CN109957579A - A method for identifying site-specific interacting RNAs based on CRISPR/cas9 and APEX2 systems - Google Patents

Abstract

The invention belongs to the field of biotechnology, and relates to a method for identifying specific site interaction RNA. The method is based on CRISPR/cas9 and peroxidase APEX2 system, and the steps include: transformation of plasmid; the plasmid includes plasmids expressing dcas9 protein, sgRNA specific sequence or peroxidase APEX2 respectively; using APEX2 to carry out protein transformation Biotin labeling; enrichment of biotin-labeled protein, DNA or RNA complexes using streptomycin magnetic beads; RNA extraction and purification; reverse transcription and analysis of enriched RNA. The method is simple, specific, and reproducible, and is suitable for studying interacting RNAs at any given chromosomal location.

Description

Identification of site-specific interacting RNAs based on CRISPR/cas9 and APEX2 systems method

technical field

The invention belongs to the field of biotechnology, and in particular relates to a method for studying specific genome site interaction RNA based on CRISPR/cas9 and peroxidase APEX2 system.

Background technique

In eukaryotic cells, DNA molecules are highly organized and tightly wrap repeating units of nucleosomes to form chromatin. However, in living cells, the structure of chromatin is dynamic and localized chromatin can be accessed by regulatory elements such as transcription factors and noncoding RNAs. In recent years, a number of mechanisms have been proposed to regulate chromatin organization. For example, each chromosome in the nucleus of eukaryotic cells is located in a specific region called a chromosomal region, which contains many structures, usually several megabases in size. domains, called topological domains; in topological domains, distal DNA elements regulate gene expression through dynamic interactions. Numerous acting factors, including CTCF, cohesin, and other DNA-binding proteins, are involved in the formation of topological domains and their long-range interactions. In addition, epigenetic modifications, such as DNA methylation and histone modifications, as well as long non-coding RNAs play an important role in controlling gene expression by regulating the higher-order structure of chromatin. These findings lead us to a new era in the study of chromatin function. However, a comprehensive understanding of chromatin function requires the identification of DNA, RNA, and transcription factors present at specific genomic loci, which is challenging due to technical difficulties.

Numerous techniques to study local chromatin composition have been proposed. For example, chromatin immunoprecipitation (ChIP) is a classic and widely used technique to study the genome-wide distribution of a given protein. However, there is currently no widely accepted method to study locally interacting molecules at a given genomic locus. Locked nucleic acid probes have been used to identify proteins that bind to telomeric regions, but this approach is limited to highly repetitive genomic regions. The LexA DNA binding site is genetically integrated into the yeast genome for site-specific chromatin purification; however, this approach requires genomic modification of the target genome, which alters the natural environment of the chromatin, and low efficiency. Improved genome editing techniques such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR/cas9) have been used to enrich for desired genomic loci. However, TALEN-based methods require the design of an amino acid sequence for each site, CRISPR-based methods require cells to be cross-linked with formaldehyde, and antibodies with high affinity and specificity are available. Additionally, these methods do not specifically provide functional analysis of native chromatin or whole genomes.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a method based on the CRISPR/cas9 and peroxidase APEX2 system to study specific genomic site interacting RNAs.

In order to explore the RNA molecules interacting at specific genomic loci, the present invention developed a method called CAPLOCUS (Combining CRISPR and peroxidase APEX2system to identify local chromatin interactions). Enrich target regions and long non-coding RNA molecules that interact with them. The present invention is completed on this basis.

The present invention provides a method for identifying specific site interaction RNA based on CRISPR/cas9 and peroxidase APEX2 system. The steps of the method include:

1) transformation of plasmids; described plasmids include plasmids expressing dcas9 protein, sgRNA specific sequence or peroxidase APEX2 respectively;

2) Biotin labeling of specific site-binding protein complexes, and biotin labeling of adjacent proteins using the APEX2 biotin labeling function;

3) Enriching biotin-labeled protein, DNA and RNA complexes using magnetic beads;

4) RNA extraction and purification;

4) Reverse transcription and analysis of enriched RNA.

The plasmid system used in this method can perform transient transfection or stable transfection, and the amount of APEX2 plasmid needs to be optimized to obtain low background during transient transfection.

Preferably, the method uses specific sgRNA to target the target genome sequence, uses peroxidase APEX2 to perform biotin labeling of the target site interacting protein complex, and fixes the target site and biotin-labeled protein by formaldehyde cross-linking. , RNA complexes.

In the present invention, the enrichment can be enrichment by streptomycin magnetic beads.

In one embodiment of the invention, the enriched RNA is detected using quantitative PCR, eg, quantitative PCR using reverse transcription.

In one embodiment of the present invention, the method can simultaneously enrich the DNA sequence of the target site and the RNA sequence that interacts with it, and analyze it by sequencing.

Specifically, the present invention adopts the following technical scheme:

1. Vector construction. The plasmid system used in the present invention includes three plasmids, which respectively express dcas9, MS2-APEX2 and sgRNA.

2. Cell transformation and biotin labeling. Transient transfection of cells was performed using polyethyleneimine (PEI) (Polysciences Inc., Warrington, PA, USA). 24h after cell transfection, cells were treated with 500uM biotin-phenol (Iris Biotech GmbH, Germany) for 30min, then hydrogen peroxide (H ₂ O ₂ ) was added to a final concentration of 1mM to treat cells for 1min, and reaction stop solution (10mM) was added immediately. Sodium azide, 10 mM Vc, 5 mM Trolox) stopped the reaction. Cells were fixed with formaldehyde for 10 minutes and the reaction was terminated with glycine.

3. The cells obtained in step 2 were enriched with streptomycin magnetic beads, and the enriched DNA was subjected to qPCR to detect the enrichment degree of the targeted region.

4. Enrich the cells obtained in step 2 with streptomycin magnetic beads, isolate and purify RNA, and perform enrichment detection after reverse transcription.

The inventive method has the following advantages:

1) CAPLOCUS can effectively enrich the target region DNA.

2) CAPLOCUS can enrich RNA molecules that interact with target regions.

This method is suitable for the analysis of any genomic locus, and the RNA that interacts with it can be obtained. At the same time, the idea of combining the CRISPR gene editing system with APEX2 for specific site DNA analysis is also applicable to the combination of other genome editing methods (such as TALEN) with APEX2.

The method is simple, specific, and reproducible, and is suitable for studying interacting RNAs at any given chromosomal location.

Description of drawings

Fig. 1 is the flow chart of CAPLOCUS system (A) and the related plasmid structure diagram (B).

Figure 2 shows that the CAPLOCUS system specifically enriches long non-coding RNAs in telomeric regions.

Detailed ways

In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, but not to limit the present invention. The following examples further illustrate the method of the present invention in conjunction with the example of human telomere regions.

Example 1 Construction and transfection of plasmid expression vector

1.1 Construction of plasmid expression vector: In order to construct MS2-APEX2_NLS fusion protein expression vector, APEX2 was amplified from pcDNA3Connexin43-GFP-APEX2 (Addgene plasmid: 49385) plasmid by PCR method, and then digested with BamHI and XhoI, Cloning into pHAGE-EFS-MCP-3XBFPnls (Addgene plasmid: 75384) vector. The pLH-sgRNA1-2XMS2 (Addgene plasmid: 75389) plasmid was digested with BbsI and then ligated with the annealed oligonucleotide fragment to obtain a plasmid expressing sgRNA. The plasmid expressing dcas9 was derived from Addgene:64107. The sgRNA sequences are listed in Table 1 below:

Table 1sgRNA sequences

Target Sequence(5'-3') sgRNA-Telomere TAGGGTTAGGGTTAGGGTTA (SEQ ID NO 1) sgRNA-Gal4 AACGACTAGTTAGGCGTGTA (SEQ ID NO 2)

1.2 HEK293T cell culture: HEK293T cells were cultured in a 5% CO2 and 37°C incubator with DMEM (Life Technologies, USA), 10% fetal bovine serum (Sigma, USA), and 1% penicillin/streptomycin. Cells were passaged at a ratio of 1:5 every two days and tested weekly for mycoplasma contamination.

1.3 Transfection of cells: Transient transfection of cells was performed with polyethyleneimine (PEI) (Polysciences Inc., USA). 900ng of dcas9 plasmid, 4.5ng of sgRNA plasmid and 120ng of MS2-APEX2_NLS plasmid were co-transferred into T75 cell culture flasks at 60%–80% confluence.

Example 2 Enrichment and detection of RNA in telomere region

2.1 After 24h of cell transfection, cells were treated with 500uM biotin-phenol (Iris Biotech GmbH, Germany) for 30min in an incubator, then hydrogen peroxide was added to a final concentration of 1mM, and cells were treated at room temperature for 1min. After that, reaction stop solution (10 mM sodium azide, 10 mM Vc, 5 mM Trolox) was added immediately to stop the reaction, and the reaction was washed three times. After terminating the reaction, 0.2% formaldehyde was fixed for 10 minutes, and the reaction was terminated by glycine.

2.2 The buffers used below were all added with 1x PIC, 100U/ml RNasin, 5mM EDTA, 0.5mM DTT. Cells were lysed with 1 mL of Hypotonic buffer (20 mM HEPES pH7.5, 10 mM KCl, 1 mM EDTA, 0.1 mM activated sodium orthostearate, 0.2% NP-40, 10% glycerol) for 15 min, centrifuged at 13000g for 1 min at 4°C, Discard the supernatant. Cell pellets were resuspended in 500ul RIPA buffer (50mM Tri s-HCl pH 8.0, 5mM EDTA, 150mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% TritonX-100), and disrupted to DNA size by Bioruptor Pico sonication device 200bp-1000bp. 4°C, 14000g, centrifugation for 10min, take the supernatant, take out 5% of the supernatant as the input sample and store it at -20°C. The supernatant was added to 50 μl of M-280 streptavidin immunomagnetic beads and incubated overnight at 4°C with rotation.

2.3 Wash the magnetic beads with the following buffers in turn, and add 100U/ml RNasin to the buffers:

RIPA buffer, High salt buffer (50mM Tris-HCl pH 7.5, 1M NaCl, 5mM EDTA, 1% Triton X-100, 0.1% SDS), Urea buffer (50mM Tris-HCl pH 8.0, 5mM EDTA, 2M urea, 1% Triton X-100, 0.1% SDS), RIPA buffer and TE buffer.

2.4 Input the sample and washed magnetic beads with 100ul Elution buffer (50mM Tris-HCl pH8.0, 10mM EDTA, 1%SDS), resuspend, add 2ul proteinase k and 20U RNasin, treat at 50℃ for 1h, reverse crosslink at 65℃ 1.5h. Add DEPC water to the anti-crosslinked sample to make up to 250ul, add 750ul of Trizol, and add 200ul of chloroform after mixing. RNA was obtained by centrifugation at 13000g for 10 min at 4°C, and the RNA pellet was resuspended in DEPC water.

2.5 The extracted RNA was reversed with TAKARA Reverse Transcription Kit (RR047A) to remove genomic contamination. The reverse primers were random primers. For specific steps, refer to the manufacturer's instructions. The reverse transcribed cDNA was subjected to QPCR to detect telomere TERRA enrichment. The primers used are as follows:

GAPDH-F CCATGTTCGTCATGGGTGTGA (SEQ ID NO3) GAPDH-R CATGGACTGTGGTCATGAGT (SEQ ID NO 4) tel-IP-F CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT (SEQ ID NO5) tel-IP-R GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT (SEQ ID NO 6) U1snRNAF GGCGAGGCTTATCCATTG (SEQ ID NO 7) U1snRNAR CCCACTACCACAAATTATGC (SEQ ID NO 8)

The results showed that there was no significant difference in the enrichment of gapd and actin in sgTelomere and sgGAL4, the enrichment of u1 in sgTelomere was slightly higher than that in sgGAL4, and the enrichment of TERRA in sgTelomere was significantly higher than that in sgGAL4 (Figure 2).

All documents mentioned herein are incorporated by reference in this application as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

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Claims (7)

1. a method for identifying specific site interaction RNA, characterized in that the method is based on CRISPR/cas9 and peroxidase APEX2 system; Its steps include: 1) Transformation of plasmid; Described plasmid comprises the plasmid that expresses dcas9 protein, sgRNA specific sequence or peroxidase APEX2 respectively; 2) Biotin labeling of proteins using APEX2; 3) Use magnetic beads to enrich biotin-labeled protein, DNA or RNA complexes; 4) RNA extraction and purification; 5) Reverse transcription and analysis of enriched RNA. 2 . The method according to claim 1 , wherein the plasmid system used in the method is transient transfection or stable transfection. 3 . 3. The method according to claim 1, wherein the method targets the target genome sequence by specific sgRNA, carries out the biotin labeling of the target site interacting protein complex by peroxidase APEX2, and is cross-linked by formaldehyde The target site and biotin-labeled protein and RNA complexes are immobilized. 4. The method according to claim 1, wherein the enrichment is performed by streptomycin magnetic beads. 5. The method of claim 1, wherein the enriched RNA is detected using quantitative PCR. 6. The method according to claim 5, wherein the quantitative PCR is reverse transcription quantitative PCR. 7 . The method according to claim 1 , further characterized in that the method simultaneously enriches the DNA sequence of the target site and the RNA sequence interacting with it, and analyzes by sequencing. 8 .