CN106399377A - Method for screening drug target genes based on CRISPR/Cas9 high-throughput technology - Google Patents

Method for screening drug target genes based on CRISPR/Cas9 high-throughput technology Download PDF

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CN106399377A
CN106399377A CN201610807508.1A CN201610807508A CN106399377A CN 106399377 A CN106399377 A CN 106399377A CN 201610807508 A CN201610807508 A CN 201610807508A CN 106399377 A CN106399377 A CN 106399377A
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蒋征
刘小乐
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Tongji University
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Abstract

The invention relates to a method for screening drug target genes based on a CRISPR/Cas9 high-throughput technology. The method comprises the steps of firstly establishing a sgRNA library; secondly packaging the sgRNA library by using slow viruses, and collecting the viruses; thirdly screening the sgRNA library in a cancer cell line; fourthly extracting cells obtained by screening and genome DNA of the cells before screening; and finally enriching sgRNA in genome DNA. Compared with the prior art, the method has the advantages that a CRISPR/Cas cell screening process is improved, the virus infection efficiency is determined with a simple and convenient method by utilizing puromycin resistance of infected cells, and MOI values of the viruses are determined; more importantly, a virus packaging method is greatly optimized, so that the virus packaging efficiency is improved to be more than 5 times that of a conventional method, and large-scale drug target screening cost can be greatly reduced; and the method is used for promoting industrialization of cancer drug target screening.

Description

A kind of method screening drug target gene based on CRISPR/Cas9 high-throughput techniques
Technical field
The invention belongs to high throughput sequencing technologies field, especially relate to one kind and be based on CRISPR/Cas9 high-throughput techniques The method of screening drug target gene.
Background technology
Oncogene and tumor suppressor gene are all the potential target spots of gene therapy for cancer, and therefore its identification is in the treatment of cancer There is huge application prospect, have been directed towards the medicine that the Data minings such as oncogene EGFR, Alk go out treating cancer at present.But It is that effectively cancer gene targeted drug also far can not meet the needs of clinical treatment at present.Based on CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered Regularly interspaced short palindromic repeats associated protein 9) extensive Functional gene triage techniques, as genetic engineering emerging technology in recent years, can screen cancer-related in full-length genome aspect Cause, thus provide drug target candidate gene for the treatment of cancer.
The only a few experiments room application at home and abroad of CRISPR/Cas9 screen technology, does not promote at present.CRISPR/ The key of Cas9 screen technology is to ensure that the coverage rate of sgRNA, the therefore validity of the structure in sgRAN library of early stage, The validity of the high efficiency of sgRAN amplified library and virus packaging becomes the key of problem.At present in International Periodicals paper Length occurs that sgRNA Library Quality is not high or later stage sgRNA is sequenced, and sgRNA library is distributed with the problem of skewness.In addition tradition Banking process is wasted time and energy, and needs substantial amounts of PCR enzyme, and financial burden is also than larger.For solving problem above, need one kind SgRNA library is set up, the method for amplification, virus packaging and sequencing, to ensure the validity of extensive cancer gene Sites Screening And high efficiency.
Content of the invention
The purpose of the present invention is exactly to overcome the defect of above-mentioned prior art presence to provide one kind to be based on CRISPR/ The method that Cas9 high-throughput techniques screen drug target gene.
The purpose of the present invention can be achieved through the following technical solutions：
A kind of method screening drug target gene based on CRISPR/Cas9 high-throughput techniques, the method includes following step Suddenly：
(1) method that electricity consumption turns sets up sgRNA library：
The method adjunction head that the oligo fragment of the sgRNA of synthesis is expanded with PCR, then uses Gibson assembly's Method connects entrance lentiCRISPRv2 plasmid, is converted connection product to competence with Bio-rad electroporation, thus obtaining Obtain sgRNA library；
(2) pack sgRNA library with slow virus：
Under aseptic condition, cultivate 293FT cell, and with transfection reagent X-tremeGENE HP DNA Transfection Lenti CRISPRv2 and other two-strain packaging plasmid psPAX2 and pMD2.G is packed slow virus by Reagent；
(3) screening in cell for the sgRNA library：
Choose suitable virus quantity, infect cancerous cell line to be detected, after infecting 48 hours, screen 2 with puromycin My god, gained positive cell collects part as a control group, and other cells continue culture and receive afterwards to 42-46 days (preferably 45 days) Take；
(4) extract screening gained cell and screen precellular genomic DNA：
Use cell pyrolysis liquid cell lysis, after piping and druming mixes, add RNase enzyme, 65 DEG C are incubated 30 minutes, then plus final concentration For the Proteinase K of 10 μ g/ml, 55 DEG C of overnight incubation, extract DNA fragmentation with refined solution；
(5) sgRNA in enrichment genomic DNA：
With restriction enzyme, genomic DNA is carried out with digestion, 37 DEG C of reactions overnight, the DNA fragmentation after digestion are placed In the low melting-point agarose glue of 0.8wt%, 80V, 1 hour electrophoresis, cut the DNA fragmentation of glue reclaim 1600-2000bp.
(6) sgRNA library construction and sequencing：
Library builds according to the storehouse kit specification of building of illumina company, carries out Hiseq2000's by building up library 50SE carries out high-flux sequence, and result carries out analysis of biological information.
Before the method that (1st) step electricity consumption turns sets up sgRNA library, first carry out sgRNA library designs, concrete operations For：Using website http://crisprscan.org and http://www.e-crisp.org Photographing On-line gene interested The sgRNA of group, designs 10 sgRNA for every kind of gene.
In step (1), the PCR amplification condition of oligo is：
In step (1), the condition of Gibson assembly is：
In step (1), the plasmid quality of described viral packaging process Lenti CRISPRv2, psPAX2, pMD2.G is pressed According to 4：3：1 ratio transfection 293FT cell；The matter of Lenti CRISPRv2, psPAX2, pMD2.G taking the culture dish of 10cm as a example Grain quality is respectively 6 μ g, 4.5 μ g, and the amount of 1.5 μ g, X-tremeGENE HP DNA Transfection Reagent is 30 μ L.Using the method for the transfection that suspends during transfection.And collect virus liquid in 48 hours afterwards and 72 hours.
Using infection virocyte, viral efficiency of infection, concrete operations are detected to the resistance of puromycin in step (2) For：
In six orifice plates, cultured cells is to degrees of fusion 70%, with 25 μ L, 50 μ L, 100 μ L, 200 μ L, 400 μ L virus liquid Infection cell, takes the virus quantity that can infect 48-52% (preferably 50% about) cell subsequently to be tried as appropriate virus amount Test.
In step (4), the composition of cell pyrolysis liquid is 400 μM of NaCl, 0.2%SDS, 2mMEDTA, 10mMTris-HCl.
In step (4), described refined solution refers to volume ratio 25:24:The mixed liquor of 1 phenol, chloroform and isoamyl alcohol.
In step (5), restriction enzyme reaction system is：In 50 μ L, containing 6 μ g DNA, 5 μ L 10 × NEB Buffer, restriction enzyme EcoN I 3 μ L.
Cancerous cell line to be detected includes lung carcinoma cell and HCC clone.
Compared with prior art, the present invention has advantages below and beneficial effect：
The present invention improves to the screening process of cell, using the puromycin resistance of infected cell, with easy Method determines viral efficiency of infection it is determined that viral MOI value；Restriction enzyme enzymatic cleavage methods and the combination of high throughput method, And the false positive that non-specific PCR amplification produces can be reduced, improve and build storehouse efficiency；Using the transfection packaging virus that suspend Method substantially increases virus titer, and transfection reagent is expensive, and the method has saved screening cost.
Specific embodiment
With reference to specific embodiment, the present invention is described in detail.
Embodiment 1
T47D thin to breast cancer carries out the screening of drug gene target spot.Concrete grammar is as follows：
(1) sgRNA library designs：
Using website http://crisprscan.org and http://www.e-crisp.org Photographing On-line is interested The sgRNA of gene group, designs 10 sgRNA for every kind of gene.
(2) method that electricity consumption turns sets up sgRNA library
The method adjunction head that the oligo fragment of the sgRNA of synthesis is expanded with PCR, then uses Gibson assembly's Method connects entrance lentiCRISPRv2 plasmid.With Bio-rad electroporation, connection product is converted to competence, thus obtaining Obtain sgRNA library.
(3) pack sgRNA library with slow virus：
Under aseptic condition, cultivate 293FT cell, and the transfection reagent X-tremeGENE HP DNA with Roche Lenti CRISPRv2 and other two-strain packaging plasmid psPAX2 and pMD2.G is packed by Transfection Reagent Slow virus.Change within 15 hours after cell transfecting liquid, and collect virus liquid in 24 hours afterwards and 48 hours；
(4) screen sgRNA library in T47D cell
Choose suitable virus quantity, infect T47D, after 48 hours, screened 2 days with puromycin, gained positive cell is received Take part as a control group, other cells continue culture and collect to after 40 days.
(5) extract screening gained cell and screen precellular genomic DNA
With cell pyrolysis liquid A cell lysis, after piping and druming mixes, add RNase enzyme, 65 DEG C are incubated 30 minutes, then plus dense eventually Spend the Proteinase K for 10 μ g/ml, 55 DEG C of overnight incubation.With refined solution (volume ratio 25:24:1 phenol, chloroform and isoamyl alcohol mixed Close liquid) extracting DNA fragmentation；
(6) sgRNA in enrichment genomic DNA
With cell pyrolysis liquid A cell lysis, after piping and druming mixes, add RNase enzyme, 65 DEG C are incubated 30 minutes, then plus dense eventually Spend the Proteinase K for 10 μ g/ml, 55 DEG C of overnight incubation.With refined solution (volume ratio 25:24:1 phenol, chloroform and isoamyl alcohol mixed Close liquid) extracting DNA fragmentation；
With restriction enzyme, genomic DNA is carried out with digestion, 37 DEG C of reactions overnight, the DNA fragmentation after digestion are placed In the low melting-point agarose glue of 0.8wt%, 80V, 1 hour electrophoresis, cut the DNA fragmentation of glue reclaim 1600-2000bp；
(7) sgRNA library construction and sequencing：
Library builds according to the storehouse kit specification of building of illumina company, carries out Hiseq2000's by building up library 50SE carries out high-flux sequence, and result carries out analysis of biological information.
Embodiment 2
The CRISPR/Cas9 being applied to prostate cancer cell line LNcap is applied to Large-scale Screening cancer gene target spot CRISPR/Cas9 screening technique, comprises the following steps：
(1) sgRNA library is set up：
Using website http://crisprscan.org and http://www.e-crisp.org Photographing On-line and full genome The sgRNA of group.The carrier of sgRNA adopts Lenti CRISPRv2.
(2) pack sgRNA library with slow virus：
Under aseptic condition, cultivate 293FT cell, and the transfection reagent X-tremeGENE HP DNA with Roche Lenti CRISPRv2 and other two-strain packaging plasmid psPAX2 and pMD2.G is packed by Transfection Reagent Slow virus.According to 4：3：1 ratio transfection 293FT cell, Lenti CRISPRv2 taking the culture dish of 10cm as a example, psPAX2, The plasmid quality of pMD2.G is respectively 6 μ g, 4.5 μ g, 1.5 μ g, X-tremeGENE HP DNA Transfection Reagent Amount be 30 μ L.Change within 15 hours after cell transfecting liquid, and collect virus liquid in 24 hours afterwards and 48 hours；
(3) sgRNA library is screened in cell：
Choose suitable virus quantity, infect cancerous cell line to be detected, after infecting 48 hours, screen 2 with puromycin My god, gained positive cell collects part as a control group, and other cells continue culture and collect to after 45 days about.For determining virus Amount, will determine the efficiency of infection of virus.Invention is detected to the resistance of puromycin using infection virocyte.In six holes In plate, cultured cells is to degrees of fusion 70%, with 50 μ L, 100 μ L, 200 μ L, 400 μ L, 500 μ L virus liquid infection cell, take energy The virus quantity of infection 50% about cell carries out follow-up test as appropriate virus amount；
(4) extract screening gained cell and screen precellular genomic DNA：
With cell pyrolysis liquid A cell lysis, after piping and druming mixes, add RNase enzyme, 65 DEG C are incubated 30 minutes, then plus dense eventually Spend the Proteinase K for 10 μ g/ml, 55 DEG C of overnight incubation.With refined solution (volume ratio 25:24:1 phenol, chloroform and isoamyl alcohol mixed Close liquid) extracting DNA fragmentation；The composition of cell pyrolysis liquid A is 400 μM of NaCl2,0.2%SDS, 2mMEDTA, 10mMTris-HCl.
(5) sgRNA in enrichment genomic DNA
With restriction enzyme, genomic DNA is carried out with digestion, 37 DEG C of reactions overnight, the DNA fragmentation after digestion are placed In the low melting-point agarose glue of 0.8wt%, 80V, 1 hour electrophoresis, cut the DNA fragmentation of glue reclaim 1600-2000bp；Restricted Restriction endonuclease reaction system is in 50 μ L：6 μ g DNA, 5 μ L 10 × NEB buffer, restriction enzyme EcoN I 3 μ L.
(6) sgRNA library construction and sequencing：
Library builds according to the storehouse kit specification of building of illumina company, carries out Hiseq2000's by building up library 50SE carries out high-flux sequence, and result carries out analysis of biological information.
The above-mentioned description to embodiment is to be understood that and use invention for ease of those skilled in the art. Person skilled in the art obviously easily can make various modifications to these embodiments, and described herein general Principle is applied in other embodiment without through performing creative labour.Therefore, the invention is not restricted to above-described embodiment, ability , according to the announcement of the present invention, the improvement made without departing from scope and modification all should be the present invention's for field technique personnel Within protection domain.

Claims (9)

1. a kind of screen the method for drug target gene it is characterised in that the method bag based on CRISPR/Cas9 high-throughput techniques Include following steps：
(1) method that electricity consumption turns sets up sgRNA library：
The method adjunction head that the oligo fragment of the sgRNA of synthesis is expanded with PCR, the method then using Gibson assembly Connecting and enter lentiCRISPRv2 plasmid, being converted connection product to competence with Bio-rad electroporation, thus obtaining SgRNA library；
(2) pack sgRNA library with slow virus：
Under aseptic condition, cultivate 293FT cell, and with transfection reagent X-tremeGENE HP DNA Transfection Lenti CRISPRv2 and other two-strain packaging plasmid psPAX2 and pMD2.G is packed slow virus by Reagent；
(3) screening in cell for the sgRNA library：
Choose suitable virus quantity, infect cancerous cell line to be detected, after infecting 48 hours, screened 2 days with puromycin, institute Obtain positive cell and collect part as a control group, other cells continue culture and collect to after 42-46 days；
(4) extract screening gained cell and screen precellular genomic DNA：
Use cell pyrolysis liquid cell lysis, after piping and druming mixes, add RNase enzyme, 65 DEG C are incubated 30 minutes, then plus final concentration of 10 The Proteinase K of μ g/ml, 55 DEG C of overnight incubation, extract DNA fragmentation with refined solution；
(5) sgRNA in enrichment genomic DNA：
With restriction enzyme, genomic DNA is carried out with digestion, 37 DEG C of reactions overnight, the DNA fragmentation after digestion are placed on In the low melting-point agarose glue of 0.8wt%, 80V, 1 hour electrophoresis, cut the DNA fragmentation of glue reclaim 1600-2000bp.
2. a kind of method screening drug target gene based on CRISPR/Cas9 high-throughput techniques according to claim 1, It is characterized in that, in step (1), the PCR amplification condition of oligo is：
3. a kind of method screening drug target gene based on CRISPR/Cas9 high-throughput techniques according to claim 1, It is characterized in that, in step (1), the condition of Gibson assembly is：
4. a kind of method screening drug target gene based on CRISPR/Cas9 high-throughput techniques according to claim 1, It is characterized in that, in step (1), the plasmid quality of described viral packaging process Lenti CRISPRv2, psPAX2, pMD2.G According to 4：3：1 ratio transfection 293FT cell.
5. a kind of method screening drug target gene based on CRISPR/Cas9 high-throughput techniques according to claim 1, It is characterized in that, using infection virocyte, viral efficiency of infection is detected to the resistance of puromycin in step (2), specifically Operate and be：
In six orifice plates, cultured cells, to degrees of fusion 70%, is infected with the virus liquid of 25 μ L, 50 μ L, 100 μ L, 200 μ L, 400 μ L Cell, takes the virus quantity that can infect 48-52% cell to carry out follow-up test as appropriate virus amount.
6. a kind of method screening drug target gene based on CRISPR/Cas9 high-throughput techniques according to claim 1, It is characterized in that, in step (4), the composition of cell pyrolysis liquid is 400 μM of NaCl, 0.2%SDS, 2mMEDTA, 10mMTris- HCl.
7. a kind of method screening drug target gene based on CRISPR/Cas9 high-throughput techniques according to claim 1, It is characterized in that, in step (4), described refined solution refers to volume ratio 25:24:The mixed liquor of 1 phenol, chloroform and isoamyl alcohol.
8. a kind of method screening drug target gene based on CRISPR/Cas9 high-throughput techniques according to claim 1, It is characterized in that, in step (5), restriction enzyme reaction system is：In 50 μ L, containing 6 μ g DNA, 5 μ L 10 × NEB delay Rush liquid, restriction enzyme EcoN I 3 μ L.
9. a kind of method screening drug target gene based on CRISPR/Cas9 high-throughput techniques according to claim 1, It is characterized in that, cancerous cell line to be detected includes lung carcinoma cell and HCC clone.
CN201610807508.1A 2016-09-07 2016-09-07 Method for screening drug target genes based on CRISPR/Cas9 high-throughput technology Pending CN106399377A (en)

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