CN106399311A - Endogenous protein marking method used for Chip-seq genome-wide binding spectrum - Google Patents
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Abstract
The invention relates to an endogenous protein marking method applied to genome-wide binding spectrum analysis. The method comprises the steps of designing sgRNA for upstream and downstream 50bp regions of an encoding termination site of an NCOA1 gene; verifying cutting efficiency of sgRNA mediated Cas9 by an experiment; building an anti-estrogen therapy tolerant MCF-7 breast cancer cell model; by utilizing a CRISPR/Cas9 homologous recombination technology, selecting and using effective sgRNA and DNA donors, and introducing a Flag tag in the 3' end of the ER alpha co-transcription factor NCOA1 gene; and establishing a stable cell line with the Flag tag. Compared with the prior art, the method has the advantages that gene editing is performed by utilizing the CRISPR/Cas9 technology, so that the tag insertion efficiency of the gene editing is greatly improved; more importantly, chromatin structure parameters are introduced for designing sgRNA, so that effective cutting of Cas9 at a target site of a genome is ensured; and positive clone screening is performed by adopting DNA sequencing and RT-PCR sequencing methods, so that the positive clone identification step is simplified and the manpower and material resources for screening are reduced.
Description
Technical field
The invention belongs to protein labeling field, it is related to one kind and is applied to Large-scale Screening cancer transcription factor transcriptional control machine
The method of system, especially relates to a kind of method of the mark of the intrinsic protein for Chip-seq full-length genome bind profile.
Background technology
CRISPR/Cas9 is the adaptive immunity defence system that a kind of bacterium and archeobacteria are formed during long-term evolution
System, emerging gene editing technology is it is considered to be change the revolutionary technology in the world.Start at the beginning of 2013, CRISPR/Cas9 conduct
Gene editing technology uses in mammalian cell.It is good that the gene editing of CRISPR/Cas9 mediation has efficiency high, accuracy
Feature, favored by numerous researchers, and be used for the functional study of specific gene in cancer, the foundation of cancer model and
The Large-scale Screening of target gene.This research team develops the analysis of the extensive knockout data of CRISPR/Cas9 within 2015
Algorithm MAGeCK.For sgRNA problem of missing the target that may be present, domestic and international researcher develop a series of based on CRISPR/
The improving technology of Cas9 and sgRNA design and algorithm.The sgRNA Photographing On-line website commonly used at present has：Zhang Feng laboratory is set up
Http:The http that //crispr.mit.edu/ and Michael Boutros laboratory is set up://e-crisp.org/E-
CRISPR/designcrispr/.
From 2014, the application of CRISPR/Cas9 became diversification.Researchers start to be used for CRISPR/Cas9
Control genetic transcription.In October, 2015, Dalvai et al. CRISPR/Cas9 technology, genome inserts target spot to label
Carry out double-strand cutting (Double-strand DNA break)；And pass through homologous recombination repair (homology-directed
Repair, HR) 3xFLAG and 2xSTREP label is marked on endogenous gene；In conjunction with affinity purification and mass-spectrometric technique (AP-MS),
It has been successfully separated the composition subunit of NuA4 protein complexes.Its result is consistent with front research, also separates first in research
The subunit (see Fig. 1) of complex MCM8/9.In the same year, Dellaire research team is by the variant of green fluorescent protein (GFP)
Clover is marked at the First Exon on endogenous gene, can strengthen the homologous recombination of CRISPR/Cas9 mediation for screening
The small molecule of reparation approach, and successfully filter out small molecule RS-1.Above CRISPR/Cas9 intrinsic protein labelling technique is transcription
The transcriptional control functional study of the factor provides new thinking.Chip-seq technology is widely used for transcription factor and combines high pass
Amount analysis, but at present due to the restriction of transcription factor antibody specificity, the transcription regulation mechanism of quite a few transcription factor
Unclear.
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 used for Chip-seq
The method of the intrinsic protein mark based on CRISPR/Cas9 technology of full-length genome bind profile.
The purpose of the present invention can be achieved through the following technical solutions：
A kind of method of the intrinsic protein mark for Chip-seq full-length genome bind profile, the method is based on CRISPR/
Cas9 technology, specifically includes following steps：
(1) design and screen the sgRNA with cutting efficiency：
Photographing On-line sgRNA, for termination encoding loci (the stop codon of the common transcription factor gene of three kinds of ER α
Site) upstream and downstream 50bp region, designs sgRNA, is simultaneously introduced DNA opening parameter, and screening further is located at degree of opening height
Genomic DNA on sgRNA；
(2) build the slow virus carrier containing sgRNA：
5 ' ends of sgRNA sequence add sequence 5 '-GACCA-3 ', and 5 ' ends of the reverse complementary sequence of sgRNA add sequence
5’-GACCA-3’.The sgRNA sequence that synthetic modification is crossed.Slow virus plasmid is from Cas9 and Puro element
LentiCRISPRv2 plasmid.With BsmBI enzyme, digestion and dephosphorylation are carried out to lentiCRISPRv2 plasmid, by linearizing matter
Grain carries out glue reclaim purifying, and is dissolved with distilled water.With annealing method by two of sgRNA single-stranded in the way of complementary pairing
In conjunction with, and with T4PNK by sgRNA phosphorylation.Finally with ligase by linearization plasmid, and it is connected with sgRNA.Felt with Stbl3
Converted by state cell, thus obtaining the lentiCRISPRv2 carrier containing sgRNA.With bacterium solution sequencing identification sgRNA insertion
Correctness.
(3) preparation is expressed the slow virus of sgRNA and is determined viral efficiency of infection：
Viral packaging plasmid selects psPAX2 and pMD2.G.By lentiCRISPRv2, psPAX2 containing sgRNA and
PMD2.G transfects 293FT cell, cultivates 48h after transfection, draws cell culture medium, harvests virus, continues second after culture 24h
Harvest virus.By the virus collected twice merging, filtered with 0.45 μm of filter, remove cell fragment.MCF-7 cell reflects
Fixed virus efficiency of infection.Cultivate MCF-7 in 6 orifice plates, after 18h, each hole is respectively with 0 μ L, 50 μ L, 100 μ L, 200 μ L, 400 μ
L, the virus of 600 μ L, the polybrene being simultaneously introduced final concentration of 10 μ g/ml strengthens viral efficiency of infection.After infection 48, use
The puromycin screening 72h of 1.5 μ g/ml, the now complete cell death of control group (0 μ L virus group), selection can make cell
The minimum virus quantity of 100% survival is made to carry out subsequent experimental.
(4) identify sgRNA efficiency：
Identify sgRNA efficiency using two methods.
A kind of is T7E1 nuclease digestion experiment.Concrete scheme is：The DNA fragmentation containing expected cleavage site for the PCR amplification,
DNA fragmentation length is made to be 700bp, PCR primer carries out cutting glue purification.Detection DNA concentration, experimental group and comparison after purification
Group takes identical amount of DNA, carries out digestion with T7E1 to DNA fragmentation.Wherein experimental group is the virus containing sgRNA to be checked for the infection
MCF-7, control group is wild type MCF-7.Detected with agarose gel electrophoresis after digestion, if experimental group DNA generates less than 700bp
Two band, then explanation sgRNA effective.
In T7E1 nuclease digestion experiment, the consumption of T7E1 restriction endonuclease is 0.5 μ L/10 μ L system.
PCR system is：
Another kind combines a generation for PCR and is sequenced.Concrete scheme is：With the DNA piece containing expected cleavage site for the PCR amplification
Section, send company to be sequenced, and plays the obvious miscellaneous peak of appearance effective for sgRNA near front 3 bases of analysis result PAM sequence (NGG)
Standard.
(5) set up the MCF-7 cell model that NCOA1 carries Flag label
So that the Flag of NCOA1 marks as a example.With CRISPR/Cas9 technology NCOA1 translational termination site (stop
Codon site) place's insertion Flag label (Flag-tag), the design of insertion position and donor is as shown in Figure 2.With lonza electricity
Turn instrument by the lentiCRISPRv2 of the sgRNA containing targeting NCOA1, be transfected into containing dox induction ER α 537 amino acids mutation
MCF-7 cell, culture 24h after, screen 24h with puromycin, with PCR combine a generation sequencing detection NCOA1 gene 3 ' end be
The no insertion having Flag-tag.If there being the insertion of Flag-tag, being separated with the method for cell dilution and culture individual cells, building
Vertical monoclonal cell strain.And the correctness of generation sequencing identification Flag-tag insertion is combined again with PCR.Resisted using Flag-tag
Body, detects the expression of Flag albumen using Western blot, confirms endogenous marker success.
In step (1) during Photographing On-line sgRNA, it is possible to use the Photographing On-line website http of this development in laboratory://
Cistrome.org/crispr/tool is designed.
In step (2), Lenti CRISPRv2, psPAX2, pMD2.G are according to mass ratio 4：3：1 ratio transfection 293FT
Cell, the proportionate relationship of Lenti CRISPRv2 and X-tremeGENE HP DNA Transfection Reagent is 6 μ g：
30μL.
In step (2), change within 15 hours after cell transfecting liquid, and collect virus respectively in 24 hours afterwards and 48 hours
Liquid, and the virus liquid collected twice is merged stand-by.
Compared with prior art, the present invention has advantages below and beneficial effect：
1. the sgRNA Photographing On-line instrument commonly used at present has the http that Zhang Feng laboratory is set up://crispr.mit.edu/
The http setting up with Michael Boutros laboratory://e-crisp.org/E-CRISPR/designcrispr/.And this
The sgRNA method for designing of bright exploitation, adds chromatin opening, can significantly improve the sgRNA improving design in design parameter
Efficiency (Fig. 3).As shown in figure 3, designing 5 kinds of sgRNA altogether for NCOA1.Wherein according to addition chromatin opening parameter
Method obtains sgRNA1 and sgRNA2, applies Photographing On-line instrument http://crispr.mit.edu/ design sgRNA3 and
SgRNA4, applies Photographing On-line instrument http://e-crisp.org/E-CRISPR/designcrispr/ designs sgRNA4,
PCR shows the Cas9 cutting adding the method for chromatin opening parameter to obtain sgRNA1 and sgRNA2 with reference to generation sequencing result
Efficiency is apparently higher than other two methods.
2. the present invention slow virus packaging in, from the transfection reagent X-tremeGENE HP DNA of Roche
Transfection Reagent is by Lenti CRISPRv2 and other two-strain packaging plasmid psPAX2 and pMD2.G corotation
Contaminate to 293FT cell, cultured cells, harvest virus liquid；And by many experiments determine CRISPRv2, psPAX2,
PMD2.G is according to mass ratio 4：3：1 ratio transfection 293FT cell, and determine Lenti CRISPRv2 and X-tremeGENE
The proportionate relationship of HP DNA Transfection Reagent is 6 μ g：30μL.Commonly use in correlation technique before
CRISPRv2, psPAX2, pMD2.G are 3 according to mass ratio：2：1.The present invention has done a series of mass ratioes on this basis, determines
4：3：The efficiency of infection highest of 1 virus of ratio packaging, and this mass ratio is closer to plasmid mol ratio 1：1：1, more accord with
Close slow virus packaging ratio in its natural state.
3rd, the present invention is marked to be described with high flux using endogenous gene and combines with reference to spectral method.Due at present for suitable
Many a part of transcription factors, because the specificity of its antibody is bad or inefficient, have drawn full-length genome bind profile and there is skill
Art bottleneck.With CRISPR/Cas9 labelling technique endogenous gene is marked, evaded the difficulty of transcription factor antibody mass
Topic.
4th, the present invention with CRISPR/Cas9 labelling technique be latest development homologous recombination labelling technique, there is skill
Art novelty.Compared with other homologous recombination labelling techniques (TALEN, ZFN), have the characteristics that accuracy is good, efficiency high.
The experimental result that PCR combines generation sequencing shows the positive rate of TALEN, ZFN homologous recombination technique typically in 3%-6%, and
CRISPR/Cas9 labelling technique is then 20%.
Brief description
Fig. 1 is endogenous gene 3xFLAG and 2xSTREP mark flow process and affinity purification analysis chart；
The homologous recombination schematic diagram that Fig. 2 mediates for CRISPR/Cas9；
Fig. 3 is the cutting efficiency comparison diagram of 5 kinds of sgRNA；
Fig. 4 is lentiCRISPRv2 plasmid construct schematic diagram.
Specific embodiment
The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings.
Embodiment 1
The mark of the gene of NCOA1 in breast cancer cell MCF7 and its subgenomic transcription spectrum are drawn.Based on the present invention's
Concrete grammar is as follows：
(1) sgRNA library is set up：
Photographing On-line website http using this development in laboratory://cistrome.org/crispr/tool, for three
Plant termination encoding loci (stop codon site) the upstream and downstream 50bp region of the common transcription factor gene of ER α, design sgRNA, draw
Enter DNA opening parameter, the sgRNA that screening is located on the high genomic DNA of degree of opening further.
(2) build the slow virus carrier containing sgRNA：
5 ' ends of sgRNA sequence add sequence 5 '-GACCA-3 ', and 5 ' ends of the reverse complementary sequence of sgRNA add sequence
5’-GACCA-3’.The sgRNA sequence that synthetic modification is crossed.Slow virus plasmid is from Cas9 and Puro element
LentiCRISPRv2 plasmid, as shown in Figure 4.With BsmBI enzyme, digestion and dephosphorylation are carried out to lentiCRISPRv2 plasmid, will
Linearizing plasmid carries out glue reclaim purifying, and is dissolved with distilled water.With annealing method by two of sgRNA single-stranded with complementation
The mode of pairing combines, and with T4PNK by sgRNA phosphorylation.Finally with ligase by linearization plasmid, and it is connected with sgRNA.
Converted with Stbl3 competent cell, thus obtaining the lentiCRISPRv2 carrier containing sgRNA.Use bacterium solution sequencing identification
The correctness of sgRNA insertion.
(3) preparation is expressed the slow virus of sgRNA and is determined viral efficiency of infection：
Viral packaging plasmid selects psPAX2 and pMD2.G.By lentiCRISPRv2, psPAX2 containing sgRNA and
PMD2.G transfects 293FT cell, cultivates 48h after transfection, draws cell culture medium, harvests virus, continues second after culture 24h
Harvest virus.By the virus collected twice merging, filtered with 0.45 μm of filter, remove cell fragment.MCF-7 cell reflects
Fixed virus efficiency of infection.Cultivate MCF-7 in 6 orifice plates, after 18h, each hole is respectively with 0 μ L, 50 μ L, 100 μ L, 200 μ L, 400 μ
L, the virus of 600 μ L, the polybrene being simultaneously introduced final concentration of 10 μ g/ml strengthens viral efficiency of infection.After infection 48, use
The puromycin screening 72h of 1.5 μ g/ml, the now complete cell death of control group (0 μ L virus group), selection can make cell
The minimum virus quantity of 100% survival is made to carry out subsequent experimental.
(4) identify sgRNA efficiency：
Identify sgRNA efficiency using two methods.
A kind of is T7E1 nuclease digestion experiment.Concrete scheme is：The DNA fragmentation containing expected cleavage site for the PCR amplification,
DNA fragmentation length is made to be 700bp, PCR primer carries out cutting glue purification.Detection DNA concentration, experimental group and comparison after purification
Group takes identical amount of DNA, carries out digestion with T7E1 to DNA fragmentation.Wherein experimental group is the virus containing sgRNA to be checked for the infection
MCF-7, control group is wild type MCF-7.Detected with agarose gel electrophoresis after digestion, if experimental group DNA generates less than 700bp
Two band, then explanation sgRNA effective.
Another kind combines a generation for PCR and is sequenced.Concrete scheme is：With the DNA piece containing expected cleavage site for the PCR amplification
Section, send company to be sequenced, and plays the obvious miscellaneous peak of appearance effective for sgRNA near front 3 bases of analysis result PAM sequence (NGG)
Standard.
Set up the MCF-7 cell model that NCOA1 carries Flag label
So that the Flag of NCOA1 marks as a example.With CRISPR/Cas9 technology NCOA1 translational termination site (stop
Codon site) place's insertion Flag label (Flag-tag).With lonza electroporation by the sgRNA's containing targeting NCOA1
LentiCRISPRv2, is transfected into the MCF-7 cell inducing the mutation of ER α 537 amino acids containing dox, after culture 24h, uses
Puromycin screens 24h, combines whether generation sequencing detection NCOA1 gene 3 ' end has the insertion of Flag-tag with PCR.If having
The insertion of Flag-tag, then separated with the method for cell dilution and culture individual cells, set up monoclonal cell strain.And use again
PCR combines the correctness of generation sequencing identification Flag-tag insertion.Using Flag-tag antibody, using Western blot detection
The expression of Flag albumen, confirms endogenous marker success.
(5) Chip-seq 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.
Also there is no the conventional method of maturation at present to the transcription factor of breast cancer cell antibody mass difference.
The present embodiment combines existing CRISPR/Cas9 methods of homologous recombination, in conjunction with Chip-seq obtained one new
Method.Compared with conventional sgRNA method for designing, step (1) chromatin degree of opening is used as the method energy of design parameter
Enough significantly more efficient design sgRNA, its sgRNAu is efficient to be more than 80%, and the sgRNA of conventional design is efficient
Less than 30%.Two methods are used to identify sgRNA efficiency it is ensured that the credibility of experiment in step (4) of the present invention, wherein another
Plant and combine generation sequencing for PCR.Concrete scheme is：With the DNA fragmentation containing expected cleavage site for the PCR amplification, company is sent to be sequenced,
Play the obvious miscellaneous peak of appearance for the effective standard of sgRNA near front 3 bases of analysis result PAM sequence (NGG).The method operates
The simple used time short (PCR only needs 2 hours), sequencing result can obtain for second day.
Embodiment 2
The CRISPR/Cas9 being applied to SMMC-7721 HepG2 is applied to the CRISPR/ of Large-scale Screening cancer gene
Cas9 is enriched with sequence measurement, comprises the following steps：
(1) design sgRNA：
Photographing On-line website http using this development in laboratory://cistrome.org/crispr/tool, for three
Plant termination encoding loci (stop codon site) the upstream and downstream 50bp region of the common transcription factor gene of ER α, design sgRNA, draw
Enter DNA opening parameter, the sgRNA that screening is located on the high genomic DNA of degree of opening further.
(2) build the slow virus carrier containing sgRNA：
5 ' ends of sgRNA sequence add sequence 5 '-GACCA-3 ', and 5 ' ends of the reverse complementary sequence of sgRNA add sequence
5’-GACCA-3’.The sgRNA sequence that synthetic modification is crossed.Slow virus plasmid is from Cas9 and Puro element
LentiCRISPRv2 plasmid.With BsmBI enzyme, digestion and dephosphorylation are carried out to lentiCRISPRv2 plasmid, by linearizing matter
Grain carries out glue reclaim purifying, and is dissolved with distilled water.With annealing method by two of sgRNA single-stranded in the way of complementary pairing
In conjunction with, and with T4PNK by sgRNA phosphorylation.Finally with ligase by linearization plasmid, and it is connected with sgRNA.Felt with Stbl3
Converted by state cell, thus obtaining the lentiCRISPRv2 carrier containing sgRNA.With bacterium solution sequencing identification sgRNA insertion
Correctness.
(3) slow virus of preparation expression sgRNA：
Viral packaging plasmid selects psPAX2 and pMD2.G.By lentiCRISPRv2, psPAX2 containing sgRNA and
PMD2.G transfects 293FT cell, cultivates 48h after transfection, draws cell culture medium, harvests virus, continues second after culture 24h
Harvest virus.By the virus collected twice merging, filtered with 0.45 μm of filter, remove cell fragment.293FT cell reflects
Fixed virus efficiency of infection.Cultivate 293FT in 6 orifice plates, after 18h, each hole is respectively with 0 μ L, 50 μ L, 100 μ L, 200 μ L, 400 μ
L, the virus of 600 μ L, the polybrene being simultaneously introduced final concentration of 10 μ g/ml strengthens viral efficiency of infection.After infection 48, use
The puromycin screening 72h of 1.5 μ g/ml, the now complete cell death of control group (0 μ L virus group), selection can make cell
The minimum virus quantity of 100% survival is made to carry out subsequent experimental.
(4) identify sgRNA efficiency：
Identify sgRNA efficiency using two methods.
A kind of is T7E1 nuclease digestion experiment.Concrete scheme is：The DNA fragmentation containing expected cleavage site for the PCR amplification,
DNA fragmentation length is made to be 700bp, PCR primer carries out cutting glue purification.Detection DNA concentration, experimental group and comparison after purification
Group takes identical amount of DNA, carries out digestion with T7E1 to DNA fragmentation.Wherein experimental group is the virus containing sgRNA to be checked for the infection
293FT, control group is wild type 293FT.Detected with agarose gel electrophoresis after digestion, if experimental group DNA generates less than 700bp
Two band, then explanation sgRNA effective.
Another kind combines a generation for PCR and is sequenced.Concrete scheme is：With the DNA piece containing expected cleavage site for the PCR amplification
Section, send company to be sequenced, and plays the obvious miscellaneous peak of appearance effective for sgRNA near front 3 bases of analysis result PAM sequence (NGG)
The screening that standard carries out cancer related gene at present to liver cancer primary cell does not also have conventional method.
Set up the HepG2 cell model that NCOA1 carries Flag label
So that the Flag of NCOA1 marks as a example.With CRISPR/Cas9 technology NCOA1 translational termination site (stop
Codon site) place's insertion Flag label (Flag-tag), the design of insertion position and donor is as shown in Figure 2.With lonza electricity
Turn instrument by the lentiCRISPRv2 of the sgRNA containing targeting NCOA1, be transfected into containing dox induction ER α 537 amino acids mutation
HepG2 cell, culture 24h after, screen 24h with puromycin, with PCR combine a generation sequencing detection NCOA1 gene 3 ' end be
The no insertion having Flag-tag.If there being the insertion of Flag-tag, being separated with the method for cell dilution and culture individual cells, building
Vertical monoclonal cell strain.And the correctness of generation sequencing identification Flag-tag insertion is combined again with PCR.Resisted using Flag-tag
Body, detects the expression of Flag albumen using Western blot, confirms endogenous marker success.
(5) Chip-seq 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.
Also there is no the conventional method of maturation at present to the transcription factor of HCC antibody mass difference.
The present embodiment combines existing CRISPR/Cas9 methods of homologous recombination, in conjunction with Chip-seq obtained one new
Method.Compared with conventional sgRNA method for designing, step (1) chromatin degree of opening is used as the method energy of design parameter
Enough significantly more efficient design sgRNA, its sgRNAu is efficient to be more than 80%, and the sgRNA of conventional design is efficient
Less than 30%.Two methods are used to identify sgRNA efficiency it is ensured that the credibility of experiment in step (4) of the present invention, wherein another
Plant and combine generation sequencing for PCR.Concrete scheme is：With the DNA fragmentation containing expected cleavage site for the PCR amplification, company is sent to be sequenced,
Play the obvious miscellaneous peak of appearance for the effective standard of sgRNA near front 3 bases of analysis result PAM sequence (NGG).The method operates
The simple used time short (PCR only needs 2 hours), sequencing result can obtain for second day.
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 method of the intrinsic protein mark for Chip-seq full-length genome bind profile is it is characterised in that the method base
In CRISPR/Cas9 technology, specifically include following steps：
(1) design and screen the sgRNA with cutting efficiency：
Photographing On-line sgRNA, for the termination encoding loci upstream and downstream 50bp region of the common transcription factor gene of three kinds of ER α, design
SgRNA, is simultaneously introduced DNA opening parameter, the sgRNA that screening is located on the high genomic DNA of degree of opening further；
(2) pack sgRNA 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) experiment screening has the sgRNA of cutting efficiency；
(4) set up the cell model that external source label is carried on NCOA1：
Based on the cell model of the abduction delivering genes of interest having built up, using CRISPR/Cas9 homologous recombination technique,
From effective sgRNA and DNA donor, introduce Flag labels at 3 ' ends of the common transcription factor NCOA1 gene of ER α, structure carries
The stable cell lines of Flag label, rotate into row plasmid transfection using electricity；
(5) resist MCF-7 cell model with anti-estrogen therapy on identification NCOA1：
Positive cell is screened using the method that PCR is sequenced with reference to a generation, and using western blot method Testing and appraisal sun
Property cell；
(6) NCOA1 full-length genome bind profile is set up and is analyzed：
Chip-seq is carried out using anti-Flag antibody, thus setting up genomic DNA to combine collection of illustrative plates, drawing and downstream target gene is turned
Record regulation and control collection of illustrative plates.
2. the method for a kind of intrinsic protein mark for Chip-seq full-length genome bind profile according to claim 1,
It is characterized in that, in step (1), according to the result of full-length genome DNase-seq, draw genomic DNA opening collection of illustrative plates, and build
The sgRNA method for designing of the vertical DNA opening parameter including binding site on DNA for the sgRNA.
3. the method for a kind of intrinsic protein mark for Chip-seq full-length genome bind profile according to claim 1,
It is characterized in that, in step (2), Lenti CRISPRv2, psPAX2, pMD2.G are according to mass ratio 4：3：1 ratio transfection
293FT cell, the proportionate relationship of Lenti CRISPRv2 and X-tremeGENE HP DNA Transfection Reagent is
6μg：30μL.
4. the method for a kind of intrinsic protein mark for Chip-seq full-length genome bind profile according to claim 1,
It is characterized in that, in step (2), change within 15 hours after cell transfecting liquid, and collect disease respectively in 24 hours afterwards and 48 hours
Venom, and the virus liquid collected twice is merged stand-by.
5. the method for a kind of intrinsic protein mark for Chip-seq full-length genome bind profile according to claim 1,
It is characterized in that, in step (3), the method for the sgRNA that experiment screening has cutting efficiency includes two kinds,
A kind of is T7E1 nuclease digestion experiment, and concrete scheme is：The DNA fragmentation containing expected cleavage site for the PCR amplification, makes
DNA fragmentation length is 700bp, and PCR primer carries out cutting glue purification, detection DNA concentration, experimental group and control group after purification
Take identical amount of DNA, with T7E1 nuclease, digestion is carried out to DNA fragmentation；
Another kind is to combine whether generation sequencing detection NCOA1 gene 3 ' end has the insertion of Flag-tag with PCR.
6. the method for a kind of intrinsic protein mark for Chip-seq full-length genome bind profile according to claim 5,
It is characterized in that, in T7E1 nuclease digestion experiment, the consumption of T7E1 restriction endonuclease is 0.5 μ L/10 μ L system.
7. the method for a kind of intrinsic protein mark for Chip-seq full-length genome bind profile according to claim 5,
It is characterized in that, PCR system is：
8. the method for a kind of intrinsic protein mark for Chip-seq full-length genome bind profile according to claim 1,
It is characterized in that, in step (4), electricity turns using bio-rad company electroporation, takes density to be 10-6The cell re-suspension liquid 100 of/mL
μ, adds 1.5 μ g sgRNA plasmids and 3 μ g DNA donors, and as in electric revolving cup after gently mixing, it is 200V, 250 μ that electricity turns condition
F.
9. the method for a kind of intrinsic protein mark for Chip-seq full-length genome bind profile according to claim 1,
It is characterized in that, in step (5), using for the DNA piece including mutational site and vector insert 5 ' flanking fragment simultaneously
Section enters performing PCR amplification.
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