CN108148835A - The sgRNA of CRISPR-Cas9 targeting knock out SLC30A1 genes and its specificity - Google Patents

The sgRNA of CRISPR-Cas9 targeting knock out SLC30A1 genes and its specificity Download PDF

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CN108148835A
CN108148835A CN201711282037.8A CN201711282037A CN108148835A CN 108148835 A CN108148835 A CN 108148835A CN 201711282037 A CN201711282037 A CN 201711282037A CN 108148835 A CN108148835 A CN 108148835A
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slc30a1
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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Abstract

The invention discloses CRISPR/Cas9 targeting knock outs human breast cancer cell SLC30A1 genes and its specificity sgRNA.It is the sgRNA for obtaining selectively targeted SLC30A1 genes first, base sequence is as shown in SEQ ID NO.1；Secondly the sgRNA of structure SLC30A1 genes, to slow virus carrier system, which contains Cas9 albumen；Finally by the CRISPR/Cas9 slow-virus infection human breast cancer cell MDA MB 231 containing the sgRNA, the significantly reduced cell strain of SLC30A1 protein expression levels is obtained.The present invention has that operating procedure is simple, sgRNA targetings are good and to SLC30A1 genes cutting efficiency height；In addition, constructed CRISPR/Cas9 slow virus system, which has, knocks out efficient advantage, and energy specific knockdown SLC30A1 genes, obtain knocking out the human breast cancer cell of SLC30A1 genes, so as to provide strong tool for the mechanism of action for further studying SLC30A1 in breast cancer cell.

Description

The sgRNA of CRISPR-Cas9 targeting knock out SLC30A1 genes and its specificity
Technical field
The invention belongs to genetic engineering field, more specifically to CRISPR-Cas9 specific knockdown people SLC30A1 The method of gene and the sgRNA for selectively targeted SLC30A1 genes.
Background technology
SLC30A1 (zinc operating body) member 1 be a kind of protein in the mankind by SLC30A1 encoding genes.Required micro member Plain zinc participates in a variety of enzymes of body and protein function by catalysis and structure function, with body development, brain function, bone growth, life It is closely related to grow health and immune function etc..Supplement zinc can prevent to a certain degree children's diarrhae, chronic hepatitis C, it is acute under The diseases such as respiratory tract infection and flu, however excessive zinc has toxicity.Therefore, there is complicated zinc ion stable state body in body It is the equilibrium process of the absorption for maintaining zinc ion, storage and loss.SLC30A families are also known as ZnT families, share 10 members, more A family member can assist zinc ion to flow out to extracellular or flow into organelle out of cytoplasm.Research prompting ZnT1, ZIP4 and ZIP5 participation small intestine zinc ion absorption processes, ZIP10 and ZnT1 participation kidney zinc ion resorption process, ZIP5, ZnT2 and ZnT1 participates in pancreas zinc ion secretion loss process.Separately prove that the albumen of SLC30A families may also participate in perhaps on evidence More diseases include the occurrence and development of tumour and diabetes.
Breast cancer is to threaten a big disease of women life and health, inquires into its pathogenesis and finds the side of being effectively prevented and treated Method is particularly important.Zinc horizontal abnormality in breast cancer increases, specific transmembrane transport protein zinc transporter and and metal matrix This known tumor correlated albumen of protease has similar structure, in addition two zinc transhipment in LIV-1 estrogen-regulated genes Body member ZIP6 and ZIP10 and the transfer of breast cancer have it is very close be associated with, therefore, inquire into the different breast of estrogen receptor character The influence that the expression of zinc transporter and zinc express zinc transporter in adenocarcinoma cell MDA-MB-231 is necessary.
CRISPR/Cas9 is that the Cas9 nucleases by guide RNA found in recent years target target gene into edlin Emerging technology.CRISPR/Cas9 has found in archeobacteria at first, is that researcher has found archeobacteria to adventive information not Break and attack and develop a kind of acquired defense mechanism come in CRISPR/Cas9 systems, crRNA (CRISPR- DerivedRNA it) is combined by DNA base pairing mechanism and tracrRNA (trans-activating RNA) and forms double-strand RNA instructs Cas9 albumen to target purpose site cut-out double-stranded DNA in the DNA sequence dna of guide crRNA.Compared to traditional zinc finger Nuclease (ZFNs) technology, transcriptional activation sample effector nuclease（TALENs）Technology etc., CRISPR-Cas technologies have only Special advantage：Simpler, specific higher is designed, speed is fast, the system genitale transfer ability is strong and the characteristics of simple economy.
Bibliography：
1)Moonhuhn Ryu, Louis A. Lichten, Juan P. Liuzzi, et al. Zinc Transporters ZnT1 (Slc30a1), Zip8 (Slc39a8), and Zip10 (Slc39a10) in Mouse Red Blood Cells Are Differentially Regulated during Erythroid Development and by Dietary Zinc Deficiency[J]. Journal of Nutrition, 2008, 138(11):2076-2083.
2)Fernandez E L, Dencker L, Tallkvist J. Expression of ZnT-1 (Slc30a1) and MT-1 (Mt1) in the conceptus of cadmium treated mice[J]. Reproductive Toxicology, 2007, 24(3–4):353-358.
3)Muraina, Issa. Reverse genetics analysis of biological functions of zinc transporters Znt1 (Slc30a1) & Zip10 (Slc39a10) in zebrafish[J]. Kings College London, 2013。
Invention content
The shortcomings that primary and foremost purpose of the present invention is to overcome the prior art and deficiency, provide a kind of targeting knock out SLC30A1 The sgRNA of gene.
It is sick slowly another object of the present invention is to provide a kind of CRISPR/Cas9 of targeting knock out SLC30A1 genes Malicious systematic difference.
Another object of the present invention is to provide people's colon-cancer cell MDA-MB-231 cells of targeting knock out SLC30A1 genes Application.
The purpose of the present invention is achieved through the following technical solutions：A kind of sgRNA of targeting knock out SLC30A1 genes, is selected from The following SLC30A1sgRNA of DNA sequence dna：
The sequence of SLC30A1sgRNA is as follows：
SLC30A1sgRNAoligo1：5’-cacc GCTCTTAACGCGAGGCCCCT-3’；
SLC30A1sgRNAoligo2：5’-aaac AGGGGCCTCGCGTTAAGAGC-3’；
A kind of CRISPR/Cas9 slow virus systems of targeting knock out SLC30A1 genes, contain above-mentioned targeting knock out SLC30A1 bases The DNA sequence dna of the sgRNA of cause.
The structure of the CRISPR/Cas9 slow virus systems of the targeting knock out SLC30A1 genes, includes 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 DNA sequence dna phosphorylation of the sgRNA of above-mentioned targeting knock out SLC30A1 genes with the CRISPR/Cas9 after digestion Slow virus carrier connects, and obtains the CRISPR/Cas9 slow virus systems of targeting knock out SLC30A1 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 the targeting knock out SLC30A1 genes is preparing knockout RITA genes Application in cell strain.
A kind of cell strain for knocking out SLC30A1 genes, is by the CRISPR/ of the targeting knock out SLC30A1 genes Cas9 slow virus system transfections aim cell strains obtain.
The cell strain of the knockout SLC30A1 genes, builds to obtain particular by following steps：
1）The CRISPR/Cas9 slow virus systems of the targeting knock out SLC30A1 genes are packed by incasing cells, Obtain lentiviral particle；
2）Lentiviral particle is infected into aim cell strain, obtains knocking out the cell strain of SLC30A1 genes.
The aim cell strain is preferably tumor cell line.
The tumor cell line is preferably breast carcinoma cell strain.
The breast carcinoma cell strain is preferably human breast carcinoma cancer immortalized cells MDA-MB-231.
The cell strain of the knockout SLC30A1 genes is the SLC30A1 gene delections or slotting in MDA-MB-231 cells Enter the cell strain that nucleotide obtains.
The present invention is had the following advantages relative to the prior art and effect：
The present invention provides the sgRNA energy efficient targeting SLC30A1 genes of SLC30A1 genes, is built into CRISPR/Cas9 Slow virus system, the system can knock out SLC30A1 genes, obtain knocking out the cell strain of SLC30A1 genes, so as to be conducive to study The mechanism of action of SLC30A1 in cell strain.
Description of the drawings
Fig. 1 is the plasmid map of vector plasmid lentiCRISPR v2 used in the embodiment of the present invention；
Fig. 2 is the plasmid figure of expression vector plasmid lentiCRISPRv2-hSLC30A1sg used in the embodiment of the present invention Spectrum；
Fig. 3 is the plasmid map of packaging plasmid pLP-VSVG used in the embodiment of the present invention；
Fig. 4 is the plasmid map of packaging plasmid psPAX2 used in the embodiment of the present invention；
Fig. 5 infects the activity identification sequencer map containing SLC30A1sgRNA for MDA-MB-231 cell virus；
Fig. 6 is the sequencer map of SLC30A1 genes in the monoclonal MDA-MB-231 cell strains 1 for knock out SLC30A1 genes；
Fig. 7 is the sequencer map of SLC30A1 genes in the monoclonal MDA-MB-231 cell strains 2 for knock out SLC30A1 genes；
Fig. 8 is the sequencer map of SLC30A1 genes in the monoclonal MDA-MB-231 cell strains 3 for knock out SLC30A1 genes；
Fig. 9 is the sequencer map of SLC30A1 genes in the monoclonal MDA-MB-231 cell strains 4 for knock out SLC30A1 genes.
Specific embodiment
With reference to embodiment and attached drawing, the present invention is described in further detail, but embodiments of the present invention are unlimited In this.
Embodiment 1
1, knocks out SLC30A1 plasmids using CRISPR/Cas9 technologies structure
1 .1sgRNA oligonucleotide chains synthesize
Use CRISPR Photographing On-line tools (http://crispr .mit .edu/) according to points-scoring system, in SLC30A1 Exon 2 on design 1 20bp sgRNA, and by BLAST verify without non-specific gene.The end of coding strand template 5 ' adds Add CACC, the cohesive end formed after the addition of the end of noncoding strand template 3 ' AAAC, with BsmBI digestions is complementary, designs 1 couple of CRISPR Oligonucleotide chain is shown in Table 1 SLC30A1 target sites and sgRNA oligonucleotide sequences
1 .2 vector constructions
1.2.1 using 2 μ g lentiCRISPRv2 plasmids (being purchased from Addgene companies) of BsmBI digestions, 2h, 37 DEG C of
1.2.2 digested plasmid product is purified using GENRY plastic recovery kits, by specification is operated
1.2.3 phosphorylation and the sgRNAoligos that anneals：
PCR instrument cycle of annealing：37 DEG C of 30min, 95 DEG C of maintenance 5min, per minute to reduce 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 tablets, It is placed in 37 DEG C of incubators and cultivates 12-16 hours, single bacterium colony may occur in which.
The expansion of 1 .3 pickings single bacterium colony, which is cultivated, and plasmid is small carries.
1.4 sequencing identification plasmid construction successes, and it is named as lentiCRISPRv2-hSLC30A1sg.
1.5 knock out efficiency verification
5%CO2 is based on the DMEM in high glucose culture containing 10% fetal calf serum, 37 DEG C of constant temperature incubation 293T cells (are purchased from the U.S. ATCC cell banks).Phase cell take the logarithm with 1 × 105/ hole is inoculated into 24 orifice plate cultures.Treat that cell fusion degree reaches 70%~8 0 Opti-MEM culture mediums are replaced with during %, corresponding CRISPR/Cas9 is knocked out into 0.8 μ g of plasmid through Lipo2000 after 1 hour Reagent is transfected into 293T cells, and as a child, cell is collected in fluorescence microscopy Microscopic observation transfection, digestion for transfection 48, is used Genome DNA extracting reagent kit extracts cell genomic dna;Using each group cell genomic dna as template, the primer of table 2 is utilized PCR amplification targets sequence.PCR is carried out with SLC30A1-4-1 and SLC30A1-4-2, the segment a (high GC) of 904bp is obtained, with a For template, PCR is carried out with SLC30A1-4-3 and SLC30A1-4-4, obtains the segment b (high GC) of 400bp, after the fragment electrophoretic Glue recycling is for sequencing primer SLC30A1-4-3, table 2
2, pack slow virus
100mm ware kinds enter 293TN cells 4X106, for 24 hours after (37 DEG C, 5%CO2), replacement fresh culture transfects for culture Incasing cells：
3 plasmids below a mixings：
12 μ g of packaging plasmid psPAX2
10 μ g of helper plasmid pLP-VSVG
22 μ g of expression plasmid lentiCRISPRv2- hSLC30A1sg
Add in CaCl2 250μl
Add in ddH2O is 500 μ l to total volume, and 37 DEG C are placed 20min；
B .2 X BES 500 μ l, 37 DEG C of placement 20min；
C 500 μ l in a, tri- plasmid mixed liquors are added dropwise in b, and gently mixing；
D. it is careful that mixed liquor addition kind in c has in the 100mm wares of 293TN；Infection liquid is removed after cultivating 12h, replaces 10ml Fresh culture produces virus liquid；After cultivating 48h, harvest in virus liquid to 50ml centrifuge tubes, and mended again into 100mm wares 10ml culture mediums is added to produce virus again；After culture for 24 hours, virus liquid, room temperature centrifugation, 1000rpm, 10min are harvested again；It uses 0.22 μm of filter filter virus supernatant；Vial supernatant is fitted into and is surpassed from pipe, 4 DEG C, 100000g, 2h；Discard supernatant liquid It is resuspended overnight with Opti-MEM afterwards；Reuse the virus that 0.22 μm of filter filtering is resuspended；After packing, -80 DEG C of preservations.
3, viruses infect
1) it in the MDA-MB-231 cell inoculations to six orifice plates that will be infected, overnight, when cell fusion degree is about 50%, moves Culture medium is removed, replaces 2ml fresh cultures；
2) 50 μ l virus liquids are added in per hole, 12 μ l polybrene polybrene are added, until final concentration of 6 μ g/ml；
3) after 37 DEG C of culture 12h, culture solution is removed, continues to cultivate 48h after replacing fresh medium.
4, screen stable cell line
After infection terminates 48h, puromycin (2 .0 μ g/ml) is added in every hole, changes liquid every other day, and keep the purine of culture medium Mycin constant concentration, screening positive clone cell, gained cell strain are named as MDA-MB-231-SLC30A1, and using limited dilute Interpretation of the law selects MDA-MB-231-SLC30A1 monoclonals and knocks out cell strain.
5, stable cell lines are identified
The monoclonal cell strain genomic DNA sequencing that each group knocks out SLC30A1 is extracted, it is active to identify sgRNA（Figure 5）, and find the stable cell line (Fig. 6-9) with SLC30A1 gene delections or insertion mutation of 4 types.
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. the sgRNA of a kind of CRISPR/Cas9 targeting knock outs human breast cancer cell SLC30A1 genes and its specificity, feature It is, design first obtains the sgRNA of selectively targeted SLC30A1 genes；Secondly the sgRNA of structure SLC30A1 genes is to slowly Virus carrier system；Then this slow virus carrier system is infected into human breast cancer cell MDA-MB-231, obtained SLC30A1 bases Because knocking out cell strain.
2. CRISPR/Cas9 targeting knock outs human breast cancer cell SLC30A1 genes according to claim 1 and its special The sgRNA of property, it is characterised in that include the following steps：
(1) sgRNA is provided, target sequences of the sgRNA on SLC30A1 genes meets the series arrangement rule of 5 '-N (19) G Then, target sequences of the sgRNA on SLC30A1 genes is located at the extron of gene, and the sgRNA is in SLC30A1 genes On target sequence be located on the common exon of different various shear patterns, targets of the sgRNA on SLC30A1 genes Sequence is unique, and target site sequences of the sgRNA on SLC30A1 such as sequence table SEQ ID NO.1 sequences institute Show, in target site sequences of the sgRNA on SLC30A1 5 ' plus CACC, synthesis obtains positive oligonucleotides i.e. Forward oligo ；The complementary strand of target site sequences of the sgRNA on SLC30A1 is obtained, and 5 ' in complementary strand add AAAC synthesizes to obtain reverse oligonucleotide i.e. Reverse oligo；By the complementary sgRNA oligonucleotides of 1 couple of synthesis Forward oligo and reverse oligo be denaturalized, anneal in pairs, annealing after formed can be connected into U6 eukaryotic expressions The double-strand sgRNA oligonucleotides of carrier；
(2) lentiCRISPRv2 plasmid of the linearisation sequence as shown in sequence table SEQ ID NO.10；By the double-strand of annealing The expression vector lentiCRISPRv2 of carrying Cas9 gene of the sgRNA oligonucleotides with linearizing, which connect acquisition and carries, to be contained The corresponding sgRNA oligonucleotides of target sequence and the expression vector lentiCRISPRv2-h SLC30A1sg matter of Cas9 genes Grain, transformed competence colibacillus bacterium simultaneously apply Amp+ tablets, and picking monoclonal is simultaneously logical as shown in sequence table SEQ ID NO.9 with sequence Positive colony is identified by sequencing with primer U6, and bacterium, extraction plasmid are shaken to the positive colony；
（3）With the expression vector lentiCRISPRv2-h for carrying sgRNA oligonucleotides and Cas9 genes SLC30A1sg 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 DEAF1 genes With the false type slow virus of Cas9；
（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 SLC30A1 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 SLC30A1 genes according to claim 1 Recombinant expression carrier lentiCRISPR v2- SLC30A1, which is characterized 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 knockdowns people SLC30A1 genes according to claim 1, 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 knockdowns people SLC30A1 genes according to claim 1, feature exist In the aim cell behaviour MDA-MB-231 cells.
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