CN109504711A - Method based on CRISPR/cas9 and peroxidase APEX2 system identification analysis specific gene group site interaction DNA - Google Patents

Method based on CRISPR/cas9 and peroxidase APEX2 system identification analysis specific gene group site interaction DNA Download PDF

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CN109504711A
CN109504711A CN201810148785.5A CN201810148785A CN109504711A CN 109504711 A CN109504711 A CN 109504711A CN 201810148785 A CN201810148785 A CN 201810148785A CN 109504711 A CN109504711 A CN 109504711A
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刘贇
邱文青
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Fudan University
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Abstract

The invention belongs to field of biotechnology, it is related to a kind of method based on CRISPR/cas9 and peroxidase APEX2 system identification analysis specific gene group site interaction DNA, including step, the conversion of plasmid, the biotin labeling of specific position binding protein compound utilizes the sequencing and analysis of strepavidin magnetic beads enriched biological element labelled protein and DNA compound and enrichment DNA；It is shown through experiment analysis results, this method is suitable for the analysis of any genomic locus, it can get the genomic locus information of interaction therewith, meanwhile CRISPR gene editing system is carried out the thinking of specific site DNA analysis in conjunction with APEX2 and is equally applicable to the combination of other genome edit methods (such as TALEN) and APEX2 by this method.This method is easy to operate, high sensitivity, specific height, favorable reproducibility, is adapted to study the requirement of chromatin local interaction DNA on any given chromosome location.

Description

Based on CRISPR/cas9 and peroxidase APEX2 system identification analysis specificity base Because of a group method of site interaction DNA
Technical field
The invention belongs to field of biotechnology, and in particular to one kind is based on CRISPR/cas9 and peroxidase APEX2 system The method of system discriminance analysis specific gene group site interaction DNA.
Background technique
Prior art discloses in eukaryotic cells, DNA molecular has the height sense of organization, and is tightly wrapped core The repetitive unit of corpusculum forms chromatin.However, chromatinic structure is dynamic change in living cells, local dyeing It is close that matter can be adjusted element such as transcription factor and non-coding RNA.In recent years, several adjusting dyes have been proposed in this field The mechanism of chromaticness tissue, for example, each chromosome in eukaryocyte nucleus, which is all located at one, is referred to as chromosomal region Specific region, it includes the structural domain that many usual sizes are millions of bases, referred to as topological structure domain；In topological structure domain In, distal end DNA element is expressed by dynamic interaction come controlling gene, many acting factors of studies have shown that, including CTCF, Laminins and other DNA binding protein, have been involved in the formation in topological structure domain and interaction long-range between them.Separately Outside, the modification of epigenetic, for example, DNA methylation and histone modification, there are also long-chain non-coding RNAs all passing through adjust Chromatinic higher structure is controlled to control the expression of gene and play an important role in the process.These discovery make the technical field into The new era of a chromatin functional study is entered, however, it is specific to need identification to be present in the overall understanding of chromatin function DNA, RNA and transcription factor of genomic locus, the research of this aspect is due to technical difficult and challenging.
In recent years, the technology formed by local chromatin has been researched and proposed, for example, chromatin immune co-precipitation (ChIP) It is one such classics, the technology for studying given albumen full-length genome distribution is widely used in, however, there is presently no one kind The method of the given genomic locus local interaction molecule of widely accepted research；The nucleic acid probe of locking is used to identify Albumen in conjunction with Telomere regions, but this method is only limitted to the duplicate genome area of height；LexA DNA binding site It is integrated into Yeast genome in gene level, the chromatin for locus specificity purifies；However, this method needs pair Target gene group carries out the transformation of genome, to can change chromatinic natural environment, and low efficiency；The genome of improvement Editing technique, such as the short palindrome repetitive sequence in interval of the similar effect nuclease (TALEN) of transcription activation factor and regular cluster (CRISPR/cas9) it is already used to be enriched with required genomic locus, however, the method based on TALEN requires to be each position One section of amino acid sequence of point design, the method based on CRISPR is needed cell formaldehyde crosslinking, and needs high-affinity It is available, etc. with the antibody of specificity；Research practice shows that above-mentioned method still cannot be provided specifically to natural dyeing The functional analysis of matter or full-length genome.
Research foundation based on the prior art, present inventor is quasi- to provide a kind of discriminance analysis specific gene group position The method of point interaction DNA.The local interaction DNA's of especially a kind of any given chromosome location of discriminance analysis is new Method.
The prior art related to the present invention has following bibliography:
1.J.and Kingston, R.E. (2009) Purification of proteins associated With specific genomic Loci.Cell, 136,175-86.
2.Byrum, S.D., Raman, A., Taverna, S.D.and Tackett, A.J. (2012) ChAP-MS:a method for identification of proteins and histone posttranslational Modifications at a single genomic locus.Cell Rep., 2,198-205.
3.Byrim, S.D., Taverna, S.D.and Tackett, A.J. (2013) Purification of a Specific native genomic locus for proteomic analysis.Nucleic Acids Res., 41, e195.
4.Fujita, T.and Fujii, H. (2013) Efficient isolation of specific genomic regions and identification of associated proteins by engineered DNA-binding molecule-mediated chromatin immunoprecipitation(enChIP)using CRISPR.Biochem.Biophys.Res.Commun., 439,132-6.
5.Waldrip, Z.J., Byrum, S.D., Storey, A.J., Gao, J., Byrd, A.K., Mackintosh, S.G., Wahls, W.P., Taverna, S.D., Raney, K.D.and Tackett, A.J. (2014) A CRISPR-based Approach for proteomic analysis of a single genomic locus.Epigenetics, 9,1207- 11.
6.Hung, V., Udeshi, N.D., Lam, S.S., Loh, K.H., Cox, K.J., Pedram, K., Carr, S.A.and Ting, A.Y. (2016) Spatially resolved proteomic mapping in living cells With the engineered peroxidase APEX2.Nat.Protoc., 11,456-75.
Summary of the invention
It is an object of the invention to the Research foundations based on the prior art, provide a kind of discriminance analysis specific gene group position The method of point interaction DNA, it is especially a kind of special based on CRISPR/cas9 and peroxidase APEX2 system identification analysis The method of specific gene group site interaction DNA.This method is easy to operate, high sensitivity, specific height, favorable reproducibility, fits Ying Yu studies the requirement of chromatin local interaction DNA on any given chromosome location.
Early-stage study basis based on the application proposes one to probe into the molecule of specific gene group site interaction Kind is known as CAPLOCUS (Combining CRISPR and peroxidase APEX2 system to identify local Chromatin interactions) method, and by capture human chromosomal telomere area, one weight of No. 13 chromosome One list in multiple region and No. 11 chromosome copies sites to verify the system；It is analyzed by genome sequencing, CAPLOCUS can To be enriched with target area and interact therewith remote genomic locus.The invention proposes one kind to be based on CRISPR/ The method of cas9 and peroxidase APEX2 system identification analysis specific gene group site interaction DNA, this method can be known The local interaction DNA of any given chromosome location is not analyzed.
To achieve the above object, the present invention adopts the following technical scheme that:
1. vector construction,
In the present invention, the pUC pUC used includes three plasmids, expresses dcas9, APEX2 and sgRNA respectively；
2. cell transformation and biotin labeling,
In the present invention, using polyethyleneimine (PEI) (Polysciences Inc., Warrington, PA, USA) into The transient transfection of row cell, cell transfecting for 24 hours after, with the biotin-phenol of 500uM (Iris Biotech GmbH, Germany cell 30min) is handled, hydrogen peroxide (H is added later2O2) to final concentration 1mM handle cell 1min after, immediately plus Enter reaction terminating liquid (10mM sodium azide, 10mM VC, 5mM Trolox) and terminate reaction, cell fixes 10 with 1% formaldehyde Minute, glycine terminates reaction；
3. the cell that step 2 is obtained carries out strepavidin magnetic beads enrichment, the DNA being enriched with carries out qPCR detection and targets area The degree of enrichment in domain simultaneously constructs DNA library；
4. the library for detecting qualified in step 3 is sequenced using illumina platform Hiseq X ten sequenator；
5. pair sequencing result carries out peak calling analysis, the genomic locus information with target sequence interaction is obtained.
The present invention has carried out experimental analysis, the results show that this method is suitable for the analysis of any genomic locus, can get The genomic locus information of interaction therewith, meanwhile, this method carries out CRISPR gene editing system specifically in conjunction with APEX2 The thinking of site DNA analysis is equally applicable to the combination of other genome edit methods (such as TALEN) and APEX2.
The method of the present invention has the advantage that
1) CAPLOCUS can effectively be enriched with duplicate genome area and high-resolution single copy gene group site；
2) the remote genomic locus that CAPLOCUS can be enriched with target area and interact therewith.
3) this method is easy to operate, high sensitivity, specific height, favorable reproducibility, is adapted in any given chromosome The requirement of chromatin local interaction DNA is studied on position.
In order to make the objectives, technical solutions, and advantages of the present invention clearer, below in conjunction with attached drawing and specific implementation Example, the present invention is described in more detail.It should be appreciated that described in following, specific examples are only used to explain the present invention, and It is not used in the restriction present invention.Raw material involved in following embodiment is commercially available, involved detection method unless otherwise instructed It unless otherwise instructed, then is conventional method.Following embodiments be combine Human chromosome 11 list copy area (chr11:5, 497,611-5,497,843) method of the invention is further described.
Detailed description of the invention
Fig. 1, it is shown that (A) CAPLOCUS system flow chart and (B) related plasmids structural diagrams.
Fig. 2, it is shown that CAPLOCUS systemic characteristic and bioaccumulation efficiency figure: where
Scheming A is the specific Immunofluorescent localization figure for repeating site telomere area (telomere) of CAPLOCUS system targeting；
Scheming B is that DNA is enriched with qPCR quantitative result figure, and wherein ga14 is negative control.
Fig. 3 show the specific single copy site of CAPLOCUS systemic characteristic capture (chr11:5,497,611-5,497, 843)。
Fig. 4 shows single copy site ChIP-seq Sync enrichment target sequence and therewith interaction site.
Specific embodiment
The building and transfection of embodiment 1, plasmid expression vector
The building of 1.1 plasmid expression vectors: in order to construct MS2-APEX2_NLS fusion protein expression vector, with PCR's Method expands from pcDNA3 Connexin43-GFP-APEX2 (Addgene plasmid:49385) plasmid and obtains APEX2, Then by its use BamHI and XhoI. digestion, be cloned into pHAGE-EFS-MCP-3XBFPnls (Addgene plasmid: 75384) on carrier, by by the oligonucleotide fragment (oligos) of annealing connection and through being connected at BbsI restriction enzyme site The building that sgRNA expression vector is completed on pLH-sgRNA1-2XMS2 (Addgene plasmid:75389) plasmid, is used Addgene plasmid 64107 expresses dCas9；All sgRNA sequence and as shown in table 1；
All sgRNA sequences of table 1
NAME Sequence(5’-3’)
sgRNA-Telomere TAGGGTTAGGGTTAGGGTTA
sgRNA-Gal4 AACGACTAGTTAGGCGTGTA
sgRNA-C20-1 CTCTTAGCTGTTATGCTGTA
sgRNA-C20-2 GGATTCCCTTCCATGCTGAA
sgRNA-C20-3 GGTGCCCAAAAAATAGTCAC
sgRNA-C20-4 ACACTGAACAAGCCGATAAG
1.2 HEK293T cell culture: HEK293T cell is cultivated in 5%CO2 and 37 DEG C of incubator, and culture medium is height Glucose Dulbecco ' s Modified Eagle ' s Medium (DMEM；Life Technologies, Carlsbad, CA, USA), the interior fetal calf serum (Sigma, St.Louis, MO, USA) for being 10% added with concentration, 1% penicillin/streptomycin (Life Technologies) with 1: 5 ratio passage cell, and detects mycoplasma in every two days；
1.3 cell transfectings: polyethyleneimine (PEI) (Polysciences Inc., Warrington, PA, USA) root is used According to producer to the step of carry out the transient transfection of cell, the dcas9 plasmid of 900ng, the sgRNA plasmid of 4.5ng and 120ng's MS2-APEX2_NLS plasmid corotation into the T75 Tissue Culture Flask of convergence degree 60%-80%,
Embodiment 2 extracts DNA progress ChIP experiment
2.1 cell transfectings for 24 hours after, when 2x107 cell be transfected target sgRNA or Gal4 control when, use 500uM Biotin-phenol (Iris Biotech GmbH, Germany) handle cell 30min, later be added hydrogen peroxide (H2O2) To final concentration 1mM, cell 1min is handled, later, reaction terminating liquid (10mM sodium azide, 10mM V are added immediatelyc, 5mM Trolox reaction) is terminated, finally, being fixed with 1% formaldehyde and being incubated at room temperature 10min.With the hypotonic buffer of 1mL (20mM HEPES pH7.5,10mM potassium chloride, the former of 1mM EDTA, 0.1mM activation consolidate sour sodium, and 0.2% NP-40,10% is sweet Oil) lytic cell 15min, for cell lysate at 4 DEG C, 13000g is centrifuged 1min, abandons supernatant；
2.2 use 500ul ChIP dilution buffer (20mM Tris-HCl pH 8.0,2mM EDTA, 150mM NaCl, 0.1%SDS, 1%TritonX-100) it is resuspended；
2.3 by the Bioruptor Pico sonication device that is deposited in of resuspension, (non-contact type ultrasonic is broken Instrument) ultrasonication is carried out, until fragment length is between 100bp-500bp；
2.4 in 4 DEG C of centrifuges, 14000g, are centrifuged 15min, take out 5% supernatant protect as input sample later In the presence of -20 DEG C；
2.5 pre-process 1h in 4 DEG C of chromatin by cutting with the Protein A of 50ul；
2.6 taking-up supernatants are added in 50 μ l M-280 Streptavidin immunomagnetic beads, and 4 DEG C of rotations are incubated overnight；
2.7 be incubated overnight after, in order use 2%SDS, high-salt buffer (50mM HEPES pH 7.5, 500mM NaCl, 1mM EDTA, 0.1% sodium deoxycholate, 1% Triton X-100), LiCl buffer (10mM Tris-HCl pH 8.0,250mM LiCl, 1mM EDTA, 0.5%NP-40,0.5%sodium Deoxycholate) and Tris-EDTA (TE) buffer clean to magnetic bead each primary；
SDS elution buffer (50mM is added in the magnetic bead for having specifically bound albumen and input sample by 2.8 Tris-HCl pH8.0,10mM EDTA, 1%SDS), 70 DEG C of water-baths, which are incubated overnight, carries out reciprocal cross connection；
2.9 are added Proteinase K and RNase A processing solution；
2.10 by 2.9 treated solution with QIAquick PCR Purification Kit (Qiagen, Hilden, Germany it) purifies；
The DNA of 2.11 purifying carries out the template of qRT-PCR to detect the degree of enrichment of each targeted area；The primer of PCR As shown in table 2.
2 QPCR primer sequence table of table
C11-IP1-F CACACAAGTGACAACACCAGC
C11-IP1-R ATTTACCCTCTGAGTGCCCC
C11-IP2-F TCCATGCTGAAGGGGTCTGA
C11-IP2-R TACCAAGTCACAAAGGAGGCT
C11-IP3-F TGAGCCTCCTTTGTGACTTGG
C11-IP3-R TTTGGGTTGGTGACATCACTG
C11-1kb up-F TGCACTTTCCCTTCACTGGG
C11-1kb up-R TCAGGCTAAGGGGTTAGGGGG
C11-0.5kb up-F GCAGCCTCACAGTACCATCTT
C11-0.5kb up-R AGGCATGAAGAGAGGGTTTGG
C11-0.5kb down-F CTGAATTTCGTGAGCGCCAG
C11-0.5kb down-R GTGCACCTCTGTGCTCATCT
C11-1kb down-F GAGCAACGCAGAAGACGGGT
C11-1kb down-R ACTCCCTGACCCCTTGTGCT
C11-interact-IP1F CGACCCAAGGAAGAAAGGGT
C11-interact-IP1R AGGGGATCTTCAGCCTCGT
C11-interact-IP2F GAGGCTGAAGATCCCCTGTG
C11-interact-IP2R GAAAGCACGCAGGGATACCA
C11-interact-0.5kb down-F TTTCCAGATCACAGTAGCCCG
C11-interact-0.5kb down-R GTCCTGGGACAAAGGATAGGC
tel-IP-F CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT
tel-IP-R GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT
2.12 QPCR reaction systems are as follows: 2XSYBR Green qPCR Mix 10ul primer R 1ul primers F 1ul template Xul deionized water supplies 20ul；Response procedures are as follows: 95 DEG C of 10s；Into the cycle stage: 95 DEG C of denaturation 10s, 56 DEG C of annealing 30s, 72 DEG C of extension 30s, totally 40 circulations, each circulate in annealing stage acquisition fluorescence signal and after amplification by identical conditions Data are analyzed, determine the Ct value of each sample；
2.13 analyze according to 2.12QPCR as a result, purifying DNA Ultra II DNA Library Prep Kit for Illumina (NEB) constructs DNA library,
2.14 sequencing libraries established illustrate to be sequenced in Hiseq X Ten instrument platform according to manufacturer.Each The sequencing depth in library is between 11.74-35.83million reads；
2.13 divide the site sgC11 and sgGal4 negative control sample sequencing data by peak calling software Analysis, the peak of overlapping is removed with bedtools.So that it is determined that target site and potential interaction sites, it is soft with IGV Part is visualized.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.

Claims (6)

1. one kind studies the interaction of specific gene group site based on CRISPR/cas9 and peroxidase APEX2 system The method of DNA, which is characterized in that itself comprising steps of
1) conversion of plasmid；
2) biotin labeling of specific position binding protein compound；
3) strepavidin magnetic beads enriched biological element labelled protein and DNA compound are utilized；
4) sequencing and analysis of enrichment DNA.
2. method according to claim 1, which is characterized in that the plasmid in the step 1) is selected from three kinds of plasmids, respectively table Up to dcas9 albumen, sgRNA specific sequence and peroxidase APEX2.
3. method according to claim 1, which is characterized in that in the step 2) using APEX2 biotin labeling function into Row closes on the biotin labeling of albumen.
4. method according to claim 1, which is characterized in that pUC pUC used in this method can be transiently transfected or be stablized Transfection, need to optimize APEX2 plasmid amount to obtain low background when wink turns.
5. method according to claim 1, which is characterized in that this method targets target gene group sequence by specificity sgRNA Column are carried out the biotin labeling of target site interaction albumen composition by peroxidase APEX2, are fixed by formaldehyde crosslinking The albumen composition of target site and biotin labeling.
6. method according to claim 1, it is further characterized in that, in this method Sync enrichment target site DNA sequence dna and with Interaction long-range DNA sequence dna, and by sequencing analyzed.
CN201810148785.5A 2018-02-14 2018-02-14 Method based on CRISPR/cas9 and peroxidase APEX2 system identification analysis specific gene group site interaction DNA Pending CN109504711A (en)

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Application publication date: 20190322