CN109957579A - Method based on CRISPR/cas9 and APEX2 system identification specific position interaction RNA - Google Patents

Method based on CRISPR/cas9 and APEX2 system identification specific position interaction RNA Download PDF

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CN109957579A
CN109957579A CN201811461038.3A CN201811461038A CN109957579A CN 109957579 A CN109957579 A CN 109957579A CN 201811461038 A CN201811461038 A CN 201811461038A CN 109957579 A CN109957579 A CN 109957579A
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rna
apex2
plasmid
albumen
dna
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刘贇
邱文青
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Fudan University
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Fudan University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/10—Vectors comprising a non-peptidic targeting moiety

Abstract

The invention belongs to field of biotechnology, are related to the method for identification specific position interaction RNA a kind of.This method is based on CRISPR/cas9 and peroxidase APEX2 system, and step includes: the conversion of plasmid；The plasmid includes the plasmid for expressing dcas9 albumen, sgRNA specific sequence or peroxidase APEX2 respectively；The biotin labeling of albumen is carried out using APEX2；Utilize strepavidin magnetic beads enriched biological element labelled protein, DNA RNA compound；The extraction and purification of RNA；It is enriched with the reverse transcription and analysis of RNA.This method is easy to operate, specific height, favorable reproducibility, is adapted to study the interaction RNA on any given chromosome location.

Description

Based on CRISPR/cas9 and APEX2 system identification specific position interaction RNA's Method
Technical field
The invention belongs to field of biotechnology, and in particular to one kind is based on CRISPR/cas9 and peroxidase APEX2 system It unites come the method for studying specific gene group site interaction RNA.
Background technique
In eukaryotic cells, DNA molecular has the height sense of organization, and is tightly wrapped the repetitive unit of nucleosome Form chromatin.However, chromatinic structure is dynamic change in living cells, local chromatin can be adjusted member Part such as transcription factor and non-coding RNA are close.In recent years it has been proposed that many mechanism for adjusting chromatin organization, for example, Each chromosome in eukaryocyte nucleus is all located at the specific region for being referred to as chromosomal region, it includes many logical Chang great little is the structural domain of millions of bases, referred to as topological structure domain；In topological structure domain, distal end DNA element passes through dynamic Interaction carrys out controlling gene expression.Many acting factors, including CTCF, laminins and other DNA binding protein, are involved in The formation in topological structure domain and interaction long-range between them.In addition, the modification of epigenetic, such as the methyl of DNA Change the modification with histone, there are also long-chain non-coding RNAs all to control the table of gene by regulating and controlling chromatinic higher structure Up to playing an important role in the process.These discoveries make us enter the new era of a chromatin functional study.However, right The overall understanding needs of chromatin function identify DNA, the RNA and transcription factor for being present in specific gene group site, this respect Research is due to technical difficult and challenging.
Many technologies for studying local chromatin composition have been suggested.For example, chromatin immune co-precipitation (ChIP) is just It is a kind of classics, is widely used for studying the technology of given albumen full-length genome distribution.However, there is presently no a kind of extensive The method of the given genomic locus local interaction molecule of the research of receiving.The nucleic acid probe of locking is used to identify and telomere The albumen that region combines, but this method is only limitted to the duplicate genome area of height.LexA DNA binding site is in gene It is integrated into Yeast genome in level, the chromatin for locus specificity purifies；However, this method is needed to target base Because of a group transformation for progress genome, so that chromatinic natural environment can be changed, and low efficiency.Genome editor's skill of improvement Art, such as the short palindrome repetitive sequence (CRISPR/ in interval of the similar effect nuclease (TALEN) of transcription activation factor and regular cluster Cas9 it) is already used to be enriched with required genomic locus.However, the method based on TALEN requires to be each site design one Section amino acid sequence, the method based on CRISPR is needed cell formaldehyde crosslinking, and needs high-affinity and specificity Antibody it is available.In addition, these methods cannot specifically provide the functional analysis to natural chromatin or full-length genome.
Summary of the invention
The purpose of the present invention is to provide one kind to study spy based on CRISPR/cas9 and peroxidase APEX2 system The method of specific gene group site interaction RNA.
In order to probe into the RNA molecule of specific gene group site interaction, the present invention develops one kind and is called CAPLOCUS (Combining CRISPR and peroxidase APEX2system to identify local chromatin Interactions method), by the specific capture and analysis to human chromosomal telomere area, CAPLOCUS can be effectively rich Collection target area and the long-chain non-coding RNA molecule interacted therewith.The present invention completes on this basis.
The present invention provides one kind to be based on CRISPR/cas9 and peroxidase APEX2 system identification specific position phase The step of method of interaction RNA, this method includes:
1) conversion of plasmid；The plasmid includes expressing dcas9 albumen, sgRNA specific sequence or peroxide respectively The plasmid of compound enzyme APEX2；
2) biotin labeling of specific position binding protein compound is faced using APEX2 biotin labeling function The biotin labeling of nearly albumen；
3) enrichment with magnetic bead biotinylated protein, DNA and RNA compound are utilized；
4) extraction and purification of RNA；
4) reverse transcription and analysis of RNA are enriched with.
PUC pUC used in this method can carry out transiently transfect or stable transfection, need to optimize when wink turns APEX2 plasmid amount with Obtain low background.
Preferably, this method by specificity sgRNA targeting target gene group sequence, by peroxidase APEX2 into The biotin labeling of row target site interaction albumen composition passes through the fixed target site of formaldehyde crosslinking and the egg of biotin labeling White, RNA compound.
In the present invention, the enrichment can be to be enriched with using strepavidin magnetic beads.
In one embodiment of the invention, the RNA of enrichment uses quantitative PCR detection, quantitative for example, by using reverse transcription PCR。
In one embodiment of the invention, this method can Sync enrichment target site DNA sequence dna and the RNA of interaction therewith Sequence, and analyzed by sequencing.
Specifically, the present invention adopts the following technical scheme that:
1. vector construction.The pUC pUC that the present invention uses include three plasmids, respectively express dcas9, MS2-APEX2 and sgRNA。
2. cell transformation and biotin labeling.Using polyethyleneimine (PEI) (Polysciences Inc., Warrington, PA, USA) carry out cell transient transfection.Cell transfecting for 24 hours after, with the biotin-phenol (Iri of 500uM S Biotech GmbH, Germany) processing cell 30min, hydrogen peroxide (H is added later2O2) to final concentration 1mM handle cell After 1min, reaction terminating liquid (10mM sodium azide, 10mM Vc, 5mM Trolox) is added immediately and terminates reaction.Cell first Aldehyde fixes 10 minutes, and 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.
4. the cell that step 2 is obtained carries out strepavidin magnetic beads enrichment, RNA is isolated and purified, carries out degree of enrichment after reverse transcription Detection.
The method of the present invention has the advantage that
1) CAPLOCUS can effectively be enriched with target area domain dna.
2) CAPLOCUS can be enriched with the RNA molecule with target area interaction.
This method is suitable for the analysis of any genomic locus, can get the RNA of interaction therewith.Meanwhile this method will The thinking that CRISPR gene editing system carries out specific site DNA analysis in conjunction with APEX2 is equally applicable to other genomes The combination of edit methods (such as TALEN) and APEX2.
This method is easy to operate, specific height, favorable reproducibility, is adapted to study the phase on any given chromosome location Interaction RNA.
Detailed description of the invention
Fig. 1 is CAPLOCUS system flow chart (A) and related plasmids structural diagrams (B).
Fig. 2 shows that CAPLOCUS systemic characteristic is enriched with telomere head of district chain non-coding RNA.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, below in conjunction with specific embodiment, to this Invention is further described.It should be appreciated that described herein, specific examples are only used to explain the present invention, is not used to limit The fixed present invention.Following example is to combine mankind's telomere area's example that method of the invention is further described.
The building and transfection of 1 plasmid expression vector of embodiment
The building of 1.1 plasmid expression vectors: in order to construct MS2-APEX2_NLS fusion protein expression vector, with the side of PCR Method expands from pcDNA3Connexin43-GFP-APEX2 (Addgene plasmid:49385) plasmid and obtains APEX2, then It is used into BamHI and XhoI digestion, is cloned into pHAGE-EFS-MCP-3XBFPnls (Addgene plasmid:75384) On carrier.PLH-sgRNA1-2XMS2 (Addgene plasmid:75389) plasmid after BbsI digestion with the oligomerization core of annealing Acid fragments connection is to obtain the plasmid for expressing sgRNA.The plasmid origin of dcas9 is expressed in Addgene:64107.SgRNA sequence Column such as the following table 1:
Table 1sgRNA sequence
Target Sequence(5'-3')
sgRNA-Telomere TAGGGTTAGGGTTAGGGTTA(SEQ ID NO1)
sgRNA-Gal4 AACGACTAGTTAGGCGTGTA(SEQ ID NO2)
1.2HEK293T cell culture: HEK293T cell is cultivated in 5%CO2 and 37 DEG C of incubator, culture medium DMEM (Life Technologies, USA), 10% fetal calf serum (Sigma, USA), 1% penicillin/strepto-.Cell every two days It is passed on the ratio of 1:5, detects mycoplasma contamination weekly.
1.3 cell transfectings: the instantaneous of cell is carried out with polyethyleneimine (PEI) (Polysciences Inc., USA) and is turned Dye.The MS2-APEX2_NLS plasmid corotation of the dcas9 plasmid of 900ng, the sgRNA plasmid of 4.5ng and 120ng are to convergence degree In 60% -80% T75 Tissue Culture Flask.
The enrichment and detection of 2 telomere area RNA of embodiment
2.1 cell transfectings for 24 hours after, trained with the biotin-phenol (Iri s Biotech GmbH, Germany) of 500uM It supports and handles cell 30min in case, hydrogen peroxide is added later to final concentration 1mM, room temperature handles cell 1min.Later, immediately plus Enter reaction terminating liquid (10mM sodium azide, 10mM Vc, 5mM Trolox) and terminate reaction, cleaning is three times.After terminating reaction, 0.2% formaldehyde fixes 10 minutes, and glycine terminates reaction.
2.2 or less the buffers used add 1x PIC, 100U/ml RNasin, 5mM EDTA, 0.5mM DTT.With (20mM HEPES pH7.5,10mM potassium chloride, the former of 1mM EDTA, 0.1mM activation consolidate acid to the Hypotonic buffer of 1mL Sodium, 0.2%NP-40,10% glycerol) lytic cell 15min, 4 DEG C, 13000g is centrifuged 1min, abandons supernatant.Cell precipitation is used (50mM Tri s-HCl pH 8.0,5mM EDTA, 150mM NaCl, 0.1%SDS, 0.5% are de- by 500ul RIPA buffer Oxycholic acid sodium, 1%TritonX-100) it is resuspended, Bioruptor Pico sonication device ultrasonication is big to DNA Small 200bp-1000bp.4 DEG C, 14000g, it is centrifuged 10min, takes supernatant, 5% supernatant is taken out and is saved as input sample At -20 DEG C.Supernatant is added in 50 μ l M-280 Streptavidin immunomagnetic beads, and 4 DEG C of rotations are incubated overnight.
2.3 magnetic beads successively use following buffer solution for cleaning 1 time, and 100U/ml RNasin is added in buffer:
RIPA buffer,High salt buffer(50mM Tri s-HCl pH 7.5,1M NaCl,5mM EDTA, 1%Triton X-100,0.1%SDS), Urea buffer (50mM Tri s-HCl pH 8.0,5mM EDTA, 2M urea, 1%Triton X-100,0.1%SDS), RIPA buffer and TE buffer.
2.4Input sample and cleaning after magnetic bead with 100ul Elution buffer (50mM Tri s-HClpH8.0, 10mM EDTA, 1%SDS) it is resuspended, 2ul protease k and 20U RNasin, 50 DEG C of processing 1h, 65 DEG C of reciprocal cross connection 1.5h is added.Instead The sample of crosslinking adds DEPC water to mend to 250ul, and Trizol 750ul is added, and 200ul chloroform is added after mixing, stands after mixing 5min, 13000g are centrifuged 10min, and isometric isopropanol to the cold, precipitation at room temperature 10min is added in supernatant.4 DEG C, 13000g centrifugation 10min obtains RNA, and RNA precipitate is resuspended with DEPC water.
The reverse transcription reagent box (RR047A) that 2.5 RNA extracted TAKARA removes genome pollution inverts, reverse primer For random primer, specific steps are referring to shop instruction.The cDNA of reverse transcription carries out Qpcr detection telomere TERRA enrichment.It is used Primer is as follows:
GAPDH-F CCATGTTCGTCATGGGTGTGA(SEQ ID NO3)
GAPDH-R CATGGACTGTGGTCATGAGT(SEQ ID NO4)
tel-IP-F CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT(SEQ ID NO5)
tel-IP-R GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT(SEQ ID NO6)
U1snRNAF GGCGAGGCTTATCCATTG(SEQ ID NO7)
U1snRNAR CCCACTACCACAAATTATGC(SEQ ID NO8)
The results show that degree of enrichment difference of the gaphd and actin in sgTelomere and sgGAL4 be not significant, u1 exists The part sgTelomere degree of enrichment is slightly larger than the part sgGAL4, and TERRA is significantly big in the part sgTelomere degree of enrichment In the part sgGAL4 (Fig. 2).
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.
Sequence table
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Claims (7)

1. it is a kind of identification specific position interaction RNA method, which is characterized in that this method be based on CRISPR/cas9 and Peroxidase APEX2 system；
Its step includes:
1) conversion of plasmid；
The plasmid includes the plasmid for expressing dcas9 albumen, sgRNA specific sequence or peroxidase APEX2 respectively；
2) biotin labeling of albumen is carried out using APEX2；
3) enrichment with magnetic bead biotinylated protein, DNA RNA compound are utilized；
4) extraction and purification of RNA；
5) reverse transcription and analysis of RNA are enriched with.
2. the method according to claim 1, wherein pUC pUC used in this method is by transiently transfecting or stablizing Transfection.
3. 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 of target site and biotin labeling, RNA compound.
4. the method according to claim 1, wherein the enrichment is enriched with using strepavidin magnetic beads.
5. the method according to claim 1, wherein the RNA of enrichment uses quantitative PCR detection.
6. according to the method described in claim 5, it is characterized in that, the quantitative PCR is reverse transcription quantitative PCR.
7. method according to claim 1, it is further characterized in that, this method Sync enrichment target site DNA sequence dna and therewith The RNA sequence of interaction, and analyzed by sequencing.
CN201811461038.3A 2018-12-01 2018-12-01 Method based on CRISPR/cas9 and APEX2 system identification specific position interaction RNA Pending CN109957579A (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003102196A1 (en) * 2002-05-30 2003-12-11 Centre National De La Recherche Scientifique (Cnrs) Vectors for expression of biotinylated proteins in mammalian cells, and their use for identification of protein-nucleic acid interactions in vivo
CN106093436A (en) * 2016-07-25 2016-11-09 高飞 A kind of simplicity detects RNA and the test kit of interactions between protein and using method thereof
US20170029831A1 (en) * 2014-05-09 2017-02-02 The Regents Of The University Of California Methods for selecting plants after genome editing

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003102196A1 (en) * 2002-05-30 2003-12-11 Centre National De La Recherche Scientifique (Cnrs) Vectors for expression of biotinylated proteins in mammalian cells, and their use for identification of protein-nucleic acid interactions in vivo
US20170029831A1 (en) * 2014-05-09 2017-02-02 The Regents Of The University Of California Methods for selecting plants after genome editing
CN106093436A (en) * 2016-07-25 2016-11-09 高飞 A kind of simplicity detects RNA and the test kit of interactions between protein and using method thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SAMUEL A MYERS等: "CRISPR/Cas9-APEX-mediated proximity labeling enables discovery of proteins associated with a predefined genomic locus in living cells", 《BIORXIV》 *

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Application publication date: 20190702