CN106566838B - A kind of miR-126 full-length gene knockout kit and its application based on CRISPR-Cas9 technology - Google Patents
A kind of miR-126 full-length gene knockout kit and its application based on CRISPR-Cas9 technology Download PDFInfo
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Abstract
The invention discloses a kind of, and the miR-126 full-length gene based on CRISPR-Cas9 technology knocks out kit and its application.The preferred miR-126 gene upstream and downstream CRISPR-Cas9 target sequence of the present invention, design synthesis is single-stranded for the sgRNA of the target sequence, and it is building up in carrier, by transfecting 293T cell strain, sgRNA and trRNA constitutes specific recognition structure, to guide Cas9 enzyme specific cleavage miR-126 gene both ends corresponding sequence, continues medicine and sieve to obtain miR-126 full-length gene knockout cell strain；Through to medicine sieve cell strain sequence verification, obtain preferred upstream and downstream sgRNA combination, the knockout efficiency of the combination is up to 90% or more, kit is constructed with this, the miR-126 full-length gene that specificity can be carried out to the multiple types cell line such as 293T, lung cancer cell line A549 and vascular endothelial cell HUVEC system knocks out.
Description
Technical field
The invention belongs to genetic engineering fields, are related to a kind of miR-126 gene knockout kit, in particular to one kind is based on
CRISPR-Cas9 technology can carry out the kit that specificity knocks out completely to miR-126 full-length gene order.In addition, this hair
It is bright to also disclose the application of the kit.
Background technique
MicroRNA (microRNA) is that a kind of non-coding for being about 22 nucleotide by the length of endogenous gene is single-stranded
RNA molecule is widely distributed in virus, plant into higher mammal, has a variety of important adjustment effects in the cell.
Each microRNA can have multiple target genes, and several microRNA also adjustable same gene.The tune of this complexity
Section network can both regulate and control the expression of multiple genes by a microRNA, can also pass through the combination of several microRNA
Carry out the expression of some gene of finely regulating, therefore the gene function of microRNA is explored and application and development is always research hotspot.
MiR-126 (microRNA126, microRNA 126) is located at the skins growth factor domain 7 of human chromosomal 9q34.3
In the introne of (epidermal growth factor like 7, EFGL7) gene.MiR-126 is that a kind of blood vessel endothelium is thin
The specific expressed microRNA of born of the same parents is highly expressed in vascularization tissue abundant, in heart, lungs, kidney.It can
By regulation Spred-1, (Sprouty-related, EVH1 domain-containing protein 1, Sprouty are related
Region protein containing EVH1 1), VCAM-1 (Vascular cell adhesion protein 1, vascular cell adhesion molecule-1),
HoxA9 (homeobox A9, hox genes A9), v-Crk (Sarcoma Virus CT10regulator of kinase,
Sarcoma virus CT10 adjusts kinases), EGFL-7 and VEGF (vascular endothelial growth factor, it is intravascular
Skin growth factor) isogenic expression participates in adjusting vascular development, new vessels are formed and the blood vessels such as vascular inflammation reaction
Physiology and pathophysiological process.Through other the study found that miR-126 is in gastrointestinal tract, genital cancers, mammary gland, thyroid gland, lung,
And it expresses and is suppressed in some other cancer.It can induce cancer cell multiplication, migration, invasion and influence after lowering miR-126
The cell survival phase, miR-126 lower with kinds cancer existence it is bad between there are correlations.These researchs are found to be gastrointestinal tract,
Genital cancers, the prevention judgement of the cancer metastasis such as mammary gland, thyroid gland, lung provide potential target, and are gastrointestinal tract, genital tract
Cancer, the biological therapy of the cancers such as mammary gland, thyroid gland, lung indicate new direction, prompt the importance and necessity of miR-126 research
Property.
Since microRNA sequence is shorter and its precursor has loop-stem structure, the method for classical RNA interference (RNAi) is not
The functional study of expression is lowered suitable for microRNA.MicroRNA studies common method at present, is applied biology technology
In vivo in cell model or external dynamic/plant model, microRNA is subjected to sequence closing or causes deletion mutant,
Negative regulation effect reduction caused by simulation microRNA afunction is removed, and then studies its function mechanism, technology
There are two main classes for method:
First kind method is the complementary closure sequence of design synthesis microRNA, and complementary series was originally specific with microRNA
In conjunction with mRNA formed competition, weaken microRNA to specific mRNA binding ability and regulating and controlling effect.Due to complementary series be with
The form of base complementrity closes microRNA, does not directly result in the downward of microRNA degradation or expression, so
The expression of microRNA does not change, and can closing efficiency to microRNA function without direct technical method
Quantization detection is carried out, cannot directly prove that the function of microRNA receives shielding, it can only be possible by detection microRNA
The function that regulation downstream gene variation carrys out side light microRNA is suppressed, thus the method is to microRNA functional study
Authenticity and validity there are large effects.
Second class method is with transcriptional activation sample effector nuclease (transcription activator-like
Effector nuclease, TALEN) TALEN technology, by DNA identification module by the position DNA of TALEN element targeting specific
It puts and combines, the shearing of specific site is then completed under the action of FokI nuclease, DNA level can be carried out to microRNA
Shearing and modification, simulate the afunction of microRNA.But TALEN technology relies on what DNA was combined to the knockout of target gene
Amino acid polymer sequence of modules, single TALEN module carry out assembling and need a large amount of molecular cloning and sequencing procedures, very
It is cumbersome, if all working, which completes basic experiment operation in laboratory internal, needs time-consuming 2 months or more, if screening, which is stablized, knocks out base
Cell line need 4 months or more of cause, time-consuming for whole process, at high cost.
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats/cas,
Cluster, regular intervals short palindrome repetitive sequence) technology is also known as CRISPR/Cas9 (CRISPR associated
Protein 9, CRISPR associated protein 9) nucleic acid zymotechnic is a kind of most emerging genome edit tool, it can be completed
The DNA identification of RNA guiding and editor.Compared with the gene technology tool of above-mentioned introduction, CRISPR technology is physically easier to perform, and is had
Stronger scalability.
CRISPR/Cas9 is found in the natural immune system of bacterium earliest, and major function is the disease of confrontation invasion
Poison and exogenous DNA, are the acquired immunity defense mechanisms of bacterium and archeobacteria.In this system, crRNA (CRISPR-
The derivative RNA of derived RNA, CRISPR) by base pairing and tracrRNA (trans-activating RNA, it is trans- to swash
Work RNA) in conjunction with double-stranded RNA is formed, this tracrRNA/crRNA binary complex instructs Cas9 nuclease protein to guide in crRNA
Sequence targets site shearing double-stranded DNA, to achieve the purpose that shear genomic DNA or modified.
In order to realize that, in mammals using CRISPR/Cas9 system, scientist is transformed CRISPR/Cas9,
TracrRNA/crRNA binary complex amalgamation and expression is formed into a single-stranded guiding RNA.And in actual design, only design
Synthesis identifies the RNA sequence of one section of 20nt (base) left and right of target sequence and referred to as sgRNA (small guide RNA, small guide
RNA), other parts guiding RNA exists because sequence is constant as the carrier sequence on sgRNA expression vector.Therefore, extensively
The CRISPR/Cas9 system of application becomes determining target gene by the Cas9 nuclease under the guiding RNA guidance containing sgRNA
To the technology of editor.
The most commonly used is for single locus design sgRNA guidance for the fixed point shearing editing technique of CRISPR/Cas9 technology
Cas9 is oriented shearing in target sequence, and (for example, disclosed in Chinese invention patent application CN105112445A, " one kind is based on
The miR-205 gene knockout kit of CRISPR-Cas9 gene Knockout "), intracellular intrinsic nonhomologous end after cutting
Connection approach (NHEJ) repair process, can introduce the gene mutation of radom insertion and deletion, this repair process is not artificially controlled
System causes all kinds of unnecessary and inaccurate sequence to be inserted into and delete, often generates indefinite or invalid cell phenotype, right
Research has an adverse effect.
Currently, there is not yet in relation to using CRISPR/Cas9 systemic characteristic the full-length gene order of miR-126 is carried out
The report of knockout.
Summary of the invention
One of the technical problem to be solved in the present invention is to provide one kind based on CRISPR/Cas9 gene editing technology to miR-
126 genes carry out the kit of gene knockout, allow CRISPR/Cas9 to target miR-126 gene coded sequence two
End while the shearing pinpointed can be avoided greatly using traditional for single locus design sgRNA guidance Cas9 in target
It is oriented non-homologous end joining approach NHEJ caused by shearing in mark sequence and introduces a possibility that random mutation occurs, it is non-
Often be conducive to generate and be directed to the single and apparent cell phenotype that the gene is deleted completely, which can efficient, quick, essence
Really, steadily realize that miR-126 complete encoding sequence completely knocks out at the genomic level.
The second technical problem to be solved by the present invention is to provide the application of the kit, which can be applied to a variety of thin
The miR-126 gene knockout of born of the same parents' type (for example, vascular endothelial cell and Several Kinds of Malignancy are such as: breast cancer, lung cancer),
These cell lines can further by nude mouse transplantable tumor construct animal model, be the research of miR-126 related pathways
And the in vitro and in vivo experiments building model of drug development etc. provides good tool.
The present invention passes through to the preferably a certain number of miR-126 upstream region of gene of target software design and downstream CRISPR-
Cas9 target sequence, the sgRNA for separately designing synthesis for upstream and downstream target sequence is single-stranded, and is building up to plasmid vector
In, by transfecting 293T cell strain, sgRNA and trRNA (trans-activating RNA) constitute special identification structure, from
And Cas9 enzyme is guided specifically to shear the corresponding sequence at miR-126 gene both ends, continue medicine sieve, obtains miR-126 full-length gene
The cell strain of knockout.By extracting cell DNA, PCR amplification, and the goldstandard skill detected with DNA level gene knockout
Art --- generation sequencing technologies are determined and verify, obtain two groups of miR-126 gene knockout CRISPR-Cas9 it is effective on
Downstream sgRNA combination, fluorescent quantitative PCR technique show that the combination realizes that miR-126 molecule knocks out efficiency and is up to 90% or more, with
Kit is applied in this building, can be to 293T, lung cancer cell line A549, breast cancer cell line T47D and vascular endothelial cell
A plurality of types of cell lines such as HUVEC system stablize accurately specific overall length miR-126 gene order knockout.
In order to solve the above technical problems, the present invention adopts the following technical scheme:
In one aspect of the invention, a kind of miR-126 full-length gene knockout reagent based on CRISPR-Cas9 technology is provided
Box,
The kit includes by plasmid pSpCas9 (BB) -2A-Puro-sgRNA1 and plasmid pSpCas9 (BB) -2A-
The sgRNA plasmid combinations of Puro-sgRNA3 composition；Plasmid pSpCas9 (the BB) -2A-Puro-sgRNA1, mutually containing one section
The DNA sequence dna of benefit, the DNA sequence dna can be transcribed into specific recognition sequence miR-126 full-length gene as shown in SEQ ID NO.2
Target sequence sgRNA, which can constitute special identification structure with trRNA, so that guidance Cas9 enzyme is specifically sheared
MiR-126 gene corresponding sequence；Plasmid pSpCas9 (the BB) -2A-Puro-sgRNA3, containing one section of complementary DNA sequence dna,
The DNA sequence dna can be transcribed into the target sequence of specific recognition sequence miR-126 full-length gene as shown in SEQ ID NO.4
SgRNA, the sgRNA can constitute special identification structure with trRNA, so that guidance Cas9 enzyme specifically shears miR-126 gene
Corresponding sequence；
Alternatively, the kit includes by plasmid pSpCas9 (BB) -2A-Puro-sgRNA2 and plasmid pSpCas9 (BB) -
The sgRNA plasmid combinations of 2A-Puro-sgRNA5 composition；Plasmid pSpCas9 (the BB) -2A-Puro-sgRNA2 contains one section
Complementary DNA sequence dna, the DNA sequence dna can be transcribed into specific recognition sequence miR-126 overall length base as shown in SEQ ID NO.3
The sgRNA of the target sequence of cause, the sgRNA can constitute special identification structure with trRNA, so that guidance Cas9 enzyme is specifically cut
Cut miR-126 gene corresponding sequence；Plasmid pSpCas9 (the BB) -2A-Puro-sgRNA5, contains one section of complementary DNA sequence
Column, the DNA sequence dna can be transcribed into the target sequence of specific recognition sequence miR-126 full-length gene as shown in SEQ ID NO.6
The sgRNA of column, the sgRNA can constitute special identification structure with trRNA, so that guidance Cas9 enzyme specifically shears miR-126
Gene corresponding sequence.
As currently preferred technical solution, complementation that plasmid pSpCas9 (the BB) -2A-Puro-sgRNA1 contains
DNA sequence dna is SEQ ID NO.7 and SEQ ID NO.8.
As currently preferred technical solution, complementation that plasmid pSpCas9 (the BB) -2A-Puro-sgRNA3 contains
DNA sequence dna is SEQ ID NO.11 and SEQ ID NO.12.
As currently preferred technical solution, complementation that plasmid pSpCas9 (the BB) -2A-Puro-sgRNA2 contains
DNA sequence dna is SEQ ID NO.9 and SEQ ID NO.10.
As currently preferred technical solution, complementation that plasmid pSpCas9 (the BB) -2A-Puro-sgRNA5 contains
DNA sequence dna is SEQ ID NO.15 and SEQ ID NO.16.
As currently preferred technical solution, plasmid pSpCas9 (the BB) -2A-Puro-sgRNA1 and plasmid
The sgRNA plasmid combinations of pSpCas9 (BB) -2A-Puro-sgRNA3 composition, plasmid pSpCas9 (the BB) -2A-Puro-
The sgRNA plasmid combinations of sgRNA2 and plasmid pSpCas9 (BB) -2A-Puro-sgRNA5 composition are with the side included the following steps
Method building:
Step 1, by the preferably a certain number of miR-126 upstream region of gene of target software design and downstream CRISPR-
Cas9 target sequence, specific as follows:
Upstream:
SgRNA-1:TAATGTCCCGTCGCCAGCGG, as shown in SEQ ID NO.2；
SgRNA-2:GCCACGCCTCCGCTGGCGAC, as shown in SEQ ID NO.3；
Downstream:
SgRNA-3:TCTCAGCGGCGTTTTCGATG, as shown in SEQ ID NO.4；
SgRNA-4:GAGTAATAATGCGCCGTCCA, as shown in SEQ ID NO.5；
SgRNA-5:TTTCGATGCGGTGCCGTGGA, as shown in SEQ ID NO.6；
Step 2 separately designs the sgRNA segment that synthesis is directed to upstream and downstream target sequence, and is building up to plasmid vector
In；The sgRNA fragment sequence is as follows:
SgRNA-1F:ccacgTAATGTCCCGTCGCCAGCGG, as shown in SEQ ID NO.7；
SgRNA-1R:aaacCCGCTGGCGACGGGACATTAc, as shown in SEQ ID NO.8；
SgRNA-2F:ccacGCCACGCCTCCGCTGGCGAC, as shown in SEQ ID NO.9；
SgRNA-2R:aaac GTCGCCAGCGGAGGCGTGGC, as shown in SEQ ID NO.10；
SgRNA-3F ccacgTCTCAGCGGCGTTTTCGATG, as shown in SEQ ID NO.11；
SgRNA-3R aaacCATCGAAAACGCCGCTGAGAC, as shown in SEQ ID NO.12；
SgRNA-4F ccacGAGTAATAATGCGCCGTCCA, as shown in SEQ ID NO.13；
SgRNA-4R aaacTGGACGGCGCATTATTACTC, as shown in SEQ ID NO.14；
SgRNA-5F ccacgTTTCGATGCGGTGCCGTGGA, as shown in SEQ ID NO.15；
SgRNA-5R aaacTCCACGGCACCGCATCGAAAc, as shown in SEQ ID NO.16；
Step 3, by transfecting 293T cell strain, sgRNA and trRNA constitute special identification structure, to guide Cas9
Enzyme specifically shears the corresponding sequence at miR-126 gene both ends, continues medicine sieve, obtains the cell of miR-126 full-length gene knockout
Strain；
Step 4, by extracting cell DNA, PCR amplification, sequencing is determined and verifies, two optimized group miR-
The sgRNA plasmid combinations of 126 gene knockout CRISPR-Cas9, i.e., described plasmid pSpCas9 (the BB) -2A-Puro-sgRNA1 and
The sgRNA plasmid combinations and the plasmid pSpCas9 (BB) -2A- of plasmid pSpCas9 (BB) -2A-Puro-sgRNA3 composition
The sgRNA plasmid combinations of Puro-sgRNA2 and plasmid pSpCas9 (BB) -2A-Puro-sgRNA5 composition.
It is described to be building up in plasmid vector in step 2 as currently preferred technical solution, specifically include following step
It is rapid:
(1) using pSpCas9 (BB) -2A-Puro plasmid as initial vector, single endonuclease digestion is carried out with Bbs I, to linearized vector
It is purified；
(2) synthetic sgRNA segment is diluted and is annealed；
(3) utilize T4DNA ligase by pSpCas9 (BB) -2A-Puro of linearisation with the sgRNA segment phase after annealing
Even, the conversion of bacillus coli DH 5 alpha competent cell is added in whole connection products；
(4) it is carried out using the single colonie that the plate that bacterium colony PCR method is coated with DH5 α competent cell bacterium solution after conversion is grown
Identification obtains expression plasmid.
In another aspect of this invention, mentioned reagent box is provided in preparation cell line miR-126 gene knockout product
Using.The cell line includes vehicles cells system 293T, and tumor cell line is (for example, lung cancer cell line A549, breast cancer cell line
) and endothelial cell HUVEC system T47D.
Compared with prior art, the invention has the following beneficial effects: the present invention is based on the miR- of CRISPR-Cas9 technology
126 full-length genes knock out kit, mainly knock out miR-126 target gene using CRISPR/Cas9 system.This is to utilize for the first time
CRISPR/Cas9 systemic characteristic knocks out miR-126 full-length gene order.The present invention has the advantage that
1. the drawbacks of overcoming tradition closing sequence method research microRNA function, miR-126, which knocks out result, can quantify to supervise
It surveys, miR-126 molecule knocks out efficiency and is up to 90% or more；
2. the cumbersome defect of the time-consuming for overcoming TALEN technology, accelerates cell screening and tests and be reduced to 1 week, stablize
Cell line experiments are constructed to 2 months；Construction step and experimental implementation are simple, while reducing reagent cost consumption；
3. done technology extending design in CRISPR-Cas9 technology, the upstream and downstream both ends of miR-126 gene order all
SgRNA is designed, fixed point shearing while two sites is carried out, is realized on DNA level complete to entire miR-126 gene order
Whole knockout (see Figure 15), generation sequencing result are shown, experiment effect can be repeated in various kinds of cell, and stability is good.Pole of the present invention
The earth avoids being led using traditional for being oriented cutting method in single locus design sgRNA guidance Cas9 target sequence
It causes non-homologous end joining approach NHEJ to introduce a possibility that random mutation occurs, can accurately realize that microRNA overall length is compiled
Code sequence completely knocks out at the genomic level.
Detailed description of the invention
Fig. 1 is that PCR method verifies pSpCas9 (BB) -2A-Puro-sgRNA vector construction schematic diagram in the embodiment of the present invention 1；
Wherein, channel 1-5 is pSpCas9 (BB) -2A-Puro-sgRNA vector construction positive colony PCR result；Channel M is DNA molecular
Amount standard.
Fig. 2 is pSpCas9 (BB) -2A-Puro-sgRNA1 and pSpCas9 (BB) -2A-Puro- in the embodiment of the present invention 2
SgRNA3 pairing sgRNA combination knocks out result schematic diagram in 293T；Wherein, channel 3,4,5 be pSpCas9 (BB) -2A-
Puro-sgRNA1 and pSpCas9 (BB) -2A-Puro-sgRNA3 combination knocks out PCR positive findings；Channel 1 and 2 is pSpCas9
(BB) -2A-Puro-sgRNA1 and pSpCas9 (BB) -2A-Puro-sgRNA5 combination knocks out PCR negative findings, and channel 6 is pair
According to 293T cell PCR result；Channel M is DNA molecular amount standard.
Fig. 3 is pSpCas9 (BB) -2A-Puro-sgRNA1 and pSpCas9 (BB) -2A-Puro- in the embodiment of the present invention 2
SgRNA3 matches sgRNA combination and knocks out verifying sequencing result schematic diagram in 293T.
Fig. 4 is pSpCas9 (BB) -2A-Puro-sgRNA2 and pSpCas9 (BB) -2A-Puro- in the embodiment of the present invention 2
SgRNA5 pairing sgRNA combination knocks out result schematic diagram in 293T；Wherein, channel 1 is pSpCas9 (BB) -2A-Puro-
SgRNA2 and pSpCas9 (BB) -2A-Puro-sgRNA5 combination knocks out the positive findings of PCR, and channel 2,3,4,5,6,7,8 is
PSpCas9 (BB) -2A-Puro-sgRNA2 and pSpCas9 (BB) -2A-Puro-sgRNA4 combination knock out PCR negative findings, lead to
Road 9 is control 293T cell PCR result；Channel M is DNA molecular amount standard.
Fig. 5 is pSpCas9 (BB) -2A-Puro-sgRNA2 and pSpCas9 (BB) -2A-Puro- in the embodiment of the present invention 2
SgRNA5 matches sgRNA combination and knocks out verifying sequencing result schematic diagram in 293T.
Fig. 6 is that the monoclonal cell PCR verifying that 293T is obtained through medicine sieve separation after transfecting in the embodiment of the present invention 2 knocks out knot
Fruit schematic diagram；Wherein, channel 1 is the PCR positive findings that 293T knocks out miR-126 monoclonal cell, and channel 2 is that control 293T is thin
Born of the same parents PCR does not knock out result；Channel M is DNA molecular amount standard.
Fig. 7 is the monoclonal cell PCR for the knockout positive that 293T is obtained through medicine sieve separation after transfecting in the embodiment of the present invention 2
Sequencing fragment result schematic diagram.
Fig. 8 is that miR-126 knocks out the stable strain fluorescent quantitation qPCR testing result schematic diagram of 293T in the embodiment of the present invention 2；
Wherein, 293Tblank is that 293T control cell does not knock out the fluorescent quantitation qPCR of miR-126 as a result, 293T cloneD2 is
The fluorescent quantitation qPCR result of 293T knockout miR-126 positive colony D2 cell.
Fig. 9 is that the monoclonal cell PCR verifying that A549 is obtained through medicine sieve separation after transfecting in the embodiment of the present invention 3 knocks out knot
Fruit schematic diagram；Wherein, channel 1,2,3,4,5,6 be that A549 knocks out PCR positive findings after miR-126, and channel 7 is control A549
The PCR result that cell is knocked out without miR-126；Channel M is DNA molecular amount standard.
Figure 10 is the monoclonal cell for the knockout positive that A549 is obtained through medicine sieve separation after transfecting in the embodiment of the present invention 3
PCR fragment sequencing result schematic diagram.
Figure 11 is that the monoclonal cell PCR verifying that HUVEC is obtained through medicine sieve separation after transfecting in the embodiment of the present invention 4 knocks out
Result schematic diagram；Wherein, channel 2 is the positive PCR results that HUVEC knocks out miR-126, and channel 1,3,4 is control HUVEC cell
The PCR result that miR-126 is not knocked out；Channel M is DNA molecular amount standard.
Figure 12 is the Dan Ke for the knockout miR-126 positive that HUVEC is obtained through medicine sieve separation after transfecting in the embodiment of the present invention 4
Grand cell PCR fragment sequencing result schematic diagram.
Figure 13 is that T47D cell PCR verifying after medicine sieves knocks out result schematic diagram after transfecting in the embodiment of the present invention 5；Its
In, channel 1,2 is the positive PCR results that T47D cell sample knocks out miR-126, and channel 3,4 is control T47D cell miR-126
The PCR result not knocked out；Channel M is DNA molecular amount standard.
Figure 14 is that T47D miR-126 after medicine sieves knocks out positive cell PCR fragment survey after transfecting in the embodiment of the present invention 5
Sequence result schematic diagram.
Figure 15 is that miR-126 gene order completely knocks out schematic diagram.
Specific embodiment
The invention is further described combined with specific embodiments below.It should be understood that particular implementation described herein
It indicates by way of example, is not intended as limitation of the present invention.Without departing from the scope of the invention, of the invention
Main feature can be used for various embodiments.
1. vector construction of embodiment
(1) miR-126 drone design
For miR-126 gene (gene name miR-126, gene ID number: 406913, gene details are shown in http: //
Www.ncbi.nlm.nih.gov/gene/406913), downloading obtains miR-126 genome sequence from the website:
5’-CGCTGGCGACGGGACATTATTACTTTTGGTACGCGCTGTGACACTTCAAACTCGTACCGTGAGT
AATAATGCGCCGTCCACGGCA-3 ', as shown in SEQ ID NO.1.
It is set using online software Feng Zhang lab's Target Finder (http://crispr.mit.edu/)
SgRNA is counted, the sequence of miR-126 genome sequence and each 50bp of its upstream and downstream is inputted, be arranged and retrieves several sgRNA sequences of acquisition
Column (are missed the target) information by the off-target of position and the sgRNA of the analysis sgRNA in gene order, are therefrom distinguished
The target sequence of optimal upstream 2 and 3, downstream target sequence are selected, specific as follows:
Upstream:
SgRNA-1:TAATGTCCCGTCGCCAGCGG, as shown in SEQ ID NO.2；
SgRNA-2:GCCACGCCTCCGCTGGCGAC, as shown in SEQ ID NO.3；
Downstream:
SgRNA-3:TCTCAGCGGCGTTTTCGATG, as shown in SEQ ID NO.4；
SgRNA-4:GAGTAATAATGCGCCGTCCA, as shown in SEQ ID NO.5；
SgRNA-5:TTTCGATGCGGTGCCGTGGA, as shown in SEQ ID NO.6；
(2) sgRNA segment is synthesized
According to sgRNA it is quasi- clone into carrier pSpCas9 (BB) -2A-Puro (purchased from Addgene) restriction enzyme site
The information of Bbs I, after above-mentioned sgRNA sequence is obtained complementary series, along with the corresponding viscosity end of Bbs I restriction enzyme
End.Obtain following sequence:
SgRNA-1F:ccacgTAATGTCCCGTCGCCAGCGG, as shown in SEQ ID NO.7；
SgRNA-1R:aaacCCGCTGGCGACGGGACATTAc, as shown in SEQ ID NO.8；
SgRNA-2F:ccacGCCACGCCTCCGCTGGCGAC, as shown in SEQ ID NO.9；
SgRNA-2R:aaac GTCGCCAGCGGAGGCGTGGC, as shown in SEQ ID NO.10；
SgRNA-3F ccacgTCTCAGCGGCGTTTTCGATG, as shown in SEQ ID NO.11；
SgRNA-3R aaacCATCGAAAACGCCGCTGAGAC, as shown in SEQ ID NO.12；
SgRNA-4F ccacGAGTAATAATGCGCCGTCCA, as shown in SEQ ID NO.13；
SgRNA-4R aaacTGGACGGCGCATTATTACTC, as shown in SEQ ID NO.14；
SgRNA-5F ccacgTTTCGATGCGGTGCCGTGGA, as shown in SEQ ID NO.15；
SgRNA-5R aaacTCCACGGCACCGCATCGAAAc, as shown in SEQ ID NO.16.
By above-mentioned sequence, send Thermofisher company synthesizing single-stranded nucleotide chain.
(3) sgRNA segment is connected and is converted with expression vector
1 μ g pSpCas9 (BB) -2A-Puro Bbs I (being purchased from NEB company, article No. R0539S) is subjected to single endonuclease digestion,
37 DEG C after water-bath 1 hour, carry linearisation using PCR product purification kit (being purchased from Tiagen company, article No. DP204)
Body is purified.
The dry powder of the F and R chain of every group of synthetic sgRNA is diluted to 100 μM, 1 μ L solution is respectively taken to be mixed in PCR pipe
It is interior, 22 DEG C gradually are down to from 95 DEG C in PCR instrument with 1.5 DEG C per minute.
PSpCas9 (the BB) -2A- for being linearized 1 μ L using T4DNA ligase (being purchased from Takara company, article No. 6022Q)
Puro is connected with the sgRNA segment after 0.5 μ L annealing, and linked system is shown in Table 1:
Table 1
| sgRNA | 0.5μL |
| PSpCas9 (BB) -2A-Puro of linearisation | 1μL |
| DNAsolution I | 5μL |
| ddH2O | up to 10μL |
After 16 DEG C connect 15 minutes, whole connection products are added to 100 μ L bacillus coli DH 5 alpha competent cell (pfu
≥108) in, after placing 30 minutes on ice, in 42 DEG C water-bath heat shock 90 seconds, then be placed in 5 minutes on ice, be added in super-clean bench
1mL LB liquid medium (Luria-Bertani culture medium), 37 DEG C shaken cultivation 1 hour, take 500 μ L to be coated on containing Amp
On the LB solid medium of (Ampicillin, ampicillin), to liquid absorption after, be inverted in 37 DEG C of overnight incubations.
(4) it is identified after converting
It is reflected using the single colonie that the plate that bacterium colony PCR method is coated with DH5 α competent cell bacterium solution after conversion is grown
It is fixed.Bacterium colony PCR identification primer is as follows:
Primer F:5'GACTATCATATGCTTACCG 3', as shown in SEQ ID NO.17；
Primer R:5'CCAAGTGGGCAGTTTACC 3', as shown in SEQ ID NO.18；
Bacterium solution PCR system is as shown in table 2:
Table 2
| Ingredient | Volume |
| 10×buffer | 2.5μL |
| primer F(10μM) | 0.2μL |
| primer R(10μM) | 0.2μL |
| dNTPs(2.5mM) | 2μL |
| RTaq enzyme (50U/ μ L) | 0.5μL |
| Bacterium colony | It picks a little |
| ddH2O | up to 25μL |
PCR program is 95 DEG C of 10min；95 DEG C of 20sec, 55 DEG C of 30sec, 72 DEG C of 30sec, totally 35 recycle；72℃
10min.Obtained PCR product is gone to detect through 1% agarose gel electrophoresis, the single colonie of 100bp or so amplified fragments occurs
It is the single colonie containing positive colony (as shown in Figure 1, PCR product clip size is big with the 100bp segment of DNA molecular amount standard
It is small close, and 1000bp segment is the most paillette section of the molecular weight standard).By the inoculation 2mL LB liquid training of positive colony single colonie
It supports in base, takes 1mL bacterium solution for conservation after 37 DEG C of overnight shaking cultures, remaining 1mL bacterium solution send sequencing company (Shanghai Mei Ji biology
Pharmaceutical Technology Co., Ltd) carry out sequence verification.By obtained sequencing result through Blast analysis shows, sgRNA segment has been cloned
Into in pSpCas9 (BB) -2A-Puro plasmid, and sequence is consistent, can be used for subsequent experimental.Carrier with sgRNA is ordered respectively
Entitled pSpCas9 (BB) -2A-Puro-sgRNA1, pSpCas9 (BB) -2A-Puro-sgRNA2, pSpCas9 (BB) -2A-
Puro-sgRNA3, pSpCas9 (BB) -2A-Puro-sgRNA4 and pSpCas9 (BB) -2A-Puro-sgRNA5.
MiR-126 knocks out activity identification in 2. plasmid transfection of embodiment and 293T cell and 293T knockout miR-126 is thin
The building of born of the same parents' model
(1) plasmid extraction
Will in embodiment 1 by sequence verification, separately include sgRNA-1, sgRNA-2, sgRNA-3, sgRNA-4,
The bacillus coli DH 5 alpha of pSpCas9 (BB) -2A-Puro plasmid of sgRNA-5 segment is respectively inoculated in 5mL LB liquid medium
In, 37 DEG C of overnight shaking cultures.Using endotoxin-free mini-scale plasmid extracts kit, (kit is Omega company product, article No.
Plasmid extraction is carried out to these bacterium solutions for D6948-02).Plasmid after extracting is again in super-clean bench after dehydrated alcohol precipitating, with nothing
The dissolution of bacterium water carries out the aseptic process to plasmid.
(2) pSpCas9 (the BB) -2A-Puro-sgRNA plasmid of 293T cell transfecting a pair containing sgRNA
1) 293T cell culture
By 293T cell with 3 × 105The density of cells/well is laid in 24 orifice plates, it is ensured that and each hole cell growth state is good,
Density is similar, is single layer to cell and is in mid-log phase, cell confluency degree is transfected up to 80% or so.
2) transfection composite is prepared:
According to the pairing of table 3, pSpCas9 (BB) -2A-Puro-sgRNA plasmid by a pair containing sgRNA passes through
2000 liposome reagent of Lipofectamine transfects 293T cell.
3 upstream and downstream of the table matching table of plasmid containing sgRNA
| Serial number | Upstream sgRNA plasmid | Downstream sgRNA plasmid |
| 1 | pSpCas9(BB)-2A-Puro-sgRNA1 | pSpCas9(BB)-2A-Puro-sgRNA3 |
| 2 | pSpCas9(BB)-2A-Puro-sgRNA1 | pSpCas9(BB)-2A-Puro-sgRNA5 |
| 3 | pSpCas9(BB)-2A-Puro-sgRNA2 | pSpCas9(BB)-2A-Puro-sgRNA4 |
| 4 | pSpCas9(BB)-2A-Puro-sgRNA2 | pSpCas9(BB)-2A-Puro-sgRNA5 |
2 sterile EP tubes are taken, dilute plasmid 0.8 in the Opti-MEM culture medium of the serum-free antibiotic-free of 50 μ L respectively
The Lipofectamine 2000 of μ g and 2 μ L are stored at room temperature 5 minutes, diluted plasmid and Lipofectamine 2000 are mixed
It closes, the transfection composite of 100 μ L is obtained.After mixing gently, it is incubated at room temperature 20 minutes.
3) cell transfecting:
Above-mentioned transfection composite is added in corresponding 24 orifice plate, tissue culture plate is softly rocked back and forth, cell is put
Return 37 DEG C of 5%CO2After incubator continues culture 6 hours, culture medium is changed to complete medium, continues for cell to be placed in 37 DEG C
5%CO2Incubator culture.
4) medicine sieves
After transfection 24 hours, culture medium is changed to the complete medium containing 2 μ g/mL puromycin.Every 24 hours more
Change the complete medium of a drug containing, continuous three days.Green florescent signal is observed under the microscope daily, is recorded cellular
State.
(3) CRISPR/Cas9 knocks out efficiency verification
1) the knockout efficiency in PCR sequencing approach verifying genomic level
293T cell after collecting medicine sieve and the 293T cell without transfection, are tried using submicrosample genotype identification
Agent box (Nanjing Yao Shunyu Biotechnology Co., Ltd, KC-101) carry out sample process, and using PCR primer as shown in table 4 into
The identification of row PCR amplification.
Table 4
| * primer sequence (5'--3') | * Primer |
| GAGGGAGGATAGGTGGGTTC, as shown in SEQ ID NO.19 | mir126-test-Fw |
| AGGCAGAGCCAGAAGACTCA, as shown in SEQ ID NO.20 | mir126-test-Rv |
| GCACTGGAATCTGGGCGGAAGG, as shown in SEQ ID NO.21 | mir126-test-2-Fw |
| AGAGCCAGGCGCTGGGTCAC, as shown in SEQ ID NO.22 | mir126-test-2-Rv |
PCR reaction system is as shown in table 5:
Table 5
94℃5min；94 DEG C of 30sec, 62 DEG C of 30sec, 72 DEG C of 30sec, totally 35 recycle；72℃10min.By PCR amplification
10 times of product dilution, the second wheel PCR is carried out by template of dilution, PCR system is as shown in table 6:
Table 6
94℃5min；94 DEG C of 30sec, 62 DEG C of 30sec, 72 DEG C of 2min, totally 35 recycle；72℃5min.PCR amplification is produced
Object detects (result is shown in Fig. 2, shown in Fig. 4) through 1% agarose gel electrophoresis, chooses with the 293T cell sample band without transfection
Sending to sequencing (Shanghai Major Biological Medical Technology Co., Ltd.) compared to obvious PCR product less than normal, (result is shown in Fig. 3, Fig. 5 institute
Show).
Sequencing result using Blast carry out analysis shows, by pSpCas9 (BB) -2A-Puro-sgRNA1 and pSpCas9
(BB) -2A-Puro-sgRNA3 and by pSpCas9 (BB) -2A-Puro-sgRNA2 and pSpCas9 (BB) -2A-Puro-
The sgRNA plasmid combinations of sgRNA5 composition can knock out miR-126 genome sequence in 293T cell, and pSpCas9 (BB)-completely
2A-Puro-sgRNA1 and pSpCas9 (BB) -2A-Puro-sgRNA3 combination, which is deleted, can obtain the more of positive findings, because
This efficiency is more preferably.It is feminine gender that other sgRNA combinations of pairs, which knock out verification result, cannot effectively knock out miR-126 in 293T cell
Genome sequence.
PSpCas9 (BB) -2A-Puro-sgRNA1 and pSpCas9 (BB) -2A-Puro-sgRNA3 is selected as a result,
PSpCas9 (BB) -2A-Puro-sgRNA2 and pSpCas9 (BB) -2A-Puro-sgRNA5 are that miR-126 full-length gene knocks out examination
The sgRNA of agent box is combined；It is preferred that pSpCas9 (BB) -2A-Puro-sgRNA1 and pSpCas9 (BB) -2A-Puro-sgRNA3 group
It closes.
(4) building miR-126 knocks out 293T cell model and qPCR test knocks out efficiency
It is miR- by selected pSpCas9 (BB) -2A-Puro-sgRNA1 and pSpCas9 (BB) -2A-Puro-sgRNA3
126 full-length genes knock out the sgRNA combination of kit, according to (1) the step of embodiment 2 --- and (3) obtain knocking out containing miR-126
293T cell line, will have verified that containing miR-126 knock out 293T cell count after, be diluted to 2/hole quantity be inoculated with 24 holes
Plate is placed in 37 DEG C, 5%CO2Incubator culture two weeks, the growth conditions of monoclonal in orifice plate were during which observed daily.After two weeks, such as
Monoclonal grows into cell mass in orifice plate, takes cell in cell mass to carry out PCR verifying sequencing, sees whether the cell mass is knockout
The 293T monoclonal cell group (result in detail as shown in Figure 6) of miR-126, the band of PCR 293T monoclonal cell group as the result is shown
It is significantly less than the PCR band of control 293T cell, send the PCR product that 293T monoclonal cell is rolled into a ball to sequencing (Shanghai Mei Ji biology
Pharmaceutical Technology Co., Ltd, as a result as shown in Figure 7), sequencing result is through Blast comparison, miR-126 genomic coding sequence
It is knocked completely in 293T monoclonal cell group (monoclonal is named as D2), correct monoclonal cell group D2 will be verified and expanded
Culture, the 293T cell model that so far miR-126 is knocked out construct successfully.
Successful 293T will be constructed to knock out miR-126 cytotostatic strain clone D2 expansion culture and utilize QIAGEN
MiRNeasy Mini Kit (QIAGEN, article No. 217004) carries out RNA (including microRNA) extracting, and the RNA after extracting is utilized
MiScript II RT Kit (QIAGEN, article No. 218160) is inverted, system such as table 7:
Table 7
37℃60min；After the cDNA inverted is diluted 10 times by 95 DEG C of 5min, qPCR is carried out using primer as shown in table 8.
Table 8
| * primer sequence (5'--3') | * Primer |
| TCGTACCGTGAGTAATAATGCG, as shown in SEQ ID NO.23 | mir126Fw |
| GATTGAATCGAGCACCAGTTAC, as shown in SEQ ID NO.24 | common q |
| TTCGTGAAGCGTTCCATATTTT, as shown in SEQ ID NO.25 | U6Fw |
PCR reaction system such as table 9:
Table 9
95℃2min；94 DEG C of 15sec, 60 DEG C of 1min are recycled for 40 totally；The channel fluorescence quantitative PCR instrument SYBR when 60 DEG C of 1min
Acquire signal.After experiment, software analysis experimental result is carried using fluorescence quantitative PCR instrument, has obtained 293T monoclonal D2 (
Verifying knocks out successful 239T monoclonal and stablizes strain) expression quantity and blank control sample of the miR-126 relative to itself U6
Expression quantity of the miR-126 relative to itself U6.Using the miR-126 relative expression quantity of blank control sample as standard, determined
It is 100% that justice, which is expression efficiency, so that the expression efficiency for calculating miR-126 in 293T monoclonal D2 is that 5.37% (result is such as
Shown in Fig. 8), show to utilize pSpCas9 (BB) -2A-Puro-sgRNA1 and pSpCas9 (BB) -2A-Puro-sgRNA3sgRNA
Combination can knock out miR-126 in 293T, and knock out efficiency up to 94.63%.
Embodiment 3 constructs the lung carcinoma cell model that miR-126 is knocked out
1) lung cell A549 cell transfecting
PSpCas9 (BB) -2A-Puro-sgRNA1, pSpCas9 (BB) -2A-Puro-sgRNA3 are utilized
2000 cotransfection lung cell A549 of Lipofectamine, after transfection 24 hours, using complete containing 2 μ g/mL puromycin
Full culture medium carries out medicine sieve, after medicine sieves three days, collects cell.
2) activity verifying is knocked out
A549 cell after collecting medicine sieve and the A549 cell without transfection, are tried using submicrosample genotype identification
Agent box (Nanjing Yao Shunyu Biotechnology Co., Ltd, KC-101) carry out sample process, and using PCR primer as shown in table 10 into
The identification of row PCR amplification.
Table 10
| * primer sequence (5'--3') | * Primer |
| GAGGGAGGATAGGTGGGTTC, as shown in SEQ ID NO.19 | mir126-test-Fw |
| AGGCAGAGCCAGAAGACTCA, as shown in SEQ ID NO.20 | mir126-test-Rv |
| GCACTGGAATCTGGGCGGAAGG, as shown in SEQ ID NO.21 | mir126-test-2-Fw |
| AGAGCCAGGCGCTGGGTCAC, as shown in SEQ ID NO.22 | mir126-test-2-Rv |
PCR reaction system such as table 11:
Table 11
94℃5min；94 DEG C of 30sec, 62 DEG C of 30sec, 72 DEG C of 30sec, totally 35 recycle；72℃10min.By PCR amplification
10 times of product dilution, the second wheel PCR is carried out by template of dilution, PCR system such as table 12:
Table 12
94℃5min；94 DEG C of 30sec, 62 DEG C of 30sec, 72 DEG C of 2min, totally 35 recycle；72℃5min.PCR amplification is produced
Object is detected through 1% agarose gel electrophoresis, is chosen the obvious PCR less than normal compared with the A549 cell sample band without transfection and is produced
Object sends to sequencing (Shanghai Major Biological Medical Technology Co., Ltd.).Sequencing result using Blast carry out analysis shows, miR-
126 segments are effectively knocked out in genome.
3) the lung cancer cell line A549 monoclonal that separation miR-126 is knocked out
By after the lung cancer cell line A549 cell count for having verified that the knockout containing miR-126, the quantity for being diluted to 2/hole is connect
24 orifice plates of kind, are placed in 37 DEG C, 5%CO2Incubator culture two weeks, the growth conditions of monoclonal in orifice plate were during which observed daily.Two
Zhou Hou takes cell in cell mass to carry out PCR verifying sequencing, whether sees the cell mass if monoclonal grows into cell mass in orifice plate
For the A549 monoclonal cell group (result in detail as shown in Figure 9) for knocking out miR-126,6 monoclonal items of PCR A549 as the result is shown
Band is significantly less than the PCR band of control A549 cell, and the PCR product for selecting monoclonal A2 in 6 monoclonals at random send sequencing
(Shanghai Major Biological Medical Technology Co., Ltd., the results are shown in Figure 10), sequencing result is through Blast comparison, miR-126
It is knocked completely in A549 monoclonal A2, the culture of correct monoclonal cell group will be verified and expand culture, so far miR-126 strikes
The A549 cell model removed constructs successfully.
Embodiment 4 constructs the endothelial cell model that miR-126 is knocked out
1) Human umbilical vein endothelial cells HUVEC cell transfecting
PSpCas9 (BB) -2A-Puro-sgRNA1, pSpCas9 (BB) -2A-Puro-sgRNA3 are utilized
2000 cotransfection Human umbilical vein endothelial cells HUVEC of Lipofectamine, after transfection 24 hours, using containing 2 μ g/mL
The complete medium of puromycin carries out medicine sieve, after medicine sieves three days, collects cell.
2) activity verifying is knocked out
HUVEC cell after collecting medicine sieve and the HUVEC cell without transfection, utilize submicrosample genotype identification
Kit (Nanjing Yao Shunyu Biotechnology Co., Ltd, KC-101) carries out sample process, and utilizes PCR primer as shown in table 13
Carry out PCR amplification identification.
Table 13
| * primer sequence (5'--3') | * Primer |
| GAGGGAGGATAGGTGGGTTC, as shown in SEQ ID NO.19 | mir126-test-Fw |
| AGGCAGAGCCAGAAGACTCA, as shown in SEQ ID NO.20 | mir126-test-Rv |
| GCACTGGAATCTGGGCGGAAGG, as shown in SEQ ID NO.21 | mir126-test-2-Fw |
| AGAGCCAGGCGCTGGGTCAC, as shown in SEQ ID NO.22 | mir126-test-2-Rv |
PCR reaction system such as table 14:
Table 14
94℃5min；94 DEG C of 30sec, 62 DEG C of 30sec, 72 DEG C of 30sec, totally 35 recycle；72℃10min.By PCR amplification
10 times of product dilution, the second wheel PCR is carried out by template of dilution, PCR system such as table 15:
Table 15
94℃5min；94 DEG C of 30sec, 62 DEG C of 30sec, 72 DEG C of 2min, totally 35 recycle；72℃5min.PCR amplification is produced
Object is detected through 1% agarose gel electrophoresis, chooses the obvious PCR less than normal compared with the HUVEC cell sample band without transfection
Product sends to sequencing (Shanghai Major Biological Medical Technology Co., Ltd.).Sequencing result using Blast carry out analysis shows, miR-
126 segments are effectively knocked out in genome.
3) the HUVEC monoclonal that separation miR-126 is knocked out
By after the HUVEC cell count for having verified that the knockout containing miR-126, the quantity for being diluted to 2/hole is inoculated with 24 orifice plates,
37 DEG C are placed in, 5%CO2Incubator culture two weeks, the growth conditions of monoclonal in orifice plate were during which observed daily.After two weeks, such as hole
Monoclonal grows into cell mass in plate, and cell in cell mass is taken to carry out PCR verifying sequencing, sees whether the cell mass is to knock out miR-
The monoclonal cell group band of 126 HUVEC monoclonal cell group (result in detail as shown in Figure 11), PCR HUVEC as the result is shown is bright
The aobvious PCR band for being less than control HUVEC cell, send the PCR product of HUVEC monoclonal cell group (being named as monoclonal H1) to survey
Sequence (Shanghai Major Biological Medical Technology Co., Ltd., as a result as shown in figure 12), sequencing result is through Blast comparison, miR-
126 are knocked completely in HUVEC monoclonal cell group H1, will verify the culture of correct monoclonal cell group and expand culture, until
The HUVEC cell model that this miR-126 is knocked out constructs successfully.
5 breast cancer cell T47D cell line miR-126 of embodiment is knocked out
1) breast cancer cell T47D cell transfecting
PSpCas9 (BB) -2A-Puro-sgRNA1, pSpCas9 (BB) -2A-Puro-sgRNA3 are utilized
2000 cotransfection breast cancer cell T47D of Lipofectamine, after transfection 24 hours, using containing 2 μ g/mL puromycin
Complete medium carries out medicine sieve, after medicine sieves three days, collects cell.
2) activity verifying is knocked out
T47D cell after collecting medicine sieve and the T47D cell without transfection, are tried using submicrosample genotype identification
Agent box (Nanjing Yao Shunyu Biotechnology Co., Ltd, KC-101) carries out sample process, and utilizes PCR primer as shown in table 16
Carry out PCR amplification identification.
Table 16
| * primer sequence (5'--3') | * Primer |
| GAGGGAGGATAGGTGGGTTC, as shown in SEQ ID NO.19 | mir126-test-Fw |
| AGGCAGAGCCAGAAGACTCA, as shown in SEQ ID NO.20 | mir126-test-Rv |
| GCACTGGAATCTGGGCGGAAGG, as shown in SEQ ID NO.21 | mir126-test-2-Fw |
| AGAGCCAGGCGCTGGGTCAC, as shown in SEQ ID NO.22 | mir126-test-2-Rv |
PCR reaction system such as table 17:
Table 17
94℃5min；94 DEG C of 30sec, 62 DEG C of 30sec, 72 DEG C of 30sec, totally 35 recycle；72℃10min.By PCR amplification
10 times of product dilution, the second wheel PCR is carried out by template of dilution, PCR system such as table 18:
Table 18
94℃5min；94 DEG C of 30sec, 62 DEG C of 30sec, 72 DEG C of 2min, totally 35 recycle；72℃5min.PCR amplification is produced
Object is detected through 1% agarose gel electrophoresis, is chosen the obvious PCR less than normal compared with the T47D cell sample band without transfection and is produced
Object (as shown in figure 13) sends to sequencing (Shanghai Major Biological Medical Technology Co., Ltd.).Sequencing result is (as shown in figure 14) to be utilized
Blast carry out analysis shows, miR-126 segment is effectively knocked out in genome.
According to above-mentioned experimental result, present invention determine that for the sgRNA-1 and sgRNA-3 of the design of miR-126 gene；
SgRNA-2 and sgRNA-5 is effective sgRNA combined sequence, wherein can be used as miR- comprising sgRNA-1, sgRNA3 plasmid combinations
The Cas9 plasmid for the kit that 126 full-length genes knock out.It is demonstrated experimentally that the kit can effectively realize the clpp gene of cellular level
It removes, and can be used for various kinds of cell system includes vehicles cells system 293T, tumor cell line: lung cancer cell line A549, breast cancer cell
It is the gene knockout of the cell lines miR-126 such as T47D and endothelial cell HUVEC system.The miR-126 gene knockout it is various thin
Born of the same parents' model can be used for vascular endothelial cell poorly differentiated and Several Kinds of Malignancy such as: the gene of breast cancer, lung cancer etc.
Functional study is laid a good foundation for the modeling of subsequent animal, the tool provided convenience for application studies such as further drug developments.
Sequence table
<110>Shanghai Biotechnology Corporation
<120>a kind of miR-126 full-length gene based on CRISPR-Cas9 technology knocks out kit and its application
<130>HJ16-12192
<160> 25
<170> PatentIn version 3.5
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<400> 21
gcactggaat ctgggcggaa gg 22
<210> 22
<211> 20
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<223>primer
<400> 22
agagccaggc gctgggtcac 20
<210> 23
<211> 22
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<223>primer
<400> 23
tcgtaccgtg agtaataatg cg 22
<210> 24
<211> 22
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<223>primer
<400> 24
gattgaatcg agcaccagtt ac 22
<210> 25
<211> 22
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<223>primer
<400> 25
ttcgtgaagc gttccatatt tt 22
Claims (9)
1. a kind of miR-126 full-length gene based on CRISPR-Cas9 technology knocks out kit, which is characterized in that the reagent
Box all designs sgRNA at the upstream and downstream both ends of miR-126 gene order, carries out fixed point shearing while two sites；
The kit includes by plasmid pSpCas9 (BB) -2A-Puro-sgRNA1 and plasmid pSpCas9 (BB) -2A-Puro-
The sgRNA plasmid combinations of sgRNA3 composition；Plasmid pSpCas9 (the BB) -2A-Puro-sgRNA1, it is complementary containing one section
DNA sequence dna, the DNA sequence dna can be transcribed into the target of specific recognition sequence miR-126 full-length gene as shown in SEQ ID NO.2
The sgRNA of sequence is marked, which can constitute special identification structure with trRNA, so that guidance Cas9 enzyme specifically shears miR-
126 gene corresponding sequences；Plasmid pSpCas9 (the BB) -2A-Puro-sgRNA3 should containing one section of complementary DNA sequence dna
DNA sequence dna can be transcribed into the target sequence of specific recognition sequence miR-126 full-length gene as shown in SEQ ID NO.4
SgRNA, the sgRNA can constitute special identification structure with trRNA, so that guidance Cas9 enzyme specifically shears miR-126 gene
Corresponding sequence；
Alternatively, the kit includes by plasmid pSpCas9 (BB) -2A-Puro-sgRNA2 and plasmid pSpCas9 (BB) -2A-
The sgRNA plasmid combinations of Puro-sgRNA5 composition；Plasmid pSpCas9 (the BB) -2A-Puro-sgRNA2, mutually containing one section
The DNA sequence dna of benefit, the DNA sequence dna can be transcribed into specific recognition sequence miR-126 full-length gene as shown in SEQ ID NO.3
Target sequence sgRNA, which can constitute special identification structure with trRNA, so that guidance Cas9 enzyme is specifically sheared
MiR-126 gene corresponding sequence；Plasmid pSpCas9 (the BB) -2A-Puro-sgRNA5, containing one section of complementary DNA sequence dna,
The DNA sequence dna can be transcribed into the target sequence of specific recognition sequence miR-126 full-length gene as shown in SEQ ID NO.6
SgRNA, the sgRNA can constitute special identification structure with trRNA, so that guidance Cas9 enzyme specifically shears miR-126 gene
Corresponding sequence.
2. kit as described in claim 1, which is characterized in that plasmid pSpCas9 (the BB) -2A-Puro-sgRNA1 contains
Some complementary dna sequences are SEQ ID NO.7 and SEQ ID NO.8.
3. kit as described in claim 1, which is characterized in that plasmid pSpCas9 (the BB) -2A-Puro-sgRNA3 contains
Some complementary dna sequences are SEQ ID NO.11 and SEQ ID NO.12.
4. kit as described in claim 1, which is characterized in that plasmid pSpCas9 (the BB) -2A-Puro-sgRNA2 contains
Some complementary dna sequences are SEQ ID NO.9 and SEQ ID NO.10.
5. kit as described in claim 1, which is characterized in that plasmid pSpCas9 (the BB) -2A-Puro-sgRNA5 contains
Some complementary dna sequences are SEQ ID NO.15 and SEQ ID NO.16.
6. kit as described in claim 1, which is characterized in that plasmid pSpCas9 (the BB) -2A-Puro-sgRNA1 and
The sgRNA plasmid combinations of plasmid pSpCas9 (BB) -2A-Puro-sgRNA3 composition, plasmid pSpCas9 (the BB) -2A-
The sgRNA plasmid combinations of Puro-sgRNA2 and plasmid pSpCas9 (BB) -2A-Puro-sgRNA5 composition are with including following step
Rapid method building:
Step 1, by the preferably a certain number of miR-126 upstream region of gene of target software design and downstream CRISPR-Cas9
Target sequence, specific as follows:
Upstream:
SgRNA-1:TAATGTCCCGTCGCCAGCGG, as shown in SEQ ID NO.2；
SgRNA-2:GCCACGCCTCCGCTGGCGAC, as shown in SEQ ID NO.3；
Downstream:
SgRNA-3:TCTCAGCGGCGTTTTCGATG, as shown in SEQ ID NO.4；
SgRNA-4:GAGTAATAATGCGCCGTCCA, as shown in SEQ ID NO.5；
SgRNA-5:TTTCGATGCGGTGCCGTGGA, as shown in SEQ ID NO.6；
Step 2 separately designs the sgRNA segment that synthesis is directed to upstream and downstream target sequence, and is building up in plasmid vector；
The sgRNA fragment sequence is as follows:
SgRNA-1 F:ccacgTAATGTCCCGTCGCCAGCGG, as shown in SEQ ID NO.7；
SgRNA-1 R:aaacCCGCTGGCGACGGGACATTAc, as shown in SEQ ID NO.8；
SgRNA-2 F:ccacGCCACGCCTCCGCTGGCGAC, as shown in SEQ ID NO.9；
SgRNA-2 R:aaac GTCGCCAGCGGAGGCGTGGC, as shown in SEQ ID NO.10；
SgRNA-3 F ccacgTCTCAGCGGCGTTTTCGATG, as shown in SEQ ID NO.11；
SgRNA-3 R aaacCATCGAAAACGCCGCTGAGAC, as shown in SEQ ID NO.12；
SgRNA-4 F ccacGAGTAATAATGCGCCGTCCA, as shown in SEQ ID NO.13；
SgRNA-4 R aaacTGGACGGCGCATTATTACTC, as shown in SEQ ID NO.14；
SgRNA-5 F ccacgTTTCGATGCGGTGCCGTGGA, as shown in SEQ ID NO.15；
SgRNA-5 R aaacTCCACGGCACCGCATCGAAAc, as shown in SEQ ID NO.16；
Step 3, by transfecting 293T cell strain, sgRNA and trRNA constitute special identification structure, to guide Cas9 enzyme
The corresponding sequence for specifically shearing miR-126 gene both ends continues medicine sieve, obtains the cell of miR-126 full-length gene knockout
Strain；
Step 4, by extracting cell DNA, PCR amplification, sequencing is determined and verifies, two optimized group miR-126
The sgRNA plasmid combinations of gene knockout CRISPR-Cas9, i.e., described plasmid pSpCas9 (the BB) -2A-Puro-sgRNA1 and matter
The sgRNA plasmid combinations and the plasmid pSpCas9 (BB) -2A- of grain pSpCas9 (BB) -2A-Puro-sgRNA3 composition
The sgRNA plasmid combinations of Puro-sgRNA2 and plasmid pSpCas9 (BB) -2A-Puro-sgRNA5 composition.
7. kit as claimed in claim 6, which is characterized in that it is described to be building up in plasmid vector in step 2, it is specific to wrap
Include following steps:
(1) using pSpCas9 (BB) -2A-Puro plasmid as initial vector, single endonuclease digestion is carried out with Bbs I, linearized vector is carried out
Purifying；
(2) synthetic sgRNA segment is diluted and is annealed；
(3) pSpCas9 (BB) -2A-Puro of linearisation is connected with the sgRNA segment after annealing using T4 DNA ligase,
The conversion of bacillus coli DH 5 alpha competent cell is added in whole connection products；
(4) it is reflected using the single colonie that the plate that bacterium colony PCR method is coated with DH5 α competent cell bacterium solution after conversion is grown
It is fixed, obtain expression plasmid.
8. such as application of the described in any item kits of claim 1-7 in preparation cell line miR-126 gene knockout product.
9. application as claimed in claim 8, which is characterized in that the cell line includes vehicles cells system 293T, tumour cell
System and endothelial cell HUVEC system.
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