CRISPR-Cas9 targeting knock outs human breast cancer cell RASSF2 genes and its specificitySgRNATechnical field
The invention belongs to genetic engineering field, more specifically, CRISPR-Cas9 targeting knock out human breast cancer cellsRASSF2 genes and its specific sgRNA.
Background technology
RASSF2 (ras association domain family 2), belongs to RASSF family members, the gene is located atHuman genome 20pl3, total length about 43.6kb, encoding proteins about 37.8kD, protein expression are mainly enriched in core.1996,Nagase etc. is found that 40 cDNA clone not being accredited fragments when studying mankind code area, then by this 40 transcriptionsOriginally it is named as KIAA0161200.Find to contain in its coded sequence in the research process for being named as one of them KIAA0168There are the familial RA domains of RASSF, be thus RASSF2 by this section of unnamed gene.
In human genome, necessary to the demethylation state on CpG islands is genetic transcription in promoter region.RASSF2Tumor suppression function forfeiture, CpG islands occur mainly due to RASSF2 gene promoter regions and methylate, causeRASSF2 transcriptional activity is reduced or lost.Ultimately resulting in RASSF2 tumor suppression function reduces.At present, research is found,In mankind tumor tissue, compared with normal structure, RASSF2 methylation level is apparently higher than normal structure, and in differenceTumor cell line in have also discovered same phenomenon.
Breast cancer is to threaten a big disease of women life and health, inquires into its pathogenesis and finds effectively prevention and treatment sideMethod is particularly important.Research shows that normal cell during to cancer cell and tumour cell transformation, modify by DNA methylationImportant effect, methylating for RASSF2 promoters is the main mechanism for losing tumor suppression function, and DNA methylation is in tumourEffect and Mechanism Study in occurrence and development are one of focuses of current biology field.Ground with reference to epigeneticsMechanism of action of the gene in breast cancer can more comprehensively be recognized with let us by studying carefully.
CRISPR/Cas9 be found in recent years by guide RNA Cas9 nucleases targeting target gene enter edlinEmerging technology.Using CRISPR-Cas9 targeting knock out RASSF2 genes, compared to traditional Zinc finger nuclease (ZFNs) technology,Transcriptional activation sample effector nuclease（TALENs）Technology etc., there is unique advantage：Design is simpler, and specificity is higher,Speed is fast, the system genitale transfer ability is strong and the characteristics of simple economy.
Bibliography：1)Sergio C,Bianca MC,Gabriella MF,et al.Isolation and molecularcharacterization of rasfadin,a novel gene in the vicinity of the bovine prionGene ［ J ］ .Mammalian Genome, 2001,12 (2):150 156.
2) Michele DV, Chad AE, Candice E, et al.RASS2 Is a Novel K Ras specificEffector and Potential Tumor Suppressor ［ J ］ .The Journal Of Biology Chemistry,2003, 278(30):28045-28051.
3) Michele DV, Chad AE, Aaron B, et al Ras uses the novel suppressor RASSF1As an effector to mediate apoptosis ［ J ］ .The Journal Of Biology Chemistry,2000, 275(46):35669-35672.
4) Anne BV, Channing JD.Increasing complexity of the Ras signalingPathway ［ J ］ J Biol Chem, 1998,273 (32): 19925-19928.
5) Guha S, Lunn JA, Santiskulvong C, et al. Neurotensin stimulates prot Ein kinase C dependent mitogenic signaling in human pancreatic carc inoma［ J ］ the Cancer Res of cell line PANC 1,2003,63 (10):2379-2387.
The content of the invention
The shortcomings that primary and foremost purpose of the present invention is to overcome prior art and deficiency, there is provided a kind of targeting knock out RASSF2 basesThe sgRNA of cause.
Another object of the present invention is to provide a kind of CRISPR/Cas9 slow virus of targeting knock out RASSF2 genesSystematic difference.
Another object of the present invention is to the human breast cancer cell MDA-MB-231 for providing targeting knock out RASSF2 genes is thinThe application of born of the same parents.
The purpose of the present invention is achieved through the following technical solutions：A kind of sgRNA of targeting knock out RASSF2 genes, is selected fromThe following RASSF2sgRNA of DNA sequence dna：
RASSF2sgRNA sequence is as follows：
RASSF2sgRNAoligo1：5’-cacc GCGTTGTCATCCTGCATCTGC-3’；
RASSF2sgRNAoligo2：5’-aaac GCAGATGCAGGATGACAACGC-3’；
A kind of CRISPR/Cas9 slow virus systems of targeting knock out RASSF2 genes, contain above-mentioned targeting knock out RASSF2 genesSgRNA DNA sequence dna.
The structure of the CRISPR/Cas9 slow virus systems of described targeting knock out RASSF2 genes, comprises the following steps：
（1）Using BsmBI digestion CRISPR/Cas9 slow virus carrier LentiCRISPRV2, the CRISPR/ after digestion is obtainedCas9 slow virus carriers；
（2）By after the sgRNA of above-mentioned targeting knock out RASSF2 genes DNA sequence dna phosphorylation with the CRISPR/Cas9 after digestionSlow virus carrier connects, and obtains the CRISPR/Cas9 slow virus systems of targeting knock out RASSF2 genes.
DNA sequence dna described in step (2) be by oligonucleotide chain 1 (oligo1) and oligonucleotide chain 2 (Oligo2) annealing obtains double-stranded sequence.
The CRISPR/Cas9 slow virus system of described targeting knock out RASSF2 genes is preparing knockout RASSF2 genesApplication in cell line.
A kind of cell line of knockout RASSF2 genes, it is by the CRISPR/Cas9 of described targeting knock out RASSF2 genesSlow virus system transfections aim cell strain obtains.
The cell line of described knockout RASSF2 genes, builds to obtain particular by following steps：
(1) the CRISPR/Cas9 slow virus systems of described targeting knock out RASSF2 genes are packed by incasing cells,Obtain lentiviral particle；
(2) lentiviral particle is infected into aim cell strain, obtains knocking out the cell line of RASSF2 genes.
Described aim cell strain is preferably tumor cell line.
Described tumor cell line is preferably breast carcinoma cell strain.
Described breast carcinoma cell strain is preferably human breast carcinoma cancer immortalized cells MDA-MB-231.
The cell line of described knockout RASSF2 genes, is RASSF2 gene delections or the insertion in MDA-MB-231 cellsThe cell line that nucleotides obtains.
The present invention is had the following advantages relative to prior art and effect：
The present invention provides the sgRNA energy efficient targeting RASSF2 genes of RASSF2 genes, and it is slow to be built into CRISPR/Cas9Virus system, the system can knock out RASSF2 genes, obtain knocking out the cell line of RASSF2 genes, so as to be advantageous to study cellThe mechanism of action of RASSF2 in strain.
Brief description of the drawings
Fig. 1 is the vector plasmid lentiCRISPR v2 used in the embodiment of the present invention plasmid map；
Fig. 2 is the expression vector plasmid lentiCRISPRv2-h RASSF2sg used in the embodiment of the present invention plasmid figureSpectrum；
Fig. 3 is the packaging plasmid pLP-VSVG used in the embodiment of the present invention plasmid map；
Fig. 4 is the packaging plasmid psPAX2 used in the embodiment of the present invention plasmid map；
Fig. 5 is that MDA-MB-231 cell virus infects the activity identification sequencer map containing RASSF2sgRNA；
Fig. 6 is the sequencer map of RASSF2 genes in the monoclonal MDA-MB-231 cell lines 1 for knock out RASSF2 genes.
Embodiment
With reference to embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are unlimitedIn this.
Embodiment 1
1. knock out RASSF2 plasmids using CRISPR/Cas9 technique constructions
1.1 sgRNA oligonucleotide chains synthesize
Use CRISPR Photographing On-line instruments (http://crispr .mit .edu/) according to points-scoring system, RASSF2's1 20bp sgRNA is designed on exon 2, and is verified by BLAST without non-specific gene.The addition of the end of coding strand template 5 'The cohesive end formed after CACC, the addition of the end of noncoding strand template 3 ' AAAC, with BsmBI digestions is complementary, designs 1 couple of CRISPR widowNucleotide chain, is shown in Table 1, the RASSF2 target sites of table 1 and sgRNA oligonucleotide sequences.
1.2 vector construction
1.2.1 the μ g lentiCRISPRv2 plasmids (being purchased from Addgene companies) of BsmBI digestions 2 are used, 2h, 37 DEG C, digestion systemIt the following is following table：
1.2.2 GENRY glue reclaim kits digested plasmid products are used, by specification is operated, under enzyme Chain System isTable：
1.2.3 phosphorylation and the sgRNAoligos that anneals, PCR instrument device cycle of annealing：37 DEG C of 30min, 95 DEG C maintenance 5min, every pointClock reduces by 5 DEG C to 25 DEG C, 4 DEG C of maintenances.
1.2.4 the lentiCRISPRv2 carriers after anneal the oligo double-strands formed and digestion are directly connected to, at room temperature,10min。
1.2.5 the plasmid after connection is converted into competent cell DH5 α, is uniformly applied in LB solid medium flat boards,Culture 12-16 hours in 37 DEG C of incubators are placed in, single bacterium colony may occur in which.
The expansion of 1 .3 pickings single bacterium colony, which is cultivated, and plasmid is small carries.
The success of 1.4 sequencing identification plasmid constructions, and it is named as lentiCRISPRv2-h RASSF2sg.
1.5 knock out efficiency checking.
1) 5%CO2 is based on the DMEM in high glucose culture containing 10% hyclone, 37 DEG C of incubated 293T cells (are purchased fromU.S.'s ATCC cell banks).Phase cell of taking the logarithm is inoculated into 24 orifice plate cultures with 1 × 105/ hole.Treat that cell fusion degree reaches 70%Opti-MEM culture mediums are replaced with during~8 0 %, corresponding CRISPR/Cas9 is knocked out into the μ g of plasmid 0.8 warps after 1 hourLipo2000 reagents, it is transfected into 293T cells, as a child, fluorescence microscopy Microscopic observation transfection, digestion is collected for transfection 48Cell, cell genomic dna is extracted using genome DNA extracting reagent kit.
2) using each group cell genomic dna as template, the primer PCR amplification targeting sequence of table 2 is utilized.Use RASSF2-4-1Enter performing PCR with RASSF2-4-2,936bp fragment a (high GC) is obtained, using a as template, with RASSF2-4-3 and RASSF2-4-4Enter performing PCR, obtain 405bp fragment b (high GC), glue reclaim is used for sequencing primer RASSF2-4-3 after the fragment electrophoretic, is shown in Table 2
2, pack slow virus
100mm ware kinds enter 293TN cells 4X106, after cultivating 24h (37 DEG C, 5%CO2), change fresh culture, transfectionIncasing cells：
1）Mix following 3 plasmids：The μ g of packaging plasmid psPAX2 12；The μ g of helper plasmid pLP-VSVG 10；Expression plasmidThe μ g of lentiCRISPRv2- h RASSF2sg 22, add CaCl2250 μ l, add ddH2O is 500 μ l to cumulative volume, and 37 DEG C putPut 20min；
2）2 X BES 500 μ l, 37 DEG C of placement 20min；
3）1）In the plasmid mixed liquors of 500 μ l tri- be added dropwise 2）In, and gently mix；
4）It is careful by 3）Middle mixed liquor, which adds kind, to be had in 293TN 100mm wares；Infection liquid is removed after cultivating 12h, is changed10ml fresh cultures produce virus liquid；After cultivating 48h, virus liquid is harvested into 50ml centrifuge tubes, and again to 100mm waresIn add 10ml culture mediums and produce virus again；After cultivating 24h, virus liquid, room temperature centrifugation, 1000rpm, 10min are harvested again；Use 0.22 μm of filter filter virus supernatant；Vial supernatant is fitted into and surpassed from pipe, 4 DEG C, 100000g, 2h；DiscardIt is resuspended overnight with Opti-MEM after clear liquid；Reuse the virus that 0.22 μm of filter filtering is resuspended；After packing, -80 DEG C of preservations.
3. virus infection
1) the MDA-MB-231 cells that will be infected are inoculated into six orifice plates, overnight, when cell fusion degree is about 50%, are removedCulture medium, change 2ml fresh cultures；
2) 50 μ l virus liquids are added per hole, add 12 μ l polybrene polybrene, to final concentration of 6 μ g/ml；
3) after 37 DEG C of culture 12h, nutrient solution is removed, continues to cultivate 48h after changing fresh medium.
4, screen stable cell line
After infection terminates 48h, puromycin (2 .0 μ g/ml) is added to every hole, changes liquid every other day, and keep the purine of culture mediumMycin constant concentration, screening positive clone cell.Gained cell line is named as MDA-MB-231- RASSF2.And using limited diluteInterpretation of the law selects MDA-MB-231- RASSF2 monoclonals and knocks out cell line.
5, stable cell lines are identified
The monoclonal cell strain genomic DNA sequencing that each group knocks out RASSF2 is extracted, it is active to identify sgRNA（Fig. 5）,And find the stable cell line (Fig. 6) with RASSF2 gene delections or insertion mutation of 1 type.
Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention are not by above-described embodimentLimitation, other any Spirit Essences without departing from the present invention with made under principle change, modification, replacement, combine, simplification,Equivalent substitute mode is should be, is included within protection scope of the present invention.
Sequence table
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