNA sequence, and 3 be ANXA7 gene sgRNA sequence；
The carrier that Fig. 5 building is completed is through BglII restriction enzyme digestion and electrophoresis result figure, and wherein M is DNA Marker15000, and 1 is
For CYP20A1 gene targeting carrier through BglII restriction enzyme digestion and electrophoresis result figure, 2 be SIRT3 genophore through BglII restriction enzyme digestion and electrophoresis result
Figure, 3 be ANXA7 gene targeting carrier through BglII restriction enzyme digestion and electrophoresis result figure；
Fig. 6 targeting vector px330-EGFP-G418-S/MAR-sgRNA schematic diagram；
Fig. 7 positive cell the selection result；
Plasmid enzyme restriction electrophoretogram is extracted in Fig. 8 cell, wherein M is DNA Marker15000, and 1-2 is CYP20A1 gene
For targeting vector through Bsp1407 restriction enzyme digestion and electrophoresis result figure, 3 be CYP20A1 gene targeting carrier electrophoretogram.
Specific embodiment
Technical term or abbreviation explanation:
G418 resistant gene: neomycin resistance gene；
S/MAR: for matrix attachment region or nuclear skeleton attachment region；
The small guide RNA of sgRNA:small guide RNA；
PBS: phosphate buffer.
Digestion connection: refer to two or more DNA fragmentation after digestion with restriction enzyme, connected by DNA
Enzyme is connect to be attached.
Homologous recombination: refer to the DNA fragmentation that two or more end is had into 20-25bp repetitive sequence, pass through DNA
DNA fragmentation is attached by recombinase.
Heretofore described:
The purchase of px330-EGFP carrier is from addgene, article No. 66581, EGFP coding region sequence described herein,
BGH poly (A) sequence, U6 promoter sequence are both from the carrier；
PEGFP-C1 carrier is bought from clontech company, article No. 6084-1；
PEI-1 carrier is documented in Stefano Manzini, Alessia Vargiolu, Isa M Stehle, Maria
Laura Bacci,Maria Grazia Cerrito,Roberto Giovannoni,Augusta Zannoni,Maria
Rosaria Bianco,Monica Forni,Pierluigi Donini,Michele Papa,Hans J Lipps,and
Marialuisa Lavitrano.Genetically modified pigs produced with a nonviral
episomal vector.PNAS November 21,2006 103(47)17672-17677；https://doi.org/
10.1073/pnas.0604938103, the public can be obtained by Northeast Agricultural University.
Pig fibroblast: with pig primary fibroblast culture acquisition, cultural method is conventional method；
Serum-free DMEM culture solution without double antibody: Gibco company DMEM culture medium (article No.: C11885500BT)；
DMEM culture solution containing 10% serum: for 10% fetal calf serum of addition and dual anti-DMEM culture solution；
Screening and culturing medium digestive juice (0.25% trypsase) refers to that trypsase mass fraction is 0.25% digestion
Liquid；
Other reagents or instrument and equipment can be bought by commercialization approach and be obtained unless otherwise specified.
1. additive type CRISPR/Cas9 expression vector of embodiment.
Additive type CRISPR/Cas9 expression vector described in the present embodiment refers to the CRISPR/Cas9 containing S/MAR sequence
Expression vector, the S/MAR sequence, nucleotide is as shown in SEQ ID NO:1；The S/MAR sequence is located at energy in the carrier
Between the coding region sequence and bGH poly (A) sequence for encoding albumen.
2. additive type CRISPR/Cas9 expression vector of embodiment.
Additive type CRISPR/Cas9 expression vector described in the present embodiment refers to the CRISPR/Cas9 containing S/MAR sequence
Expression vector, the S/MAR sequence, nucleotide contain in the CRISPR/Cas9 expression vector as shown in SEQ ID NO:1
There are EGFP coding region sequence, the EGFP coding region sequence such as in px330-EGFP carrier, the S/MAR sequence is located at
In CRISPR/Cas9 expression vector between EGFP coding region sequence and bGH poly (A) sequence.
3. additive type CRISPR/Cas9 expression vector of embodiment.
Additive type CRISPR/Cas9 expression vector described in the present embodiment refers to the CRISPR/Cas9 containing S/MAR sequence
Expression vector, the S/MAR sequence, nucleotide is as shown in SEQ ID NO:1；The S/MAR sequence is located at energy in the carrier
Between the coding region sequence and bGH poly (A) sequence for encoding albumen, sgRNA sequence is in the CRISPR/Cas9 expression vector
One of SEQ ID NO:2-4.
4. additive type CRISPR/Cas9 expression vector of embodiment.
Additive type CRISPR/Cas9 expression vector described in the present embodiment refers to the CRISPR/Cas9 containing S/MAR sequence
Expression vector, the S/MAR sequence, nucleotide is as shown in SEQ ID NO:1；The S/MAR sequence is located at energy in the carrier
Between the coding region sequence and bGH poly (A) sequence for encoding albumen.
In order to further analyze target practice efficiency, additive type CRISPR/Cas9 expression vector described in the present embodiment also contains
There is G418 resistant gene, the nucleotide sequence of the G418 resistant gene is as shown in SEQ ID NO:5.
The construction method of 5. additive type CRISPR/Cas9 expression vector of embodiment.
The construction method of additive type CRISPR/Cas9 expression vector described in the present embodiment refers to through digestion connection or same
SMAR sequence construct can be encoded the coding region sequence and bGH of albumen by the method for source recombination into CRISPR/Cas9 expression vector
Between poly (A) sequence.
Citing is described as follows below:
1. G418 resistant gene is building up in px330-EGFP carrier, carrier framework px330-EGFP-G418 is obtained；Institute
The nucleotide sequence of G418 resistant gene is stated as shown in SEQ ID NO:5；It is specific as follows:
1) G418 resistance gene sequences obtain.
PCR amplification is carried out according to G418 resistance gene sequences design primer in pEGFP-C1 carrier, obtains G418 resistance base
Because of sequence.
PCR reaction system is 50 μ L systems (TaKaRa, RR902): 25 μ L of Ex Taq；Upstream primer Cas9-Kana-F 1
μL；1 μ L of downstream primer Cas9-Kana-R；1 μ L (10ng) of total DNA template；ddH2O 22μL；PCR reaction condition are as follows: 94 DEG C pre-
It is denaturalized 5min, 94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 30s, totally 33 circulations, later 72 DEG C of extension 10min.
The PCR product of G418 resistant gene is verified through electrophoresis respectively, as shown in Figure 1, utilizing TIANGEN company
TIANgel MidiPurification Kit (TIANGEN DP209-03) carries out Ago-Gel DNA recycling.Specific steps
It operates and carries out according to kit.
1 vector construction primer of table
2) BsmBI digestion G418 resistant gene and px330-EGFP plasmid vector.
By px330-EGFP plasmid vector and G418 resistance gene fragment DNA use respectively BsmBI digestion (NEB company,
R0134L), digestion system is 10 μ L systems: 1 μ L of BsmBI enzyme；10xBuffer 1μL；Plasmid DNA (or G418 resistant gene piece
Section) 5 μ L (200ng)；ddH2O 3μL.The 8h digestion at 55 DEG C of optimal reactive temperature of BsmBI enzyme.Electrophoresis detection digestion as a result,
Endonuclease bamhi uses Ago-Gel DNA kit recycling G418 resistant gene and px330-EGFP carrier framework large fragment.
3) G418 resistant gene and px330-EGFP carrier are connected.
T4Ligase (NEB company, M0202L) linked system is 10 μ L systems: 1 μ L of px330-EGFP digestion products；G418
7 μ L of resistant gene digestion products；T41 μ L of ligase；SolutionI 1μL.After linked system is mixed under the conditions of 16 DEG C mistake
Night connection forms new carrier framework px330-EGFP-G418, and in next day conversion DH5 α competent bacteria and massive amplification company
It practices midwifery object, obtains the carrier bone of high concentration by using plasmid small extraction reagent kit (TIANGEN, DP103-03) small amount plasmid to extract
Frame.It serves Hai Yingjun sequencing company and carries out sequence verification using G418 sequencing primer.
2 sequencing primer of table
2. on the carrier framework px330-EGFP-G418 that S/MAR sequence construct to step 1 is obtained, obtaining carrier framework
px330-EGFP-G418-S/MAR；Specific step is as follows:
1) S/MAR sequence obtains
PCR amplification is carried out according to S/MAR primers in pEI-1 carrier, obtains S/MAR sequence.PCR reaction system
(TaKaRa,RR902)
50 μ L systems: 25 μ L of Ex Taq；1 μ L of upstream primer Cas9-SMAR-F；1 μ L of downstream primer Cas9-SMAR-R；
1 μ L (10ng) of total DNA template；
ddH2O 22μL；PCR reaction condition are as follows: 94 DEG C of 5min, 94 DEG C of 30s, 60 DEG C of 45s, 72 DEG C of 1min40s, totally 33
It recycles, later 72 DEG C of extension 10min.
The PCR rear electrophoresis of S/MAR is verified, as shown in Fig. 2, using Ago-Gel DNA QIAquick Gel Extraction Kit (TIANGEN
DP209-03 product recycling) is carried out, specific experiment step is carried out by kit operating instruction.
3 vector construction primer of table
2) Bsp1407 digestion S/MAR sequence and px330-EGFP-G418 carrier framework.
Px330-EGFP-G418 plasmid vector and S/MAR sequence DNA are used into Bsp1407 digestion, digestion system 20 respectively
μ L system: 2 μ L of Bsp1407；10xLoading Buffer 2μL；8 μ L (200ng) of DNA fragmentation (plasmid)；ddH2O8μL.It will
Digestion system mixing after under the conditions of 37 DEG C digestion 3h.
3) S/MAR is connect with px330-EGFP-G418 skeleton.
Linked system is 10 μ L systems: 1 μ L of px330-EGFP-G418 digestion products；7 μ L of S/MAR digestion products；
T41 μ L of Ligase (NEB company M0202L)；SolutionI 1μL.The 8h connection under the conditions of 16 DEG C after linked system is mixed,
New carrier framework px330-EGFP-G418-S/MAR is formed, and in next day conversion DH5 α competent cell and massive amplification company
It practices midwifery object, small amount plasmid is extracted, and the carrier that DH5 α amplification generates is passed through, this carrier framework Primary Construction is completed, protected in 4 DEG C of refrigerators
It deposits spare.
4 sequencing primer of table
3. by sgRNA sequence construct into carrier framework px330-EGFP-G418-SMAR, BbsI digestion after U6 promoter
In site, additive type CRISPR/Cas9 expression vector px330-EGFP-G418-S/MAR-sgRNA, the sgRNA sequence are obtained
Such as one of SEQ ID NO:2-4.It is specific as follows:
1) sgRNA sequence design
Design sgRNA sequence.According to the design principle of sgRNA, website (http is used；//crispr.mit.edu/), root
Target practice site is designed according to the code area the MC4R exon sequence of pig.SgRNA has two sections of different regions, and total length is 80bp left
It is right.Firstly, there is the base sequence of 20bp that can be complementary with target DNA or so at its 5 ' end, formed using annealing processing
Hairpin structure instructs Cas9 albumen to carry out gene knockout to the target site of 200 bases before the area CDS of target DNA and cuts.
There are the PAM structures (protospacer adjacent motif, PAM) in one and target site downstream to contain on sgRNA simultaneously
There are two guanine residue (- NGG) or contain a guanine residue, what adenine residue (- NAG) segment combined contains
The segment of two cytosine residues (- NCC) selects then according to the sequence used that provides on the net and to synthesize off-target rate lower
Sequence design sgRNA sequence.Target gene is respectively as follows: cytochrome P450, family 20, subfamily A,
Polypeptide 1 (CYP20A1), NAD-dependent deacetylase sirtuin-3 (SIRT3) and Annexin A7
(ANXA7) gene.
Table 5sgRNA sequence
According to designed sgRNA sequence, 2 single-stranded DNA sequences are separately designed, are sgRNA positive-sense strand and antisense strand
Complementary single strand, two of them complementary strand both ends all have the restriction enzyme site of BbsI.Positive-sense strand is digestion position according to 5 ' -3 ' sequence
Point (BbsI), sgRNA sequence, restriction enzyme site (BbsI).Antisense strand is restriction enzyme site (BbsI), antisense according to 3 ' -5 ' sequence
SgRNA sequence, restriction enzyme site (BbsI).The subsequent annealed formation complementary DNA double-strand of two chains, both ends are BbsI restriction enzyme site,
It can directly be connect with the px330-EGFP-G418-S/MAR after BbsI digestion.Respectively CYP20A1-gRNA-1-F1,
CYP20A1-gRNA-1-R1；SIRT3-gRNA-2-F1,SIRT3gRNA-2-R1；ANXA7-gRNA-3-F1,ANXA7-gRNA-
3-R1.Every group of single-stranded anneal through 55 DEG C forms the double-strand of hairpin structure, and electrophoresis detection result is as shown in Figure 4.
Table 6 synthesizes the sgRNA sequence after double-strand
7 sequencing primer of table
2) targeting vector constructs
By targeting vector skeleton px330-EGFP-G418-SMAR and sgRNA by BbsI single endonuclease digestion (NEB company,
R0539V), the product after digestion is connected after glue recycles, forms complete cyclic annular recombinant plasmid.
(1) the BbsI digestion of px330-EGFP-G418-SMAR and sgRNA.
Digestion system is 50 μ L systems: 5 μ L of BsmbI enzyme；NE Buffer 2.1 25μL；20 μ of plasmid vector (or sgRNA)
L(200ng).It is placed in 37 DEG C of PCR instrument overnight digestions.Electrophoresis, the recycling of digestion products glue.
(2) px330-EGFP-G418-SMAR is connect with gRNA.
Linked system is 10 μ L: 1 μ L (10ng) of plasmid vector；7 μ L (100ng) of sgRNA digestion products；T4Ligase 1μ
L；SolutionI 1μL.It is placed in thermostat water bath, 16 DEG C of connections overnight obtain targeting vector px330-EGFP-G418-S/
MAR-sgRNA, Vector map are as shown in Figure 6.
(3) it converts in recombinant plasmid px330-EGFP-G418-S/MAR-sgRNA to DH5 α.
Water bath with thermostatic control temperature is adjusted to 42 DEG C in advance, and opens the ultraviolet illuminator of super-clean bench and penetrates 30min, from -80 DEG C of refrigerators
Take out pipe (100 μ L) competence bacteria, ice bath 5-10min to molten condition.The plasmid mixed liquor of 10 μ L connected is added
In the competence bacteria of molten condition, after gently shaking, ice bath 30min takes out the competent cell of ice bath, 42 DEG C of water-bath 45s, so
It puts back to rapidly afterwards on ice, ice bath 2min, it is light that 500 μ L SOC culture solutions (without antibiotic) is added into above-mentioned pipe on the super-clean bench
It is light to mix, 37 DEG C are then attached on constant-temperature table, and 200rpm shakes 1h, takes 500 μ L clear liquids, uniformly flat to be coated in SOC culture medium
On, it is placed in 37 DEG C of insulating boxs and is incubated overnight.
(4) plasmid extracts.
Using the small extraction reagent kit TIANprep Mini Plasmid Kit of the plasmid of Tiangeng extract plasmid (TIANGEN,
DP103-03) specific experiment operating procedure is carried out by kit operating instruction.Identification is sequenced through digestion and obtains positive plasmid.
One, the target practice effect and free property verifying of additive type CRISPR/Cas9 expression vector of the present invention are investigated.
1. the recovery of cell
The pig freezing fibroblast pipe frozen is taken out from liquid nitrogen container, is put into rapidly fast instant in 37~40 DEG C of water-baths
Solution.Suitable DMEM culture solution for containing 10% serum is added into cell precipitation, after gently piping and druming mixes, cell suspension is moved into
In culture dish, completion culture solution is placed in 37 DEG C, 5%CO2It is cultivated in incubator, continues to cultivate thawing to carry out changing liquid for 24 hours afterwards,
With cell recovery, the ratio evaluation of attached cell freezes efficiency afterwards for 24 hours.
2. the secondary culture of cell
(1) super-clean bench will be put into after required culture apparatus cleaning and sterilizing, ultraviolet disinfection 30min will have been formed fine and close single
The culture bottle of confluent monolayer cells is put into superclean bench from taking-up in incubator, is lighted alcolhol burner, is fallen culture solution by alcolhol burner
Enter in small beaker.
(2) digestive juice (0.25% trypsase) 500 μ L is added into culture bottle, jog culture bottle keeps digestive juice wet
Moisten entire cell monolayer, sets 2-3min at room temperature.Overturning culture bottle makes its bottom downward, cell monolayer is visually seen, to single layer
Occur that digestive juice can be removed when gap (about aperture size).
(3) 3.5mL culture solution is added into culture bottle to terminate the digestion of trypsase, is drawn in bottle with suction pipe
Cell in culture solution repeated stock bottle wall mixes gently and cell suspension is made until whole cells are swept away.
(4) a small amount of cell suspension is drawn in the cell of cell counter, is counted under light microscopic, according to result by cell concentration
It adjusts to every milliliter 5 × 105A cell.
(5) cell suspension for drawing 2mL moves into another culture bottle, and 2mL cell suspension (remaining house is left in original culture bottle
Go), and to new culture solution 4mL is added in every bottle, bottle stopper is covered, gently shakes up to be placed in 37 DEG C of constant incubators and cultivate.
3. liposome transfection
Liposome transfection illustrates to be operated (Shanghai Bo Yao biotech firm 11668-019 1.5mL) to cell by kit
When length to 75% density, prepare transfection.2 EP pipes are taken, 100 μ L serum-frees DMEM culture solution without double antibody is separately added into, A pipe adds
Enter the px330-EGFP-G418-S/MAR-sgRNA plasmid of 4 μ g, B pipe is added 8 μ L liposomes, is incubated for 5min respectively.Then, will
A, liquid mixes in B pipe, is blown and beaten uniformly with liquid-transfering gun, 37 DEG C of incubation 15min of compound.Cell to be transfected is taken, old training is discarded
Nutrient solution cleans cell twice with serum-free DMEM culture solution without double antibody, and it is unparalleled to add 1.8mL serum-free in the every hole of 6 well culture plates
Anti- DMEM.A/B compound is slowly added into serum-free DMEM culture medium without double antibody, tissue culture plate is gently shaken when being added dropwise, sets
The 8h in 37 DEG C of incubators, the DMEM culture solution that replacement 2mL contains 10% serum continue to cultivate.
4. the screening of target practice cell
Drug screening target practice cell: at cell transfecting second day, for pig fibroblast according to 1:10 (volume ratio)
It passes on and is incubated overnight in new culture dish, then PBS cleaning replacement screening and culturing medium, grope concentration addition G418 by preliminary experiment
(Sigma), concentration 1000ng/mL, cellular control unit are not transfection carrier plasmid, but addition G418 drug.It is right after general 4 days
After group cell death, experimental group cell is changed to screening and culturing medium (G418 concentration is 200ng/mL).Every 1 day,
Clean and replace screening and culturing medium with sterile PBS solution, it is five days or so long to enough to cell, monoclonal can be carried out and chosen
It takes.
5. the verifying of target practice efficiency
(1) attached cell Genome DNA extraction.
With genome DNA extracting reagent kit TaKaRa MiniBEST Universal Genomic DNA Extraction
Kit Ver.5.0 (TaKaRa 9765) attached cell total DNA.(agents useful for same is both from kit) specific as follows:
Culture solution to the greatest extent is abandoned, to every 10cm2Attached cell in 1mLPBS is added, blown off with the pipette tips of liquid-transfering gun adherent thin
Born of the same parents are transferred in new 1.5mLEP pipe, 12000rpm, are centrifuged 1min, absorb supernatant, add 10 μ L deionized waters, blown with rifle
It beats uniform.The RNaseA9 (10mg/mL) of the BufferGL of 180 μ L, the ProteinaseK of 20 μ L and 10 μ L is added, in 56 DEG C of water
Bath temperature is bathed to tissue cracking completely, and elder generation 12000rpm is centrifuged 2min after cracking, carries out subsequent operation again after removing impurity.To cracking
200 μ LBufferGB and 200 μ L100% ethyl alcohol are added in liquid, sufficiently inhales and plays mixing.Spin Column is placed in
On Collection Tube, solution is moved in Spin Column, and 12000rpm is centrifuged 2min.The BufferWA of 500 μ L is added
Enter in Spin Column, 12000rpm is centrifuged 1min, abandons filtrate.Spin is added in the BufferWB (containing ethyl alcohol) of 700 μ L
In Column, 12000rpm is centrifuged 1min, abandons filtrate, is repeated once.Spin Column is placed in Collection Tube
On, 12000rpm is centrifuged 2min.Spin Column is placed on the centrifuge tube of new 1.5mL, in Spin Column film
The deionized water of 100 μ L is added in centre, is stored at room temperature 5min.12000rpm is centrifuged 2min eluted dna.Spectrophotometer quantitative determination
And sample is put into 4 DEG C of showcases and is saved.
(2) PCR amplification CYP20A1, SIRT3, ANXA7 target practice sequence.
It is expanded using the primer of the code area CYP20A1, SIRT3, ANXA7 exon for pig, is reacted by PCR
Sequence information is obtained after sequencing, is compared with former sequence, and target practice efficiency is examined.
8 gene DNA sequence amplimer of table
Table 9CYP20A1 gene targeting genomic DNA sequencing result
| Original series CYP20A1 | 5’-CTTCCACTGTTCACAATATCTGG-3’ |
| Mutant nucleotide sequence 1 | 5’-CTTCCACTGTTCACAAT-TCTGG-3’ |
| Mutant nucleotide sequence 2 | 5’-CTTCCACTGTTCACAA--TCTGG-3’ |
| Mutant nucleotide sequence 3 | 5’-CTTCCACTGTTCAC------TGG-3’ |
| Mutant nucleotide sequence 4 | 5’-CTTCCACTGTTCACAA----TGG-3’ |
Table 10SIRT3 target practice genomic DNA sequencing result
| Original series SIRT3 | 5’-AAGCCCGACATCGTGTTCTT TGG-3’ |
| Mutant nucleotide sequence 1 | 5’-AAGCCCGACATCGT-TTCTT TGG-3’ |
| Mutant nucleotide sequence 2 | 5’-AAGCCCGACATCG--TTCTT TGG-3’ |
| Mutant nucleotide sequence 3 | 5’-AAGCCCGACA----GTTCTT TGG-3’ |
| Mutant nucleotide sequence 4 | 5’-AAGCCCGACA---TGTTCTT TGG-3’ |
Table 11ANXA7 target practice genomic DNA sequencing result
| Original series ANXA7 | 5’-GGTCAGTATCCTTATCCTAG TGG-3’ |
| Mutant nucleotide sequence 1 | 5’-GGTCAGTATCCTTA-CCTAG TGG-3’ |
| Mutant nucleotide sequence 2 | 5’-GGTCAGTATCCT---CCTAG TGG-3’ |
| Mutant nucleotide sequence 3 | 5’-GGTCAGTAT-----TCCTAG TGG-3’ |
| Mutant nucleotide sequence 4 | 5’-GGTCAGTAT---TATCCTAG TGG-3’ |
Target practice result is as in the table below: sequencing result shows to generate mutation in sgRNA target target site, it was demonstrated that
CRISPR/Cas9 carrier system works orderly in cell.
6. carrier free property is verified
(1) extraction of plasmid DNA in cell.
Px330-EGFP-G418-SMAR-sgRNA (targeting CYP20A1 gene) transfection pig fibroblast of above-mentioned preparation
Afterwards, adenovirus extracts kit Vader- is pressed in picking monoclonal, relevant operation after cell screening in progress step 4 as above
TrapTMHigh Purity MiniSpin Plasmid Purification Kit (VIRONGY company NO.VR8880628)
Operating instruction carries out, and extracts the existing vector plasmid that dissociates in cell.
(2) it converts.
Water bath with thermostatic control temperature is adjusted to 42 DEG C in advance, and opens the ultraviolet illuminator of super-clean bench and penetrates 30min, from -80 DEG C of refrigerators
Take out pipe (100 μ L) DH5 α competence bacteria, ice bath 5-10min to molten condition.Extract in the previous step of 10 μ L is added
In the competence bacteria of molten condition, after gently shaking, ice bath 30min takes out the competent cell of ice bath, 42 DEG C of water-bath 45s, so
It puts back to rapidly afterwards on ice, ice bath 2min, it is light that 500 μ LSOC culture solutions (without antibiotic) is added into above-mentioned pipe on the super-clean bench
It is light to mix, 37 DEG C are then attached on constant-temperature table, and 200rpm shakes 1h, takes 500 μ L clear liquids, uniformly flat to be coated in SOC culture medium
On, it is placed in 37 DEG C of insulating boxs and is incubated overnight.
(3) plasmid extracts.
The positive colony bacterium that previous step is obtained, massive amplification culture.Utilize the small extraction reagent kit TIAN of the plasmid of Tiangeng
Prep Mini Plasmid Kit extracts plasmid (TIANGEN, DP103-03) specific experiment operating procedure and says by kit operation
Bright progress.
(4) plasmid of extraction is subjected to digestion identification with Bsp1407 restriction endonuclease, as shown in figure 8, simultaneously, plasmid is served
Hai Yingjun sequencing company carries out sequence verification using G418 sequencing primer.
The result shows that: there are circular plasmid vectors in the pig fibroblast through neomycin screening, as shown in figure 8, with target
Digestion qualification result shows plasmid size and is expected consistent for the targeting vector of CYP20A1 gene.Sequencing result shows matter
Grain is px330-EGFP-G418-S/MAR-sgRNA (targeting CYP20A1 gene) carrier.These are the result shows that long through neomycin
Cyclic annular px330-EGFP-G418-SMAR-sgRNA (targeting CYP20A1 base is still remained in the pig fibroblast of time screening
Cause) plasmid vector, illustrate the presence that S/MAR can make plasmid vector in dyeing stabilization in vitro.
The targeting vector that other two is directed to target gene SIRT3, ANXA7 respectively dissociates verification result with above-mentioned through carrier
CYP20A1 targeting vector result is similar, is also demonstrated that the presence that S/MAR can make plasmid vector in dyeing stabilization in vitro.
Nucleotides sequence list
<110>Northeast Agricultural University
<120>a kind of additive type CRISPR/Cas9 expression vector and its construction method and application
<130>
<160> 39
<170> PatentIn version 3.5
<210> 1
<211> 1995
<212> DNA
<213>S/MAR sequence
<400> 1
agatctaaat aaacttataa attgtgagag aaattaatga atgtctaagt taatgcagaa 60
acggagagac atactatatt catgaactaa aagacttaat attgtgaagg tatactttct 120
tttcacataa atttgtagtc aatatgttca ccccaaaaaa gctgtttgtt aacttgtcaa 180
cctcatttca aaatgtatat agaaagccca aagacaataa caaaaatatt cttgtagaac 240
aaaatgggaa agaatgttcc actaaatatc aagatttaga gcaaagcatg agatgtgtgg 300
ggatagacag tgaggctgat aaaaagagta gagctcagaa acagacccat tgatatatgt 360
aagtgaccta tgaaaaaaat atggcatttt acaatgggaa aatgatgatc tttttctttt 420
ttagaaaaac agggaaatat atttatatgt aaaaaataaa agggaaccca tatgtcatac 480
catacacaca aaaaaattcc agtgaattat aagtctaaat ggagaaggca aaactttaaa 540
tcttttagaa aataatatag aagcatgcca tcatgacttc agtgtagaga aaaatttctt 600
atgactcaaa gtcctaacca caaagaaaag attgttaatt agattgcatg aatattaaga 660
cttattttta aaattaaaaa accattaaga aaagtcaggc catagaatga cagaaaatat 720
ttgcaacacc ccagtaaaga gaattgtaat atgcagatta taaaaagaag tcttacaaat 780
cagtaaaaaa taaaactaga caaaaatttg aacagatgaa agagaaactc taaataatca 840
ttacacatga gaaactcaat ctcagaaatc agagaactat cattgcatat acactaaatt 900
agagaaatat taaaaggcta agtaacatct gtggcaatat tgatggtata taaccttgat 960
atgatgtgat gagaacagta ctttacccca tgggcttcct ccccaaaccc ttaccccagt 1020
ataaatcatg acaaatatac tttaaaaacc attaccctat atctaaccag tactcctcaa 1080
aactgtcaag gtcatcaaaa ataagaaaag tctgaggaac tgtcaaaact aagaggaacc 1140
caaggagaca tgagaattat atgtaatgtg gcattctgaa tgagatccca gaacagaaaa 1200
agaacagtag ctaaaaaact aatgaaatat aaataaagtt tgaactttag ttttttttaa 1260
aaaagagtag cattaacacg gcaaagtcat tttcatattt ttcttgaaca ttaagtacaa 1320
gtctataatt aaaaattttt taaatgtagt ctggaacatt gccagaaaca gaagtacagc 1380
agctatctgt gctgtcgcct aactatccat agctgattgg tctaaaatga gatacatcaa 1440
cgctcctcca tgttttttgt tttcttttta aatgaaaaac tttatttttt aagaggagtt 1500
tcaggttcat agcaaaattg agaggaaggt acattcaagc tgaggaagtt ttcctctatt 1560
cctagtttac tgagagattg catcatgaat gggtgttaaa ttttgtcaaa tgctttttct 1620
gtgtctatca atatgaccat gtgattttct tctttaacct gttgatggga caaattacgt 1680
taattgattt tcaaacgttg aaccaccctt acatatctgg aataaattct acttggttgt 1740
ggtgtatatt ttttgataca ttcttggatt ctttttgcta atattttgtt gaaaatgttt 1800
gtatctttgt tcatgagaga tattggtctg ttgttttctt ttcttgtaat gtcattttct 1860
agttccggta ttaaggtaat gctggcctag ttgaatgatt taggaagtat tccctctgct 1920
tctgtgttct gaaagagatt gtagaaagtt gatacaattt ttttttcttt aaatatcttg 1980
atagaattct gtaca 1995
<210> 2
<211> 23
<212> DNA
<213> sgRNA-CYP20A1
<400> 2
cttccactgt tcacaatatc tgg 23
<210> 3
<211> 23
<212> DNA
<213> sgRNA-SIRT3
<400> 3
aagcccgaca tcgtgttctt tgg 23
<210> 4
<211> 23
<212> DNA
<213> sgRNA-ANXA7
<400> 4
ggtcagtatc cttatcctag tgg 23
<210> 5
<211> 1771
<212> DNA
<213>G418 resistant gene
<400> 5
tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat 60
gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagtc ctgaggcgga 120
aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca 180
ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca 240
ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc 300
ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc 360
catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta 420
ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaga tcgatcaaga 480
gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc 540
cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga 600
tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct 660
gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac 720
gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct 780
attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt 840
atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt 900
cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt 960
cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag 1020
gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt 1080
gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg 1140
tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg 1200
cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg 1260
catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg 1320
accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat 1380
gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg 1440
gatctcatgc tggagttctt cgcccaccct agggggaggc taactgaaac acggaaggag 1500
acaataccgg aaggaacccg cgctatgacg gcaataaaaa gacagaataa aacgcacggt 1560
gttgggtcgt ttgttcataa acgcggggtt cggtcccagg gctggcactc tgtcgatacc 1620
ccaccgagac cccattgggg ccaatacgcc cgcgtttctt ccttttcccc accccacccc 1680
ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg gggcggcagg ccctgccata 1740
gcctcaggtt actcatatat actttagatt g 1771
<210> 6
<211> 33
<212> DNA
<213> Cas9-Kana-F
<400> 6
ggcgtctccg ggtgtgcgcg gaacccctat ttg 33
<210> 7
<211> 38
<212> DNA
<213> Cas9-Kana-R
<400> 7
ggcgtctcgc gcgcaatcta aagtatatat gagtaacc 38
<210> 8
<211> 25
<212> DNA
<213>G418 sequencing primer Cas9-G418-F
<400> 8
acccgctgac gcgccctgac gggct 25
<210> 9
<211> 33
<212> DNA
<213> Cas9-SMAR-F
<400> 9
gctgtacaag atctaaataa acttataaat tgt 33
<210> 10
<211> 33
<212> DNA
<213> Cas9-SMAR-R
<400> 10
gctgtacaga attctatcaa gatatttaaa gaa 33
<210> 11
<211> 28
<212> DNA
<213>SMAR sequencing primer-Cas9-SMAR-R
<400> 11
ttattaggaa aggacagtgg gagtggca 28
<210> 12
<211> 24
<212> DNA
<213> CYP20A1-gRNA-1-F1
<400> 12
cacccttcca ctgttcacaa tatc 24
<210> 13
<211> 24
<212> DNA
<213> CYP20A1-gRNA-1-R1
<400> 13
aaacgatatt gtgaacagtg gaag 24
<210> 14
<211> 24
<212> DNA
<213> SIRT3-gRNA-2-F1
<400> 14
caccaagccc gacatcgtgt tctt 24
<210> 15
<211> 24
<212> DNA
<213> SIRT3-gRNA-2-R1
<400> 15
aaacaagaac acgatgtcgg gctt 24
<210> 16
<211> 24
<212> DNA
<213> ANXA7-gRNA-3-F1
<400> 16
caccggtcag tatccttatc ctag 24
<210> 17
<211> 24
<212> DNA
<213> ANXA7-gRNA-3-R1
<400> 17
aaacctagga taaggatact gacc 24
<210> 18
<211> 25
<212> DNA
<213>SgRNA sequencing primer Cas9-SgRNA-F
<400> 18
ctgttagaga gataattgga attaa 25
<210> 19
<211> 24
<212> DNA
<213> ANAX7-S
<400> 19
ttagaaggat tatgtttctc aatg 24
<210> 20
<211> 22
<212> DNA
<213> ANAX7-A
<400> 20
gaagtggaaa gtagcctaag tg 22
<210> 21
<211> 18
<212> DNA
<213> CYP20A1-S
<400> 21
acagctagtt cagaatca 18
<210> 22
<211> 17
<212> DNA
<213> CYP20A1-A
<400> 22
tgtatggtct tcctttt 17
<210> 23
<211> 21
<212> DNA
<213> SIRT3-S
<400> 23
gagttttccc ttttccccta a 21
<210> 24
<211> 15
<212> DNA
<213> SIRT3-A
<400> 24
agccccgcct gcttt 15
<210> 25
<211> 23
<212> DNA
<213>original series CYP20A1
<400> 25
cttccactgt tcacaatatc tgg 23
<210> 26
<211> 22
<212> DNA
<213>CYP20A1- mutant nucleotide sequence 1
<400> 26
cttccactgt tcacaattct gg 22
<210> 27
<211> 21
<212> DNA
<213>CYP20A1 mutant nucleotide sequence 2
<400> 27
cttccactgt tcacaatctg g 21
<210> 28
<211> 17
<212> DNA
<213>CYP20A1- mutant nucleotide sequence 3
<400> 28
cttccactgt tcactgg 17
<210> 29
<211> 19
<212> DNA
<213>CYP20A1- mutant nucleotide sequence 4
<400> 29
cttccactgt tcacaatgg 19
<210> 30
<211> 23
<212> DNA
<213>original series SIRT3
<400> 30
aagcccgaca tcgtgttctt tgg 23
<210> 31
<211> 22
<212> DNA
<213>SIRT3- mutant nucleotide sequence 1
<400> 31
aagcccgaca tcgtttcttt gg 22
<210> 32
<211> 21
<212> DNA
<213>SIRT3- mutant nucleotide sequence 2
<400> 32
aagcccgaca tcgttctttg g 21
<210> 33
<211> 19
<212> DNA
<213>SIRT3- mutant nucleotide sequence 3
<400> 33
aagcccgaca gttctttgg 19
<210> 34
<211> 20
<212> DNA
<213>SIRT3- mutant nucleotide sequence 4
<400> 34
aagcccgaca tgttctttgg 20
<210> 35
<211> 23
<212> DNA
<213>original series ANXA7
<400> 35
ggtcagtatc cttatcctag tgg 23
<210> 36
<211> 22
<212> DNA
<213>ANXA7- mutant nucleotide sequence 1
<400> 36
ggtcagtatc cttacctagt gg 22
<210> 37
<211> 20
<212> DNA
<213>ANXA7- mutant nucleotide sequence 2
<400> 37
ggtcagtatc ctcctagtgg 20
<210> 38
<211> 18
<212> DNA
<213>ANXA7- mutant nucleotide sequence 3
<400> 38
ggtcagtatt cctagtgg 18
<210> 39
<211> 20
<212> DNA
<213>ANXA7- mutant nucleotide sequence 4
<400> 39
ggtcagtatt atcctagtgg 20
Claims (10)
1. a kind of additive type CRISPR/Cas9 expression vector, which is characterized in that refer to the CRISPR/Cas9 containing S/MAR sequence
Expression vector, the S/MAR sequence, nucleotide is as shown in SEQ ID NO:1；The S/MAR sequence is located at energy in the carrier
Between the coding region sequence and bGH poly (A) sequence for encoding albumen.
2. additive type CRISPR/Cas9 expression vector according to claim 1, which is characterized in that described to encode albumen
Coding region sequence be EGFP coding region sequence.
3. additive type CRISPR/Cas9 expression vector according to claim 1, which is characterized in that the CRISPR/Cas9
The sgRNA sequence of expression vector is one of SEQ ID NO:2-4.
4. additive type CRISPR/Cas9 expression vector according to claim 1, which is characterized in that the carrier also contains
G418 resistant gene, the nucleotide sequence of the G418 resistant gene is as shown in SEQ ID NO:5.
5. the construction method of additive type CRISPR/Cas9 expression vector described in claim 1, which is characterized in that pass through digestion
S/MAR sequence construct can be encoded the code area of albumen by the method for connection or homologous recombination into CRISPR/Cas9 expression vector
Between sequence and bGH poly (A) sequence.
6. construction method according to claim 5, which is characterized in that specific step is as follows:
1) G418 resistant gene is building up in px330-EGFP carrier, obtains carrier framework px330-EGFP-G418；It is described
The nucleotide sequence of G418 resistant gene is as shown in SEQ ID NO:5；
2) on the carrier framework px330-EGFP-G418 for obtaining S/MAR sequence construct to step 1), carrier framework is obtained
px330-EGFP-G418-S/MAR；
3) by sgRNA sequence be inserted into carrier px330-EGFP-G418-S/MAR expression skeleton U6 promoter after BbsI digestion position
In point, additive type CRISPR/Cas9 expression vector px330-EGFP-G418-S/MAR-sgRNA is obtained, the sgRNA sequence is such as
One of SEQ ID NO:2-4.
7. construction method according to claim 6, which is characterized in that G418 resistant gene described in step 1) and
Px330-EGFP carrier uses BsmBI digestion, recycling px330-EGFP carrier framework digestion large fragment and G418 resistant gene respectively
Digestion products, through T4DNA ligase obtains carrier framework px330-EGFP-G418.
8. construction method according to claim 6, which is characterized in that S/MAR sequence and px330- described in step 2)
EGFP-G418 carrier uses Bsp1407 digestion, recycling px330-EGFP-G418 carrier framework digestion large fragment and S/MAR sequence respectively
Column digestion products, through T4DNA enzymatic connection, obtains carrier framework px330-EGFP-G418-S/MAR.
9. construction method according to claim 6, which is characterized in that sgRNA sequence described in step 3) and carrier framework
For px330-EGFP-G418-S/MAR respectively after BbsI digestion, recycling carrier framework px330-EGFP-G418-S/MAR digestion is big
Segment and sgRNA sequence digestion products, through T4DNA enzymatic connection, obtains additive type CRISPR/Cas9 expression vector px330-
EGFP-G418-S/MAR-sgRNA。
10. the answering in gene knockout research of additive type CRISPR/Cas9 expression vector described in claim 1-4 any one
With.
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