CN107893076A - CRISPR Cas9 targeting knock outs human breast cancer cell RASSF2 genes and its specific sgRNA - Google Patents

CRISPR Cas9 targeting knock outs human breast cancer cell RASSF2 genes and its specific sgRNA Download PDF

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CN107893076A
CN107893076A CN201711179781.5A CN201711179781A CN107893076A CN 107893076 A CN107893076 A CN 107893076A CN 201711179781 A CN201711179781 A CN 201711179781A CN 107893076 A CN107893076 A CN 107893076A
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rassf2
genes
sgrna
cas9
crispr
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马佩敏
孙子豪
杨兴林
潘讴东
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Yuan Biotechnology (shanghai) Ltd By Share Ltd
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Yuan Biotechnology (shanghai) Ltd By Share Ltd
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Abstract

The invention discloses CRISPR/Cas9 targeting knock outs human breast cancer cell RASSF2 genes and its specific sgRNA.It is the sgRNA for obtaining selectively targeted RASSF2 genes first, its base sequence is as shown in SEQ ID NO.1；Next to that the sgRNA of structure RASSF2 genes contains Cas9 albumen to slow virus carrier system, the system；Finally by the CRISPR/Cas9 slow-virus infection human breast cancer cell MDA MB 231 containing the sgRNA, the significantly reduced cell line of RASSF2 protein expression levels is obtained.The present invention has that operating procedure is simple, sgRNA targetings are good and high to RASSF2 genes cutting efficiency；In addition, constructed CRISPR/Cas9 slow virus system has the advantage for knocking out efficiency high, and energy specific knockdown RASSF2 genes, obtain knocking out the human breast cancer cell of RASSF2 genes, so as to provide strong instrument further to study the mechanism of action of RASSF2 in breast cancer cell.

Description

CRISPR-Cas9 targeting knock outs human breast cancer cell RASSF2 genes and its specificity SgRNA
Technical field
The invention belongs to genetic engineering field, more specifically, CRISPR-Cas9 targeting knock out human breast cancer cells RASSF2 genes and its specific sgRNA.
Background technology
RASSF2 (ras association domain family 2), belongs to RASSF family members, the gene is located at Human 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 transcriptions Originally it is named as KIAA0161200.Find to contain in its coded sequence in the research process for being named as one of them KIAA0168 There 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.RASSF2 Tumor suppression function forfeiture, CpG islands occur mainly due to RASSF2 gene promoter regions and methylate, cause RASSF2 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 difference Tumor 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 side Method is particularly important.Research shows that normal cell during to cancer cell and tumour cell transformation, modify by DNA methylation Important effect, methylating for RASSF2 promoters is the main mechanism for losing tumor suppression function, and DNA methylation is in tumour Effect and Mechanism Study in occurrence and development are one of focuses of current biology field.Ground with reference to epigenetics Mechanism 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 edlin Emerging 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 molecular characterization of rasfadin,a novel gene in the vicinity of the bovine prion Gene ［ J ］ .Mammalian Genome, 2001,12 (2):150  156.
2) Michele DV, Chad AE, Candice E, et al.RASS2 Is a Novel K  Ras  specific Effector 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 RASSF1 As 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 signaling Pathway ［ 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 bases The sgRNA of cause.
Another object of the present invention is to provide a kind of CRISPR/Cas9 slow virus of targeting knock out RASSF2 genes Systematic 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 thin The 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 from The 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 genes SgRNA 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 obtained Cas9 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 digestion Slow 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 genes Application in cell line.
A kind of cell line of knockout RASSF2 genes, it is by the CRISPR/Cas9 of described targeting knock out RASSF2 genes Slow 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 cells The 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/Cas9 Virus system, the system can knock out RASSF2 genes, obtain knocking out the cell line of RASSF2 genes, so as to be advantageous to study cell The 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 figure Spectrum；
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 unlimited In 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's 1 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 widow Nucleotide 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 system It 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 is Table：
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 point Clock 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 from U.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 hour Lipo2000 reagents, it is transfected into 293T cells, as a child, fluorescence microscopy Microscopic observation transfection, digestion is collected for transfection 48 Cell, 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 RASSF2-4-1 Enter performing PCR with RASSF2-4-2,936bp fragment a (high GC) is obtained, using a as template, with RASSF2-4-3 and RASSF2-4-4 Enter 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, transfection Incasing cells：
1）Mix following 3 plasmids：The μ g of packaging plasmid psPAX2 12；The μ g of helper plasmid pLP-VSVG 10；Expression plasmid The μ g of lentiCRISPRv2- h RASSF2sg 22, add CaCl2250 μ l, add ddH2O is 500 μ l to cumulative volume, and 37 DEG C put Put 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 changed 10ml fresh cultures produce virus liquid；After cultivating 48h, virus liquid is harvested into 50ml centrifuge tubes, and again to 100mm wares In 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；Discard It 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 removed Culture 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 medium Mycin constant concentration, screening positive clone cell.Gained cell line is named as MDA-MB-231- RASSF2.And using limited dilute Interpretation 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 embodiment Limitation, 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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Claims (5)

1. a kind of CRISPR/Cas9 targeting knock outs human breast cancer cell RASSF2 genes and its specific sgRNA, its feature It is, design first obtains the sgRNA of selectively targeted RASSF2 genes；Secondly the sgRNA of structure RASSF2 genes is to slow sick Poisonous carrier system；Then this slow virus carrier system is infected into human breast cancer cell MDA-MB-231, obtained RASSF2 genes Knock out cell line.
2. CRISPR/Cas9 targeting knock outs human breast cancer cell RASSF2 genes according to claim 1 and its special The sgRNA of property, it is characterised in that comprise the following steps：
(1) sgRNA is provided, target sequences of the sgRNA on RASSF2 genes meets 5 '-N (19) G series arrangement rule Then, target sequences of the sgRNA on RASSF2 genes is located at the extron of gene, and the sgRNA is on RASSF2 genes Target sequence be located on the common exon of different various shear patterns, target sequences of the sgRNA on RASSF2 genes It is unique, and target site sequences of the sgRNA on RASSF2 be as shown in sequence table SEQ ID NO.1 sequences, The 5 ' of target site sequences of the sgRNA on RASSF2 synthesize to obtain positive oligonucleotides i.e. Forward plus CACCG oligo ；The complementary strand of target site sequences of the sgRNA on RASSF2 is obtained, and AAAC is added the 5 ' of complementary strand, 3 ' mend C, synthesis obtain reverse oligonucleotide i.e. Reverse oligo；By the complementary sgRNA oligonucleotides of 1 couple of synthesis Forward oligo and reverse oligo are denatured, annealed in pairs, and U6 eukaryotic expressions load can be connected into by being formed after annealing The double-strand sgRNA oligonucleotides of body；
(2) lentiCRISPRv2 plasmid of the sequence as shown in sequence table SEQ ID NO.10 is linearized；By the double-strand of annealing The expression vector lentiCRISPRv2 of carrying Cas9 gene of the sgRNA oligonucleotides with linearizing is connected acquisition carrying and contained The corresponding sgRNA oligonucleotides of target sequence and the expression vector lentiCRISPRv2-h RASSF2sg matter of Cas9 genes Grain, transformed competence colibacillus bacterium simultaneously apply Amp+ flat boards, and picking monoclonal is simultaneously logical as shown in sequence table SEQ ID NO.9 with sequence Positive colony is gone out by sequencing identification with primer U6,37 DEG C of shaking tables shake positive colony bacterium and stayed overnight, with article No. AP-MN-P-250's AxyPrep Plasmid Miniprep Kit extract plasmid；
（3）With the expression vector lentiCRISPRv2-h for carrying sgRNA oligonucleotides and Cas9 genes RASSF2sg plasmids, pVSVg (AddGene 8454) and that sequence is SEQ ID NO.11 and SEQ ID NO.12 PsPAX2 (AddGene 12260) packaging plasmids and package cell line are packed out while carry the sgRNA of targeting RASSF2 genes With Cas9 false type slow virus；
（4）Using the false type slow-virus infection aim cell, and further cultivate；Then infected aim cell is collected, The genetic fragment of the target sequence, T7EN1 digestions detection and TA cloning and sequencings are included using its genomic DNA as template amplification Confirm that RASSF2 genes have been knocked and have obtained the cell of gene knockout.
3. being used in the method for CRISPR-Cas9 specific knockdown people's RASSF2 genes according to claim 1 Recombinant expression carrier lentiCRISPR v2- RASSF2, it is characterised in that the sequence of the skeleton carrier of the recombinant expression carrier SEQ ID NO in row such as sequence table：Shown in 10.
4. the method for CRISPR-Cas9 specific knockdown people's RASSF2 genes according to claim 1, its feature exist In the packaging plasmid is pVSVg (AddGene 8454) and psPAX2 (AddGene 12260)；The packaging is thin Born of the same parents system is HEK293TN cells.
5. the method for CRISPR-Cas9 specific knockdown people's RASSF2 genes according to claim 1, its feature exist In the aim cell behaviour MDA-MB-231 cells.
CN201711179781.5A 2017-11-23 2017-11-23 CRISPR Cas9 targeting knock outs human breast cancer cell RASSF2 genes and its specific sgRNA Pending CN107893076A (en)

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CN108642053A (en) * 2018-04-28 2018-10-12 和元生物技术（上海）股份有限公司 The sgRNA of CRISPR-Cas9 targeting knock out people colon-cancer cell PPP1R1C genes and its specificity
US10113163B2 (en) 2016-08-03 2018-10-30 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US10323236B2 (en) 2011-07-22 2019-06-18 President And Fellows Of Harvard College Evaluation and improvement of nuclease cleavage specificity
US10465176B2 (en) 2013-12-12 2019-11-05 President And Fellows Of Harvard College Cas variants for gene editing
US10508298B2 (en) 2013-08-09 2019-12-17 President And Fellows Of Harvard College Methods for identifying a target site of a CAS9 nuclease
US10597679B2 (en) 2013-09-06 2020-03-24 President And Fellows Of Harvard College Switchable Cas9 nucleases and uses thereof
US10682410B2 (en) 2013-09-06 2020-06-16 President And Fellows Of Harvard College Delivery system for functional nucleases
US10704062B2 (en) 2014-07-30 2020-07-07 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US10745677B2 (en) 2016-12-23 2020-08-18 President And Fellows Of Harvard College Editing of CCR5 receptor gene to protect against HIV infection
US10858639B2 (en) 2013-09-06 2020-12-08 President And Fellows Of Harvard College CAS9 variants and uses thereof
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US11542509B2 (en) 2016-08-24 2023-01-03 President And Fellows Of Harvard College Incorporation of unnatural amino acids into proteins using base editing
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US11661590B2 (en) 2016-08-09 2023-05-30 President And Fellows Of Harvard College Programmable CAS9-recombinase fusion proteins and uses thereof
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US11912985B2 (en) 2020-05-08 2024-02-27 The Broad Institute, Inc. Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence

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Application publication date: 20180410