CN106566838A - MiR-126 full-length gene knockout kit based on CRISPR-Cas9 technology and application thereof - Google Patents

MiR-126 full-length gene knockout kit based on CRISPR-Cas9 technology and application thereof Download PDF

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CN106566838A
CN106566838A CN201611002317.4A CN201611002317A CN106566838A CN 106566838 A CN106566838 A CN 106566838A CN 201611002317 A CN201611002317 A CN 201611002317A CN 106566838 A CN106566838 A CN 106566838A
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徐晓晶
赵莹
孙冰玉
姚琴琴
陆凌佳
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SHANGHAI BIOTECHNOLOGY Corp
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Abstract

The invention discloses a MiR-126 full-length gene knockout kit based on a CRISPR-Cas9 technology and an application thereof. CRISPR-Cas9 target sequences upstream and downstream a MiR-126 gene are preferably selected, and sgRNA single strands are designed and synthesized for the target sequences and built into a carrier. Through 293T cell strain transfection, sgRNA and trRNA constitute a specific recognition structure. Thus, Cas9 enzyme is guided to specifically shear the corresponding sequences at the two ends of the MiR-126 gene. Drug sieving is carried out constantly to get a MiR-126 full-length gene knockout cell strain. An optimal upstream and downstream sgRNA combination is obtained through drug sieving cell strain sequencing verification. The knockout efficiency of the combination is as high as 90% above. The kit built based on the combination can be used to carry out specific MiR-126 full-length gene knockout on a variety of cell lines such as 293T, a lung cancer cell line A 549 and a vascular endothelial cell HUVEC line.

Description

A kind of miR-126 full-length genes based on CRISPR-Cas9 technologies knock out test kit and Its application
Technical field
The invention belongs to genetic engineering field, is related to a kind of miR-126 gene knockouts test kit, more particularly to one kind is based on CRISPR-Cas9 technologies can carry out the test kit that specificity is knocked out completely to miR-126 full-length gene orders.Additionally, this The bright application for also disclosing the test kit.
Background technology
MicroRNA (microRNA) is that a class is single-stranded by the non-coding that the length of endogenous gene is about 22 nucleotide RNA molecule, is widely distributed in virus, plant in higher mammal, and it has in the cell various important adjustment effects. Each microRNA can have multiple target genes, and several microRNA can also adjust same gene.This complicated tune Section network both can regulate and control the expression of multiple genes by a microRNA, it is also possible to by the combination of several microRNA Carry out the expression of certain gene of finely regulating, therefore the gene function of microRNA is explored and application and development is always study hotspot.
MiR-126 (microRNA126, microRNA 126) is positioned at the skins somatomedin domain 7 of human chromosomal 9q34.3 In the intron 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 the abundant tissue of vascularization, such as in heart, lungs, kidney.It can By regulating and controlling Spred-1, (Sprouty-related, EVH1 domain-containing protein 1, Sprouty is related Region protein containing EVH1 1), VCAM-1 (Vascular cell adhesion protein 1, vascular cell adhesion molecule-1), HoxA9 (homeobox A9, hox geneses A9), v-Crk (Sarcoma Virus CT10regulator of kinase, Sarcoma virus CT10 adjusts kinases), EGFL-7 and VEGF (vascular endothelial growth factor, Ink vessel transfusing Skin growth factor) isogenic expression participates in adjusting vascular development, new vesselses and formed and the blood vessel such as vascular inflammation reaction Physiology and pathophysiological process.Jing other researchs find, miR-126 in gastrointestinal tract, genital cancers, mammary gland, thyroid, lung, And express suppressed in some other cancer.Lower and can induce after miR-126 cancer cell multiplication, migration is attacked and affected The cell survival phase, there is dependency between miR-126 downwards are bad with kinds cancer existence.These researchs are found to be gastrointestinal tract, The prevention of the cancer metastasiss such as genital cancers, mammary gland, thyroid, lung judges to provide potential target, and for gastrointestinal tract, reproductive tract The Biotherapeutics of the cancers such as cancer, mammary gland, thyroid, lung indicate new direction, point out the importance and necessity of miR-126 researchs Property.
Because microRNA sequences are shorter and its precursor has loop-stem structure, classical RNA disturbs the method for (RNAi) not The functional study of expression is lowered suitable for microRNA.The conventional method of microRNA researchs at present, is Applied Biology technology In vivo in cell model or external dynamic/plant model, microRNA is carried out into sequence closing or deletion mutant is caused, Negative regulation effect reduction or remove that simulation microRNA afunction causes, and then study its function mechanism, technology Method mainly has two classes:
First kind method is the complementary closure sequence of design synthesis microRNA, and complementary seriess were originally specific with microRNA With reference to mRNA formed competition, reduction microRNA to specific mRNA binding abilities and regulating and controlling effect.Due to complementary seriess be with The form of base complementrity is closed to microRNA, does not directly result in microRNA degradeds or the downward expressed, so The expression of microRNA does not change, and can be to the closing efficiency of microRNA functions without direct technical method Carry out quantization detection, it is impossible to directly prove that the function of microRNA receives shielding, can only be possible by detection microRNA Regulation and control downstream gene change carrys out the function of side light microRNA and is suppressed, thus the method is to microRNA functional studies Verity and effectiveness there is large effect.
Equations of The Second Kind method is with transcriptional activation sample effector nuclease (transcription activator-like Effector nuclease, TALEN) TALEN technologies, by DNA identification modules by the DNA positions of TALEN element targeting specifics Put and combine, the shearing of specific site is then completed in the presence of FokI nucleases, DNA level can be carried out to microRNA Shearing and modification, simulate microRNA afunction.But TALEN technologies rely on what DNA was combined to the knockout of target gene Amino acid polymer sequence of modules, single TALEN modules carry out assembling needs substantial amounts of molecular cloning and sequencing procedures, very It is loaded down with trivial details, if all working completes basic experiment operation in laboratory internal to need to take more than 2 months, if screening stably knocks out base The cell line of cause is needed more than 4 months, and time-consuming for whole process, high cost.
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats/cas, Cluster, regular intervals short palindrome repetitive sequence) technology is also called 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 DNA identifications and edit that RNA is oriented to.Compared with the gene technology instrument of above-mentioned introduction, CRISPR technologies are physically easier to perform, and have Higher extensibility.
CRISPR/Cas9 is found in the natural immune system of antibacterial, and its major function is the disease of antagonism invasion Poison and foreign DNA, are the acquired immunity defense mechanisms of antibacterial and archeobacteria.In this system, crRNA (CRISPR- Derived RNA, CRISPR derive RNA) by base pairing and tracrRNA (trans-activating RNA, it is trans to swash RNA living) double-stranded RNA is combined to form, this tracrRNA/crRNA binary complex instructs Cas9 nuclease proteins to guide in crRNA Double-stranded DNA is sheared in the site that targets of sequence, so as to reach the purpose sheared to genomic DNA or modified.
In order to realize that, using CRISPR/Cas9 systems in mammal, scientist is transformed CRISPR/Cas9, TracrRNA/crRNA binary complex amalgamation and expression is formed into single-stranded guiding RNA.And in actual design, only design The RNA sequence of one section of 20nt (base) left and right of synthesis identification target sequence and referred to as sgRNA (small guide RNA, little guide RNA), other parts are oriented to RNA and exist as the carrier sequence on sgRNA expression vectors because sequence is constant.Therefore, extensively Using CRISPR/Cas9 systems become and target gene carried out calmly into the Cas9 nucleases under being instructed by the guidings RNA containing sgRNA To the technology of editor.
The fixed point shearing editing technique of CRISPR/Cas9 technologies is most commonly for Single locus design sgRNA guiding Cas9 is oriented shearing in target sequence, and (for example, " one kind is based on disclosed in Chinese invention patent application CN105112445A The miR-205 gene knockout test kits of CRISPR-Cas9 gene Knockouts "), intracellular intrinsic nonhomologous end after cutting Connection approach (NHEJ) repair process, can introduce radom insertion and the gene mutation deleted, and this repair process is not artificially controlled System, causes all kinds of unnecessary and inaccurate sequence to insert and delete, and Jing often produces indefinite or invalid cell phenotype, right Research has a negative impact.
At present, there is not yet about being carried out the full-length gene order of miR-126 using CRISPR/Cas9 systemic characteristics ground The report of knockout.
The content of the invention
One of the technical problem to be solved in the present invention is to provide a kind of CRISPR/Cas9 gene editing technologies that are based on to miR- 126 genes carry out the test kit of gene knockout, and it allows CRISPR/Cas9 to target miR-126 gene coded sequences two The shearing that end is pinpointed simultaneously, can greatly avoid guiding Cas9 in target for Single locus design sgRNA using traditional The probability that non-homologous end joining approach NHEJ caused by shearing institute introduces random mutation generation is oriented in mark sequence, it is non- Often be conducive to producing and be directed to the single and obvious cell phenotype that the gene is deleted completely, the test kit can efficient, quick, essence Really, stably realize that miR-126 complete encoding sequences are completely knocked out in genomic level.
The two of the technical problem to be solved in the present invention are to provide the application of the test kit, and the test kit can be applicable to various thin Born of the same parents' type (for example, vascular endothelial cell, and Several Kinds of Malignancy is such as:Breast carcinoma, pulmonary carcinoma etc.) miR-126 gene knockouts, These cell lines further can build animal model by the transplanted tumor in nude mice body, be the research of miR-126 related pathways And the internal and external experimental construction model of drug development etc. provides good tool.
The present invention is by target software design preferably a number of miR-126 upstream region of gene 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 strains, sgRNA and trRNA (trans-activating RNA) constitutes special identification structure, from And guide Cas9 enzymes specifically to shear the corresponding sequence at miR-126 genes two ends, and continue medicine sieve, obtain miR-126 full-length genes The cell strain of knockout.By extracting cell DNA, PCR amplifications, 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 is combined, and fluorescent quantitative PCR technique shows that the combination realizes that miR-126 molecules knock out efficiency high up to more than 90%, with This structure applies test kit, can be to 293T, lung cancer cell line A549, breast cancer cell line T47D and vascular endothelial cell Polytype cell line such as HUVEC systems carries out the total length miR-126 gene order of stable specificity exactly and knocks out.
To solve above-mentioned technical problem, the present invention is adopted the following technical scheme that:
In one aspect of the invention, there is provided a kind of miR-126 full-length genes based on CRISPR-Cas9 technologies knock out reagent Box,
The test kit is included by plasmid pSpCas9 (BB) -2A-Puro-sgRNA1 and plasmid pSpCas9 (BB) -2A- The sgRNA plasmid combinations of Puro-sgRNA3 compositions;Plasmid pSpCas9 (the BB) -2A-Puro-sgRNA1, containing one section mutually The DNA sequence of benefit, the DNA sequence can be transcribed into miR-126 full-length gene of the specific recognition sequence as shown in SEQ ID NO.2 Target sequence sgRNA, the sgRNA can constitute special identification structure with trRNA, so that guiding Cas9 enzymes are specifically sheared MiR-126 gene pairss answer sequence;Plasmid pSpCas9 (the BB) -2A-Puro-sgRNA3, the DNA sequence containing one section of complementation, The DNA sequence can be transcribed into the target sequence of miR-126 full-length gene of the specific recognition sequence as shown in SEQ ID NO.4 SgRNA, the sgRNA can constitute special identification structure with trRNA, so that guiding Cas9 enzymes specifically shear miR-126 genes Correspondence sequence;
Or, the test kit include by plasmid pSpCas9 (BB) -2A-Puro-sgRNA2 and plasmid pSpCas9 (BB) - The sgRNA plasmid combinations of 2A-Puro-sgRNA5 compositions;Plasmid pSpCas9 (the BB) -2A-Puro-sgRNA2, containing one section Complementary DNA sequence, the DNA sequence can be transcribed into miR-126 total length base of the specific recognition sequence 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 guiding Cas9 enzymes are specifically cut Cut miR-126 gene pairss and answer sequence;Plasmid pSpCas9 (the BB) -2A-Puro-sgRNA5, the DNA sequences containing one section of complementation Row, the DNA sequence can be transcribed into the target sequence of miR-126 full-length gene of the specific recognition sequence as shown in SEQ ID NO.6 The sgRNA of row, the sgRNA can constitute special identification structure with trRNA, so that guiding Cas9 enzymes specifically shear miR-126 Gene pairss answer sequence.
As currently preferred technical scheme, the complementation that plasmid pSpCas9 (the BB) -2A-Puro-sgRNA1 contains DNA sequence is SEQ ID NO.7 and SEQ ID NO.8.
As currently preferred technical scheme, the complementation that plasmid pSpCas9 (the BB) -2A-Puro-sgRNA3 contains DNA sequence is SEQ ID NO.11 and SEQ ID NO.12.
As currently preferred technical scheme, the complementation that plasmid pSpCas9 (the BB) -2A-Puro-sgRNA2 contains DNA sequence is SEQ ID NO.9 and SEQ ID NO.10.
As currently preferred technical scheme, the complementation that plasmid pSpCas9 (the BB) -2A-Puro-sgRNA5 contains DNA sequence is SEQ ID NO.15 and SEQ ID NO.16.
As currently preferred technical scheme, plasmid pSpCas9 (the BB) -2A-Puro-sgRNA1 and plasmid The sgRNA plasmid combinations of pSpCas9 (BB) -2A-Puro-sgRNA3 compositions, plasmid pSpCas9 (the BB) -2A-Puro- The sgRNA plasmid combinations of sgRNA2 and plasmid pSpCas9 (BB) -2A-Puro-sgRNA5 compositions are with the side for comprising the steps What method built:
Step one, by target software design preferably a number of miR-126 upstream region of gene and downstream CRISPR- Cas9 target sequence, it is 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 sgRNA fragment of the synthesis for upstream and downstream target sequence, and is building up to plasmid vector In;The sgRNA fragment sequences are 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 strains, sgRNA and trRNA constitutes special identification structure, so as to guide Cas9 Enzyme specifically shears the corresponding sequence at miR-126 genes two ends, continues medicine sieve, obtains the cell of miR-126 full-length genes knockout Strain;
Step 4, by extracting cell DNA, PCR amplifications, sequencing is determined and verifies, obtains the two groups of miR- for optimizing The sgRNA plasmid combinations of 126 gene knockout CRISPR-Cas9, i.e., described plasmid pSpCas9 (BB) -2A-Puro-sgRNA1 and The sgRNA plasmid combinations of plasmid pSpCas9 (BB) -2A-Puro-sgRNA3 compositions, and the plasmid pSpCas9 (BB) -2A- The sgRNA plasmid combinations of Puro-sgRNA2 and plasmid pSpCas9 (BB) -2A-Puro-sgRNA5 compositions.
It is described to be building up in plasmid vector in step 2 as currently preferred technical scheme, specifically include following step Suddenly:
(1) with pSpCas9 (BB) -2A-Puro plasmids as initial vector, single endonuclease digestion is carried out with Bbs I, to linearized vector Carry out purification;
(2) synthetic sgRNA fragments are diluted and is annealed;
(3) the sgRNA fragment phases after linearizing pSpCas9 (BB) -2A-Puro is annealed together using T4DNA ligases Even, whole connection products are added into the conversion of bacillus coli DH 5 alpha competent cell;
(4) single bacterium colony that the flat board of DH5 α competent cells bacterium solution coating after conversion grows is carried out using bacterium colony PCR methods Identification, obtains expression plasmid.
In another aspect of this invention, there is provided mentioned reagent box is in cell line miR-126 gene knockout product is prepared Using.The cell line includes vehicles cells system 293T, tumor cell line (for example, lung cancer cell line A549, breast cancer cell line ) and endotheliocyte HUVEC systems T47D.
Compared with prior art, the present invention has the advantages that:MiR- of the present invention based on CRISPR-Cas9 technologies 126 full-length genes knock out test kit, mainly knock out miR-126 genes of interest using CRISPR/Cas9 systems.This is to utilize first CRISPR/Cas9 systemic characteristics are knocked out miR-126 full-length gene orders.The invention has the advantages that:
1. the drawbacks of overcoming tradition closing sequence method research microRNA functions, miR-126 knocks out result can quantify prison Survey, miR-126 molecules knock out efficiency high up to more than 90%;
2. the time-consuming loaded down with trivial details defect of TALEN technologies is overcome, cell screening is accelerated and is tested and be reduced to 1 week, it is stable Cell line experiments are built to 2 months;Construction step and experimental implementation are simple, while reducing reagent cost consumption;
3. technology extending design has been done in CRISPR-Cas9 technologies, at the upstream and downstream two ends of miR-126 gene orders all Design sgRNA, carries out fixed point shearing while two sites, realizes on DNA level complete to whole miR-126 gene orders Whole knockout (see Figure 15), generation sequencing result shows, experiment effect, good stability can be repeated in various kinds of cell.Pole of the present invention The earth avoids being led for being oriented cutting method in Single locus design sgRNA guiding Cas9 target sequence using traditional Cause non-homologous end joining approach NHEJ to introduce the probability that random mutation occurs, can accurately realize that microRNA total lengths are compiled Code sequence is completely knocked out in genomic level.
Description of the drawings
Fig. 1 is PCR methods checking pSpCas9 (BB) -2A-Puro-sgRNA vector construction schematic diagrams in the embodiment of the present invention 1; Wherein, passage 1-5 is pSpCas9 (BB) -2A-Puro-sgRNA vector construction positive colony PCR results;Passage 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 combinations knock out result schematic diagram in 293T;Wherein, passage 3,4,5 is pSpCas9 (BB) -2A- Puro-sgRNA1 and pSpCas9 (BB) -2A-Puro-sgRNA3 combinations knock out PCR positive findingses;Passage 1 and 2 is pSpCas9 (BB) -2A-Puro-sgRNA1 and pSpCas9 (BB) -2A-Puro-sgRNA5 combinations knock out PCR negative findingses, and passage 6 is right According to 293T cell PCR results;Passage 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 pairing sgRNA combinations knock out checking 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 combinations knock out result schematic diagram in 293T;Wherein, passage 1 is pSpCas9 (BB) -2A-Puro- SgRNA2 and pSpCas9 (BB) -2A-Puro-sgRNA5 combinations knock out the positive findingses of PCR, and passage 2,3,4,5,6,7,8 is PSpCas9 (BB) -2A-Puro-sgRNA2 and pSpCas9 (BB) -2A-Puro-sgRNA4 combinations knock out PCR negative findingses, lead to Road 9 is control 293T cell PCR results;Passage 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 pairing sgRNA combinations knock out checking sequencing result schematic diagram in 293T.
Fig. 6 is that the monoclonal cell PCR checkings that 293T Jing medicine sieve separations are obtained after transfection in the embodiment of the present invention 2 knock out knot Fruit schematic diagram;Wherein, passage 1 is the PCR positive findingses that 293T knocks out miR-126 monoclonal cells, and passage 2 is thin for control 293T Born of the same parents PCR does not knock out result;Passage M is DNA molecular amount standard.
Fig. 7 is the monoclonal cell PCR of the knockout positive that 293T Jing medicine sieve separations are obtained after transfection 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 diagrams of 293T in the embodiment of the present invention 2; Wherein, 293Tblank is the fluorescent quantitation qPCR results that 293T compared with control cells does not knock out miR-126, and 293T cloneD2 are 293T knocks out the fluorescent quantitation qPCR results of miR-126 positive colony D2 cells.
Fig. 9 is that the monoclonal cell PCR checkings that A549 Jing medicine sieve separations are obtained after transfection in the embodiment of the present invention 3 knock out knot Fruit schematic diagram;Wherein, passage 1,2,3,4,5,6 are A549 knocks out PCR positive findingses after miR-126, and passage 7 is control A549 The PCR results that cell is knocked out without miR-126;Passage M is DNA molecular amount standard.
Figure 10 is the monoclonal cell of the knockout positive that A549 Jing medicine sieve separations are obtained after transfection in the embodiment of the present invention 3 PCR fragment sequencing result schematic diagram.
Figure 11 is that the monoclonal cell PCR checkings that HUVEC Jing medicine sieve separations are obtained after transfection in the embodiment of the present invention 4 are knocked out Result schematic diagram;Wherein, passage 2 is the positive PCR results that HUVEC knocks out miR-126, and passage 1,3,4 is control HUVEC cells The PCR results that miR-126 is not knocked out;Passage M is DNA molecular amount standard.
Figure 12 is the Dan Ke of the knockout miR-126 positives that HUVEC Jing medicine sieve separations are obtained after transfection in the embodiment of the present invention 4 Grand cell PCR fragment sequencing result schematic diagram.
Figure 13 is that T47D cell PCR checkings Jing after medicine sieve knock out result schematic diagram after transfection in the embodiment of the present invention 5;Its In, passage 1,2 is the positive PCR results that T47D cell samples knock out miR-126, and passage 3,4 is control T47D cell miR-126 The PCR results not knocked out;Passage M is DNA molecular amount standard.
Figure 14 is that T47D miR-126 Jing after medicine sieve knock out positive cell PCR fragment survey after transfection in the embodiment of the present invention 5 Sequence result schematic diagram.
Figure 15 is that miR-126 gene orders completely knock out schematic diagram.
Specific embodiment
This invention is expanded on further with reference to specific embodiment.It should be understood that particular implementation described here By way of example representing, limitation of the present invention is not intended as.Without departing from the scope of the invention, the present invention Principal character can be used for various embodiments.
The vector construction of embodiment 1.
(1) miR-126 drone designs
For miR-126 genes (gene name miR-126, gene I/D number:406913, gene details are shown in http:// Www.ncbi.nlm.nih.gov/gene/406913), download from the website and obtain miR-126 genome sequences:
5’-CGCTGGCGACGGGACATTATTACTTTTGGTACGCGCTGTGACACTTCAAACTCGTACCGTGAGT AATAATGCGCCGTCCACGGCA-3 ', as shown in SEQ ID NO.1.
Using online software Feng Zhang lab's Target Finder (http://crispr.mit.edu/) set Meter sgRNA, is input into the sequence of miR-126 genome sequences and each 50bp of its upstream and downstream, arranges and retrieve some sgRNA sequences of acquisition Row, by off-target (missing the target) information for analyzing positions and the sgRNA of the sgRNA in gene order, therefrom distinguish The optimum target sequence of upstream 2, and 3, downstream target sequence are selected, it is 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 fragments are synthesized
According to sgRNA intend cloning into carrier pSpCas9 (BB) -2A-Puro (purchased from Addgene) restriction enzyme site The information of Bbs I, above-mentioned sgRNA sequences are obtained after complementary seriess, along with the corresponding viscosity end of Bbs I restricted 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 companies synthesizing single-stranded nucleotide chain.
(3) sgRNA fragments connect with expression vector and convert
1 μ g pSpCas9 (BB) -2A-Puro are carried out into single endonuclease digestion with Bbs I (being purchased from NEB companies, article No. R0539S), After 37 DEG C of water-baths 1 hour, linearisation is carried using PCR primer purification kit (being purchased from Tiagen companies, article No. is DP204) Body carries out purification.
The dry powder of the F and R chains of every group of synthetic sgRNA is diluted to into 100 μM, 1 μ L solution is respectively taken and is mixed in PCR pipe It is interior, gradually it is down to 22 DEG C from 95 DEG C with 1.5 DEG C per minute in PCR instrument.
Using T4DNA ligases (be purchased from Takara companies, article No. 6022Q) by linearizing pSpCas9 (the BB) -2A- of 1 μ L Puro is connected with the sgRNA fragments after 0.5 μ L annealing, and linked system is shown in Table 1:
Table 1
sgRNA 0.5μL
Linearizing pSpCas9 (BB) -2A-Puro 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 of water-bath heat shocks 90 seconds, then it is placed in 5 minutes on ice, add in super-clean bench 1mL LB fluid mediums (Luria-Bertani culture medium), 37 DEG C of shaken cultivation 1 hour, take 500 μ L and coat containing Amp On the LB solid mediums of (Ampicillin, ampicillin), after liquid absorption is finished, 37 DEG C of overnight incubations are inverted in.
(4) identify after converting
The single bacterium colony that the flat board of DH5 α competent cells bacterium solution coating after conversion grows is reflected using bacterium colony PCR methods It is fixed.Bacterium colony PCR identifications are as follows with primer:
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
Composition Volume
10×buffer 2.5μL
primer F(10μM) 0.2μL
primer R(10μM) 0.2μL
dNTPs(2.5mM) 2μL
RTaq enzymes (50U/ μ L) 0.5μL
Bacterium colony Pick a little
ddH2O up to 25μL
PCR programs are 95 DEG C of 10min;95 DEG C of 20sec, 55 DEG C of 30sec, 72 DEG C of 30sec, totally 35 circulations;72℃ 10min.The agarose gel electrophoresiies of PCR primer Jing 1% for obtaining are gone into detection, the single bacterium colony of 100bp or so amplified fragments occurs It is the single bacterium colony containing positive colony (as shown in figure 1, PCR primer clip size is big with the 100bp fragments of DNA molecular amount standard It is little close, and 1000bp fragments are the most paillette section of the molecular weight standard).By the inoculation 2mL LB liquid trainings of positive colony single bacterium colony In foster base, 1mL bacterium solutions are taken after 37 DEG C of overnight shaking cultures for conservation, remaining 1mL bacterium solutions send sequencing company, and (Shanghai Mei Ji is biological Pharmaceutical Technology Co., Ltd) carry out sequence verification.The sequencing result Jing Blast analysis shows for obtaining, sgRNA fragments are cloned In entering pSpCas9 (BB) -2A-Puro plasmids, 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 and 293T knockout miR-126 are thin in the plasmid transfection of embodiment 2. and 293T cells The structure of born of the same parents' model
(1) plasmid extraction
By in embodiment 1 through sequence verification, respectively comprising sgRNA-1, sgRNA-2, sgRNA-3, sgRNA-4, The bacillus coli DH 5 alpha of pSpCas9 (the BB) -2A-Puro plasmids of sgRNA-5 fragments is each inoculated in 5mL LB fluid mediums In, 37 DEG C of overnight shaking cultures.Using endotoxin-free mini-scale plasmid extracts kit, (test kit is Omega companies product, article No. For D6948-02) plasmid extraction is carried out to these bacterium solutions.Plasmid after extracting again in super-clean bench dehydrated alcohol precipitation after, with nothing Bacterium water dissolution carries out the aseptic process to plasmid.
(2) a pair pSpCas9 (BB) -2A-Puro-sgRNA plasmids containing sgRNA of 293T cell transfectings
1) 293T cell culture
By 293T cells with 3 × 105The density of cells/well is laid in 24 orifice plates, it is ensured that each hole cell growth state is good, Density is similar, treats cell for monolayer and in mid-log phase, and cell confluency degree is transfected up to 80% or so.
2) transfection composite is prepared:
According to the pairing of table 3, a pair pSpCas9 (BB) -2A-Puro-sgRNA plasmids containing sgRNA are passed through The liposome reagents of Lipofectamine 2000 transfect 293T cells.
Upstream and downstream plasmid containing the sgRNA matching table of table 3
Sequence number Upstream sgRNA plasmids Downstream sgRNA plasmids
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 aseptic EP pipes are taken, plasmid 0.8 is diluted 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, is stored at room temperature 5 minutes, and the plasmid of dilution and Lipofectamine 2000 are mixed Close, the transfection composite of 100 μ L is obtained.After gently mixing, 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 to cultivate 6 hours, culture medium is replaced by into complete medium, cell is placed in 37 DEG C by continuation 5%CO2Incubator culture.
4) medicine sieve
After transfection 24 hours, culture medium is replaced by into the complete medium containing 2 μ g/mL puromycin.Per 24 hours more Change the complete medium of a drug containing, continuous three days.Green florescent signal is examined under a microscope daily, is recorded cellular State.
(3) CRISPR/Cas9 knocks out efficiency checking
1) the knockout efficiency in PCR sequence measurements checking genomic level
293T cells and the 293T cells without transfection after medicine sieve are collected, is tried using submicrosample genotype identification Agent box (Nanjing Yao Shunyu bio tech ltd, KC-101) carries out sample process, and is entered using PCR primer as shown in table 4 Performing PCR amplification identification.
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 systems are 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 circulations;72℃10min.PCR is expanded 10 times of product dilution, by template of dilution the second wheel PCR is carried out, and 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 circulations;72℃5min.PCR amplifications are produced The agarose gel electrophoresiies of thing Jing 1% detect (result is shown in Fig. 2, shown in Fig. 4), choose with the 293T cell sample bands without transfection Compare PCR primer substantially less than normal to send to sequencing (Shanghai Major Biological Medical Technology Co., Ltd.) (result is shown in Fig. 3, Fig. 5 institutes Show).
Sequencing result is analyzed using Blast and is shown, 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 compositions can completely knock out miR-126 genome sequences in 293T cells, and pSpCas9 (BB)- 2A-Puro-sgRNA1 and pSpCas9 (BB) -2A-Puro-sgRNA3 combinations are deleted and can obtain the more of positive findingses, because This efficiency is more preferably.Other sgRNA combinations of pairs knock out the result for feminine gender, it is impossible to effectively knock out miR-126 in 293T cells Genome sequence.
Thus, pSpCas9 (BB) -2A-Puro-sgRNA1 and pSpCas9 (BB) -2A-Puro-sgRNA3 are selected, PSpCas9 (BB) -2A-Puro-sgRNA2 and pSpCas9 (BB) -2A-Puro-sgRNA5 are that miR-126 full-length genes knock out examination The sgRNA combinations of agent box;It is preferred that pSpCas9 (BB) -2A-Puro-sgRNA1 and pSpCas9 (BB) -2A-Puro-sgRNA3 groups Close.
(4) build miR-126 and knock out 293T cell models and qPCR test knockout 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 combinations of test kit, according to (1) the step of embodiment 2 --- (3) obtain being knocked out containing miR-126 293T cell lines, will have verified that containing miR-126 knock out 293T cell countings after, be diluted to 2/hole quantity be inoculated with 24 holes Plate, is placed in 37 DEG C, 5%CO2Incubator culture two weeks, period observes daily monoclonal growth conditions in orifice plate.After two weeks, such as Monoclonal grows into cell mass in orifice plate, takes cell in cell mass and enters performing PCR checking sequencing, sees whether the cell mass is knockout The 293T monoclonal cells group (the results detailed in shown in Fig. 6) of miR-126, PCR results show the band of 293T monoclonal cells group The PCR bands of control 293T cells are significantly less than, (Shanghai Mei Ji is biological to send sequencing by the PCR primer that 293T monoclonal cells are rolled into a ball Pharmaceutical Technology Co., Ltd, as a result as shown in Figure 7), sequencing result Jing Blast comparisons, miR-126 genomic coding sequences It is knocked completely in 293T monoclonal cells group (monoclonal is named as D2), will verifies that correct monoclonal cell group D2 expands Culture, the 293T cell models that so far miR-126 is knocked out are successfully constructed.
The 293T for successfully constructing is knocked out into miR-126 cytotostatics strain clone D2 amplification culture and QIAGEN is utilized MiRNeasy Mini Kit (QIAGEN, article No. 217004) carry out RNA (comprising 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 for having inverted is diluted 10 times by 95 DEG C of 5min, using primer as shown in table 8 qPCR is carried out.
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 systems such as table 9:
Table 9
95℃2min;94 DEG C of 15sec, 60 DEG C of 1min totally 40 circulations;Quantitative real time PCR Instrument SYBR passages during 60 DEG C of 1min Collection signal.After experiment terminates, software analysis experimental result is carried using quantitative real time PCR Instrument, obtained 293T monoclonals D2 ( Checking knocks out the stable strain of successful 239T monoclonals) miR-126 relative to itself U6 expression, and blank sample MiR-126 relative to itself U6 expression.MiR-126 relative expression quantities with blank sample are determined as standard Justice is 100% for expression efficiency, so as to calculate 293T monoclonal D2 in the expression efficiency of miR-126 be that 5.37% (result is such as Shown in Fig. 8), show using 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 builds 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 The cotransfection lung cell A549s of Lipofectamine 2000, after transfecting 24 hours, using complete containing 2 μ g/mL puromycin Full culture medium carries out medicine sieve, after medicine is sieved three days, collects cell.
2) activity checking is knocked out
A549 cells and the A549 cells without transfection after medicine sieve are collected, is tried using submicrosample genotype identification Agent box (Nanjing Yao Shunyu bio tech ltd, KC-101) carries out sample process, and is entered using PCR primer as shown in table 10 Performing PCR amplification identification.
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 systems 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 circulations;72℃10min.PCR is expanded 10 times of product dilution, by template of dilution the second wheel PCR, PCR system such as table 12 are carried out:
Table 12
94℃5min;94 DEG C of 30sec, 62 DEG C of 30sec, 72 DEG C of 2min, totally 35 circulations;72℃5min.PCR amplifications are produced The agarose gel electrophoresiies of thing Jing 1% detect, choose PCR substantially less than normal compared with the A549 cell sample bands without transfection and produce Thing sends to sequencing (Shanghai Major Biological Medical Technology Co., Ltd.).Sequencing result is analyzed using Blast and is shown, miR- 126 fragments are effectively knocked out in genome.
3) the lung cancer cell line A549 monoclonals that miR-126 is knocked out are separated
To have verified that the quantity for being diluted to 2/hole connects after the lung cancer cell line A549 cell countings containing miR-126 knockouts 24 orifice plates are planted, 37 DEG C is placed in, 5%CO2Incubator culture two weeks, period observes daily monoclonal growth conditions in orifice plate.Two Monoclonal grows into cell mass in Zhou Hou, such as orifice plate, takes cell in cell mass and enters performing PCR checking sequencing, whether sees the cell mass To knock out the A549 monoclonal cells group (the results detailed in shown in Fig. 9) of miR-126, PCR results show 6 monoclonal bars of A549 Band is significantly less than the PCR bands of control A549 cells, and the PCR primer of monoclonal A2 send sequencing in 6 monoclonals of random choose (Shanghai Major Biological Medical Technology Co., Ltd., as a result as shown in Figure 10), sequencing result Jing Blast comparisons, miR-126 It is knocked completely in A549 monoclonal A2, correct monoclonal cell group culture amplification culture will be verified, so far miR-126 strikes The A549 cell models for removing are successfully constructed.
Embodiment 4 builds 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 Cotransfection Human umbilical vein endothelial cells HUVEC of Lipofectamine 2000, after transfecting 24 hours, using containing 2 μ g/mL The complete medium of puromycin carries out medicine sieve, after medicine is sieved three days, collects cell.
2) activity checking is knocked out
HUVEC cells and the HUVEC cells without transfection after medicine sieve are collected, using submicrosample genotype identification Test kit (Nanjing Yao Shunyu bio tech ltd, KC-101) carries out sample process, and using PCR primer as shown in table 13 Enter performing 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 systems 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 circulations;72℃10min.PCR is expanded 10 times of product dilution, by template of dilution the second wheel PCR, PCR system such as table 15 are carried out:
Table 15
94℃5min;94 DEG C of 30sec, 62 DEG C of 30sec, 72 DEG C of 2min, totally 35 circulations;72℃5min.PCR amplifications are produced The detection of the agarose gel electrophoresiies of thing Jing 1%, chooses PCR substantially less than normal compared with the HUVEC cell sample bands without transfection Product sends to sequencing (Shanghai Major Biological Medical Technology Co., Ltd.).Sequencing result is analyzed using Blast and is shown, miR- 126 fragments are effectively knocked out in genome.
3) the HUVEC monoclonals that miR-126 is knocked out are separated
To have verified that after the HUVEC cell countings containing miR-126 knockouts, 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, period observes daily monoclonal growth conditions in orifice plate.After two weeks, such as hole Monoclonal grows into cell mass in plate, takes cell in cell mass and enters performing PCR checking sequencing, sees whether the cell mass is to knock out miR- 126 HUVEC monoclonal cells group (the results detailed in shown in Figure 11), PCR results show that the monoclonal cell group band of HUVEC is bright The aobvious PCR bands less than control HUVEC cells, by the PCR primer of HUVEC monoclonal cells group (being named as monoclonal H1) survey is sent Sequence (Shanghai Major Biological Medical Technology Co., Ltd., as a result as shown in figure 12), sequencing result Jing Blast comparisons, miR- 126 are knocked completely in HUVEC monoclonal cells group H1, will verify correct monoclonal cell group culture amplification culture, extremely The HUVEC cell models that this miR-126 is knocked out are successfully constructed.
Breast cancer cell T47D cell lines miR-126 of embodiment 5 are knocked out
1) breast cancer cell T47D cell transfectings
PSpCas9 (BB) -2A-Puro-sgRNA1, pSpCas9 (BB) -2A-Puro-sgRNA3 are utilized The cotransfection breast cancer cell T47D of Lipofectamine 2000, after transfecting 24 hours, utilize containing 2 μ g/mL puromycin Complete medium carries out medicine sieve, after medicine is sieved three days, collects cell.
2) activity checking is knocked out
T47D cells and the T47D cells without transfection after medicine sieve are collected, is tried using submicrosample genotype identification Agent box (Nanjing Yao Shunyu bio tech ltd, KC-101) carries out sample process, and using PCR primer as shown in table 16 Enter performing 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 systems 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 circulations;72℃10min.PCR is expanded 10 times of product dilution, by template of dilution the second wheel PCR, PCR system such as table 18 are carried out:
Table 18
94℃5min;94 DEG C of 30sec, 62 DEG C of 30sec, 72 DEG C of 2min, totally 35 circulations;72℃5min.PCR amplifications are produced The agarose gel electrophoresiies of thing Jing 1% detect, choose PCR substantially less than normal compared with the T47D cell sample bands without transfection and produce Thing (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 is analyzed and shows, miR-126 fragments are effectively knocked out in genome.
According to above-mentioned experimental result, present invention determine that for the sgRNA-1 and sgRNA-3 of miR-126 gene design; SgRNA-2 and sgRNA-5 is effective sgRNA combined sequence, wherein can be used as miR- comprising sgRNA-1, sgRNA3 plasmid combinations The Cas9 plasmids of the test kit that 126 full-length genes are knocked out.It is demonstrated experimentally that the test kit can effectively realize the clpp gene of cellular level Remove, and can be used for various kinds of cell system including vehicles cells system 293T, tumor cell line:Lung cancer cell line A549, breast cancer cell It is the gene knockout of cell line miR-126 such as T47D and endotheliocyte HUVEC systems.The miR-126 gene knockouts it is various thin Born of the same parents' model, can be used for vascular endothelial cell poorly differentiated, and Several Kinds of Malignancy is such as:The gene of the aspects such as breast carcinoma, pulmonary carcinoma Functional study, is that follow-up animal modeling is laid a good foundation, and is the instrument that the applied researcies such as further drug development are provided convenience.
Sequence table
<110>Shanghai Biotechnology Corporation
<120>A kind of miR-126 full-length genes based on CRISPR-Cas9 technologies knock out test kit and its application
<130>HJ16-12192
<160> 25
<170> PatentIn version 3.5
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<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 genes based on CRISPR-Cas9 technologies knock out test kit, it is characterised in that
The test kit is included by plasmid pSpCas9 (BB) -2A-Puro-sgRNA1 and plasmid pSpCas9 (BB) -2A-Puro- The sgRNA plasmid combinations of sgRNA3 compositions;Plasmid pSpCas9 (the BB) -2A-Puro-sgRNA1, containing one section of complementation DNA sequence, the DNA sequence can be transcribed into the target of miR-126 full-length gene of the specific recognition sequence as shown in SEQ ID NO.2 The sgRNA of mark sequence, the sgRNA can constitute special identification structure with trRNA, so that guiding Cas9 enzymes specifically shear miR- 126 gene pairss answer sequence;Plasmid pSpCas9 (the BB) -2A-Puro-sgRNA3, the DNA sequence containing one section of complementation should DNA sequence can be transcribed into the target sequence of miR-126 full-length gene of the specific recognition sequence as shown in SEQ ID NO.4 SgRNA, the sgRNA can constitute special identification structure with trRNA, so that guiding Cas9 enzymes specifically shear miR-126 genes Correspondence sequence;
Or, the test kit is included by plasmid pSpCas9 (BB) -2A-Puro-sgRNA2 and plasmid pSpCas9 (BB) -2A- The sgRNA plasmid combinations of Puro-sgRNA5 compositions;Plasmid pSpCas9 (the BB) -2A-Puro-sgRNA2, containing one section mutually The DNA sequence of benefit, the DNA sequence can be transcribed into miR-126 full-length gene of the specific recognition sequence as shown in SEQ ID NO.3 Target sequence sgRNA, the sgRNA can constitute special identification structure with trRNA, so that guiding Cas9 enzymes are specifically sheared MiR-126 gene pairss answer sequence;Plasmid pSpCas9 (the BB) -2A-Puro-sgRNA5, the DNA sequence containing one section of complementation, The DNA sequence can be transcribed into the target sequence of miR-126 full-length gene of the specific recognition sequence as shown in SEQ ID NO.6 SgRNA, the sgRNA can constitute special identification structure with trRNA, so that guiding Cas9 enzymes specifically shear miR-126 genes Correspondence sequence.
2. test kit as claimed in claim 1, it is characterised 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. test kit as claimed in claim 1, it is characterised 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. test kit as claimed in claim 1, it is characterised 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. test kit as claimed in claim 1, it is characterised 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. test kit as claimed in claim 1, it is characterised in that plasmid pSpCas9 (the BB) -2A-Puro-sgRNA1 and The sgRNA plasmid combinations of plasmid pSpCas9 (BB) -2A-Puro-sgRNA3 compositions, plasmid pSpCas9 (the BB) -2A- The sgRNA plasmid combinations of Puro-sgRNA2 and plasmid pSpCas9 (BB) -2A-Puro-sgRNA5 compositions are with including following step What rapid method built:
Step one, by target software design preferably a number of miR-126 upstream region of gene and downstream CRISPR-Cas9 targets Mark sequence, it is 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 sgRNA fragment of the synthesis for upstream and downstream target sequence, and is building up in plasmid vector; The sgRNA fragment sequences are 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 strains, sgRNA and trRNA constitutes special identification structure, so as to guide Cas9 enzymes special The corresponding sequence at strange land shearing miR-126 genes two ends, continues medicine sieve, obtains the cell strain of miR-126 full-length genes knockout;
Step 4, by extracting cell DNA, PCR amplifications, sequencing is determined and verifies, obtains the two groups of miR-126 bases for optimizing SgRNA plasmid combinations because knocking out CRISPR-Cas9, i.e., described plasmid pSpCas9 (BB) -2A-Puro-sgRNA1 and plasmid The sgRNA plasmid combinations of pSpCas9 (BB) -2A-Puro-sgRNA3 compositions, and the plasmid pSpCas9 (BB) -2A- The sgRNA plasmid combinations of Puro-sgRNA2 and plasmid pSpCas9 (BB) -2A-Puro-sgRNA5 compositions.
7. test kit as claimed in claim 6, it is characterised in that described to be building up in plasmid vector in step 2, concrete bag Include following steps:
(1) with pSpCas9 (BB) -2A-Puro plasmids as initial vector, single endonuclease digestion is carried out with Bbs I, linearized vector is carried out Purification;
(2) synthetic sgRNA fragments are diluted and is annealed;
(3) linearizing pSpCas9 (BB) -2A-Puro is connected with the sgRNA fragments after annealing using T4DNA ligases, will Whole connection products add the conversion of bacillus coli DH 5 alpha competent cell;
(4) single bacterium colony that the flat board of DH5 α competent cells bacterium solution coating after conversion grows is identified using bacterium colony PCR methods, Obtain expression plasmid.
8. application of the test kit as described in any one of claim 1-7 in cell line miR-126 gene knockout product is prepared.
9. application as claimed in claim 8, it is characterised in that the cell line includes vehicles cells system 293T, tumor cell System and endotheliocyte HUVEC systems.
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