搜索

In vitro Engineered DNA-binding Molecule-mediated Chromatin Immunoprecipitation (in vitro enChIP) Using CRISPR Ribonucleoproteins in Combination with Next-generation Sequencing (in vitro enChIP-Seq) for the Identification of Chromosomal Interactions
使用CRISPR核糖核蛋白的体外工程DNA结合分子介导的染色质免疫沉淀(体外enChIP)结合下一代测序(体外enChIP-Seq)用于染色体相互作用的鉴定   

评审
匿名评审
下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

We have developed locus-specific chromatin immunoprecipitation (locus-specific ChIP) technologies consisting of insertional ChIP (iChIP) and engineered DNA-binding molecule-mediated ChIP (enChIP). Locus-specific ChIP is a method to isolate a genomic region of interest from cells while it also identifies what binds to this region using mass spectrometry (for protein) or next generation sequencing (for RNA or DNA) as described in Fujita et al. (2016a). Recently, we identified genomic regions that physically interact with a locus using an updated form of enChIP, in vitro enChIP, in combination with NGS (in vitro enChIP-Seq) (Fujita et al., 2017a). Here, we describe a protocol on in vitro enChIP to isolate a target locus for identification of genomic regions that physically interact with the locus.

Keywords: Chromatin immunoprecipitation(染色质免疫沉淀), ChIP(ChIP), locus-specific ChIP(基因座特异性ChIP), enChIP(enChIP), in vitro enChIP(体外enChIP), NGS(下一代测序), in vitro enChIP-Seq(体外enChIP-Seq)

Background

Elucidation of molecular mechanisms underlining genome functions requires the identification of molecules that interact with the genomic region of interest in vivo. To this end, we have developed locus-specific chromatin immunoprecipitation (locus-specific ChIP) technologies consisting of insertional ChIP (iChIP) and engineered DNA-binding molecule-mediated ChIP (enChIP) (Fujita et al., 2016a). Locus-specific ChIP is a method to biochemically isolate a genomic region of interest from cells. Molecules interacting with that isolated genomic region are identified by biochemical analyses, such as mass spectrometry (MS) and next generation sequencing (NGS). In iChIP, an exogenous DNA-binding protein and its recognition DNA sequence are used for ‘in cell’ locus-tagging. In enChIP, engineered DNA-binding molecules, such as transcription activator-like (TAL) proteins and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), are used for ‘in cell’ locus-tagging. The tagged loci are isolated by affinity-purification. We recently developed in vitro enChIP, in which the locus-tagging was performed in vitro (in a test tube) using recombinant and/or synthetic engineered DNA-binding molecules (Figure 1) (Fujita and Fujii, 2014a and 2016b). Here, we describe a protocol of in vitro enChIP using CRISPR ribonucleoproteins (RNPs) followed by NGS (in vitro enChIP-Seq) for the identification of genomic regions that physically interact with the locus of interest (Fujita et al., 2017a).


Figure 1. Scheme of in vitro enChIP using CRISPR RNPs. Figure 1 reproduced under the Creative Commons Attribution License from: Fujita et al. Efficient sequence-specific isolation of DNA fragments and chromatin by in vitro enChIP technology using recombinant CRISPR ribonucleoproteins. Genes Cells. 2016; 21: 370-377.

Scheme of in vitro enChIP using CRISPR RNPs (Figure 1)
1. A nuclease-dead form of Cas9 (dCas9) fused to an epitope-tag(s) (e.g., 3xFLAG-dCas9) is prepared as a recombinant protein. Guide RNA (gRNA) that recognizes the DNA sequence of the genomic region of interest is generated chemically. As to gRNA, single gRNA (sgRNA) or a complex of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) can be used.
2. Cells to be analyzed are crosslinked, if necessary, and lysed, and DNA is fragmented.
3. The 3xFLAG-dCas9/gRNA complex is incubated with the fragmented chromatin DNA in a test tube. The genomic DNA bound to the 3xFLAG-dCas9/gRNA complex is affinity-purified using antibody against the epitope-tag(s) or dCas9 itself. Alternatively, the complex can be purified using tags fused to gRNA (e.g., biotin). The isolated complexes retain molecules that interact with the target locus.
4. Reverse crosslinking, if necessary, and subsequent purification of DNA, RNA, or proteins allows identification and characterization of these molecules. For example, NGS or MS can be combined to identify genomic regions or proteins that physically bind to the target locus.

We previously described a detailed protocol of ‘in cell’ enChIP for the identification of proteins that interact with a locus of interest in Bio-protocol (Fujita and Fujii, 2014b). Procedures for the preparation of sonicated chromatin in in vitro enChIP are the same as those for ‘in cell’ enChIP. In addition, other procedures (e.g., preparation of Dynabeads) are similar between them. Therefore, we recommend to refer to the ‘in cell’ enChIP protocol (Fujita and Fujii, 2014b) in parallel.

Materials and Reagents

  1. 1.5 ml centrifuge tube (SARSTEDT, catalog number: 72.690.001 )
  2. Pipettes tips (DNase/RNase-free)
  3. DT40 cell (RIKEN BioResource Center) (an example)
  4. 10 µM crRNA (FASMAC, diluted in DNase/RNase-free water) (see step B1)
  5. 10 µM tracrRNA (FASMAC, diluted in DNase/RNase-free water) (see step B1)
  6. 3xFLAG-dCas9-D (Sysmex, ProCube)
  7. 37% formaldehyde (NACALAI TESQUE, catalog number: 16223-55 )
  8. Anti-FLAG M2 antibody (Sigma-Aldrich, catalog number: F1804 )
  9. Normal mouse IgG (Santa Cruz Biotechnology, catalog number: sc-2025 )
  10. Dynabeads-Protein G (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10004D )
  11. RNasin plus RNase inhibitor (Promega, catalog number: N261A )
  12. 10 mg/ml RNase A (Sigma-Aldrich, catalog number: R6513 )
  13. 10% SDS solution (NACALAI TESQUE, catalog number: 30562-04 )
  14. 20 mg/ml Proteinase K (Roche Diagnostics, catalog number: 3115828001 )
  15. ChIP DNA Clean and Concentrator (ZYMO RESEARCH, catalog number: D5205 )
  16. TruSeq ChIP Sample Prep Kit (Illumina, catalog number: IP-202-1012 )
  17. Potassium chloride (KCl) (NACALAI TESQUE, catalog number: 28538-62 )
  18. Dithiothreitol (DTT) (NACALAI TESQUE, catalog number: 14128-91 )
  19. 1 M HEPES (pH 7.1-7.5) (NACALAI TESQUE, catalog number: 17557-94 )
  20. 0.5 M ethylenediaminetetraacetate acid (EDTA) (pH 8.0) (NACALAI TESQUE, catalog number: 06894-85 )
  21. 1 M magnesium chloride hexahydrate (MgCl2·6H2O) (NACALAI TESQUE, catalog number: 20942-34 )
  22. cOmplete, Mini, EDTA-free Protease Inhibitor (Roche Diagnostics, catalog number: 4693159001 )
  23. UltraPure DNase/RNase-Free Distilled Water (DDW) (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10977015 )
  24. 10x phosphate buffered saline (PBS) (pH 7.4) (NACALAI TESQUE, catalog number: 27575-31 )
  25. 1x PBS (10x dilution of 10x PBS with distilled water)
  26. Tween-20 (Sigma-Aldrich, catalog number: P5927 )
    Note: This product has been discontinued.
  27. BSA fraction V (7.5%) (Thermo Fisher Scientific, catalog number: 15260037 )
  28. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9625 )
  29. 1 M Tris (pH 8.0) (AppliChem, catalog number: A4577 )
  30. 1 M Tris (pH 7.5) (AppliChem, catalog number: A4263 )
  31. Polyethylene Glycol Mono-p-isooctylphenyl Ether (Triton X-100) (NACALAI TESQUE, catalog number: 12967-45 )
  32. IGEPAL CA-630 (Sigma-Aldrich, catalog number: I8896 )
  33. 3xFLAG peptide (Sigma-Aldrich, catalog number: F4799 )
  34. 1 M KCl (see Recipes)
  35. 0.1 M DTT (see Recipes)
  36. In vitro CRISPR buffer (+ 0.5 mM DTT) (see Recipes)
  37. PBS-T (see Recipes)
  38. PBS-T-BSA (see Recipes)
  39. 5 M NaCl (see Recipes)
  40. Modified low salt buffer (+ 0.04% SDS) (see Recipes)
  41. TBS (see Recipes)
  42. TBS-IGEPAL CA-630 (see Recipes)
  43. Elution buffer (see Recipes)

Equipment

  1. Pipettes
  2. Ultrasonic Disruptor (TOMY SEIKO, model: UD-201 )
  3. Block heater (Eppendorf, catalog number: 5355 000.046 )
  4. Magnetic stand (Magical Trapper) (TOYOBO, catalog number: MGS-101 )
  5. Centrifuge (Eppendorf, catalog number: 5427 R )
  6. Vortex mixer (Fisher Scientific, catalog number: 128101 )
  7. Rotator (AS ONE, catalog number: TR-118 )
  8. HiSeq 2500 system (Illumina, model: HiSeq 2500 System )

Software

  1. Integrative Genomics Viewer, http://software.broadinstitute.org/software/igv/IGV

Procedure

  1. Preparation of sonicated chromatin
    1. Formaldehyde crosslinking of cells (e.g., 2 x 107 DT40 cells, 1% formaldehyde in culture medium at 37 °C for 5 min) and preparation of chromatin are performed as described previously (steps B and C in Fujita and Fujii, 2014b).
    2. Sonicate the prepared chromatin (e.g., Output: 3, Duty: 100%, Time: Free, 10 sec x 15 cycles using Ultrasonic Disruptor UD-201 (TOMY)) as described previously (step D in Fujita and Fujii, 2014b) except for substituting 800 µl of in vitro CRISPR buffer (+ 0.5 mM DTT) (see Recipes) instead of MLB3.
    3. Evaluate fragmentation of chromatin (the average length of fragments is about 2 kbp) as described previously (step E in Fujita and Fujii, 2014b).

  2. Preparation of gRNA
    1. Mix 2 µl of 10 µM crRNA and 2 µl of 10 µM tracrRNA. In our previous study (Fujita et al., 2017a), a crRNA (cggcaggcucgggugcgccuguuuuagagcuaugcuguuuug) and a tracrRNA (aacagcauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuuuu) were used for targeting the chicken Pax5 promoter region; the underlined sequence is corresponding to the target site.
    2. Incubate at 100 °C for 2 min in a block heater. Incubation for 2 min does not make considerable evaporation. After heating, put the RNAs out of the heater to naturally cool down to room temperature. The RNAs are ready for step D6.

  3. Preparation of Dynabeads conjugated with antibody (anti-FLAG antibody or normal mouse IgG)
    1. Prepare two 1.5 ml tubes; one is for anti-FLAG antibody and the other is for normal mouse IgG. Add 20 µl slurry of Dynabeads-Protein G in each tube (20 µl x 2 tubes). In this regard, we usually use 20 µl slurry of the beads for 2 µg of an antibody (see step C7). In this protocol, we describe preparation of one sample for NGS analysis. In this regard, normal mouse IgG is used for pre-clear in step E1 and anti-FLAG antibody is used in Procedure D. If you handle another sample (e.g., affinity purification without gRNA as a negative control), you may prepare chromatin and Dynabeads for it separately.
    2. Place the tubes on the magnetic stand on ice for 3 min. Discard the supernatant by pipetting. Detailed instructions on the use of the magnetic stand are illustrated in Figure 2 in Fujita and Fujii, 2014b.
    3. Resuspend the pellet in 0.5 ml of PBS-T (see Recipes). Place the tubes on the magnetic stand on ice for 3 min. Discard the supernatant by pipetting.
    4. Repeat step F3 (total of twice).
    5. Resuspend the pellet in 0.5 ml of PBS-T-BSA (see Recipes).
    6. Add 2 µg of antibody, to the beads either anti-FLAG or normal mouse IgG. Rotate at 4 °C overnight. As to Dynabeads conjugated with anti-FLAG antibody, proceed to Procedure D. As to Dynabeads conjugated with normal mouse IgG, proceed to the next step (C7).
    7. Centrifuge briefly (400 x g for 2-3 sec). Place the tubes on the magnetic stand on ice for 3 min. Discard the supernatant by pipetting.
    8. Resuspend the pellet in 0.5 ml of PBS-T. Invert several times and centrifuge briefly (400 x g for 2-3 sec). Place the tubes on the magnetic stand on ice for 3 min. Discard the supernatant by pipetting.
    9. Repeat step C8, twice (total of three times). The Dynabeads conjugated with normal mouse IgG are ready for step E1.

  4. Preparation of Dynabeads conjugated with recombinant CRISPR RNPs
    1. Add 2 µg of recombinant 3xFLAG-dCas9-D to the tube, in which Dynabeads are conjugated with anti-FLAG antibody in 0.5 ml of PBS-T-BSA (step C6). Rotate at 4 °C for 2-3 h.
    2. Centrifuge briefly (400 x g for 2-3 sec). Place the tubes on the magnetic stand on ice for 3 min. Discard the supernatant by pipetting.
    3. Resuspend the pellet in 0.5 ml of PBS-T. Invert several times and centrifuge briefly (400 x g for 2-3 sec). Place the tubes on the magnetic stand on ice for 3 min. Discard the supernatant by pipetting.
    4. Repeat step D3 (total of twice).
    5. Resuspend the pellet in 0.5 ml of in vitro CRISPR buffer (+ 0.5 mM DTT). Invert several times and centrifuge briefly (400 x g for 2-3 sec). Place the tubes on the magnetic stand on ice for 3 min. Discard the supernatant by pipetting.
    6. Add 100 µl of in vitro CRISPR buffer (+ 0.5 mM DTT) and 4 µl of the gRNA prepared in Procedure B.
    7. Incubate at 37 °C for 10 min. The Dynabeads conjugated with recombinant CRISPR RNPs are ready for step E3.

  5. Affinity purification (chromatin immunoprecipitation)
    1. Take 800 µl of the fragmented chromatin (Procedure A), which corresponds to chromatin extracted from 2 x 107 cells, and add to the tube containing the Dynabeads conjugated with normal mouse IgG (step C9). Rotate at 4 °C for 1 h.
    2. Place the tubes on the magnetic stand on ice for 3 min.
    3. Transfer the cleared supernatant into the tube, in which the Dynabeads conjugated with recombinant CRISPR RNPs were prepared (step D7). Add RNase inhibitor (40 U/ml, final concentration).
    4. Incubate at 37 °C for 20 min at 650 rpm in a block heater.
    5. Place the tubes on the magnetic stand on ice for 3 min. Discard the supernatant by pipetting.
    6. Wash the beads with 1 ml of modified low salt buffer (+ 0.04% SDS) (see Recipes). Rotate at 4 °C for 10 min. Place the tubes on the magnetic stand on ice for 3 min. Discard the supernatant by pipetting.
    7. Repeat step E6, three times (total of 4 times).
    8. Add 1 ml of TBS-IGEPAL CA-630 (see Recipes). Rotate at 4 °C for 10 min. Place the tubes on the magnetic stand on ice for 3 min. Discard the supernatant by pipetting.
    9. Elution 1: Resuspend the beads in 60 µl of elution buffer (see Recipes). Incubate at 37 °C for 20 min at 850 rpm in a block heater. Place the tubes on the magnetic stand on ice for 3 min. Transfer the supernatant into a 1.5 ml tube.
    10. Elution 2: Resuspend the beads in 40 µl of elution buffer. Incubate at 37 °C for 5 min at 850 rpm in a block heater. Place the tubes on the magnetic stand on ice for 3 min. Transfer the supernatant into the 1.5 ml tube, in which 60 µl of the eluate was collected (total 100 µl).
    11. Add 4 μl of 5 M NaCl (see Recipes). Incubate at 65 °C overnight.
    12. Add 2 µl of 10 mg/ml RNase A. Incubate at 37 °C for 1 h.
    13. Add 5 µl of 10% SDS and 5 µl of 20 mg/ml Proteinase K. Incubate at 45 °C for 2 h.
    14. Purify DNA with ChIP DNA Clean and Concentrator (Zymo Resarch) according to the manufacturer’s protocol (e.g., 0.6 ml of ChIP DNA binding buffer and 60 µl of elution buffer in the kit can be used).

  6. Preparation of NGS libraries and NGS analysis
    1. Prepare NGS libraries from the DNA samples (step E14) using TruSeq ChIP Sample Prep Kit (Illumina) according to the manufacturer’s protocol.
    2. Subject the DNA libraries to DNA sequencing using the HiSeq 2500 system (36 bp single end, 20-50 million reads).
    3. You can visualize the NGS data in an appropriate viewer (e.g., Integrative Genomics Viewer, http://software.broadinstitute.org/software/igv/IGV) (Figures 2 and 3).


      Figure 2. Isolation of a target genomic region by in vitro enChIP. A. Target position of a gRNA; B. NGS peak images around the target region. NGS data were visualized in IGV. Figure 2 reproduced under the Creative Commons Attribution License from: Fujita et al. Locus-specific ChIP combined with NGS analysis reveals genomic regulatory regions that physically interact with the Pax5 promoter in a chicken B cell line. DNA Res. 2017, 24: 537-548.


      Figure 3. Identification of genomic regions interacting with the Pax5 promoter region. Genomic regions that physically interact with the Pax5 promoter region. NGS data were visualized in IGV. Figure 3 reproduced under the Creative Commons Attribution License from: Fujita et al. Locus-specific ChIP combined with NGS analysis reveals genomic regulatory regions that physically interact with the Pax5 promoter in a chicken B cell line. DNA Res. 2017, 24: 537-548.

Data analysis

  1. For visualization in IGV, the NGS data (BAM file) is uploaded to IGV. A reference genome information (e.g., galGal4) is also loaded to map the NGS data.
  2. Enlarge the position of the target genomic region in the browser to confirm isolation of a target genomic region (Figure 2B). Also check the NGS data with a negative control NGS data (e.g., in vitro enChIP-Seq without gRNA) to judge if the signal at the target genomic region is not noise.
  3. Viewing on a genome-wide scale, you can manually identify genomic regions that interact with the target genomic region (Figure 3). In this regard, the peak regions detected in the NGS data but not in a negative control NGS data can be judged as interacting genomic regions. Narrow peaks of each in vitro enChIP-Seq dataset can be automatically detected using MACS2 or other software (Fujita et al., 2017b).

Notes

  1. It is also possible to combine conventional ‘in cell’ enChIP using CRISPR with NGS (enChIP-Seq) (Fujita et al., 2017b). In this case, the CRISPR components (e.g., 3xFLAG-dCas9 and sgRNA) are expressed in cells and enChIP is performed to isolate a target locus as described previously (Fujita and Fujii, 2014b and 2015). After isolation of the locus, DNA is purified and analyzed as shown in steps E12-F3.
  2. It is possible to confirm isolation of the target genomic region by qPCR before NGS analysis. To this end, the DNA sample (step E14) is subjected to qPCR with a primer set specific to the target genomic region. Examples are shown previously (Fujita et al., 2016b and 2017a).
  3. Off-target binding sites may be listed as candidates of interacting genomic regions. To eliminate such potential off-target sites, it would be necessary to check whether DNA sequences of the candidate genomic regions do not contain sequences similar to the target sequences of gRNA.
  4. To increase the reliability of extraction of interacting genomic regions, it would be useful to perform in vitro enChIP with two or more gRNAs to the same genomic region. In this regard, genomic regions commonly detected by in vitro enChIP using different gRNAs (except for the target genomic region) can be judged as bona fide interacting genomic regions. Details are described previously (Fujita et al., 2017b)

Recipes

  1. 1 M KCl solution (50 ml)
    3.73 g KCl (MW: 74.55)
    DDW to 50 ml
  2. 0.1 M DTT solution (10 ml) (store at -20 °C)
    0.15 g DTT (MW: 154.25)
    DDW to 10 ml
  3. in vitro CRISPR buffer (+ 0.5 mM DTT)
    20 mM HEPES, pH 7.1-7.5
    150 mM KCl
    0.1 mM EDTA
    10 mM MgCl2
    0.5 mM DTT
    1x protease inhibitors
    For preparing 50 ml:
    1 ml 1 M HEPES (pH 7.1-7.5)
    7.5 ml 1 M KCl
    10 µl 0.5 M EDTA
    500 µl 1 M MgCl2
    250 µl 0.1 M DTT (add just before use)
    5 tablets cOmplete, Mini, EDTA-free (add just before use)
    41 ml DDW
  4. PBS-T (10 ml)
    PBS (pH 7.4), 0.01% Tween-20
    10 ml PBS
    10 µl 10% Tween-20
  5. PBS-T-BSA
    PBS (pH 7.4)
    0.01% Tween-20
    0.1% BSA
    For preparing 10 ml:
    10 ml PBS
    10 µl 10% Tween-20
    133 µl 7.5% BSA fraction V
  6. 5 M NaCl solution (50 ml)
    14.61 g NaCl (MW: 58.44)
    DDW to 50 ml
  7. Modified low salt buffer (+ 0.04% SDS)
    20 mM Tris, pH 8.0
    2 mM EDTA
    150 mM NaCl
    0.1% Triton X-100
    0.04% SDS
    1x protease inhibitors
    5 U/ml RNase inhibitor
    For preparing 50 ml:
    1 ml 1 M Tris (pH 8.0)
    200 µl 0.5 M EDTA
    1.5 ml 5 M NaCl
    50 µl Triton X-100
    200 µl 10% SDS (add before use)
    5 tablets cOmplete-Mini (add before use)
    6.25 µl RNasin plus (40 U/ml) (add before use)
    47.05 ml DDW
  8. TBS
    50 mM Tris (pH 7.5)
    150 mM NaCl
    For preparing 10 ml:
    500 µl 1 M Tris (pH 7.5)
    300 µl 5 M NaCl
    9.2 ml DDW
  9. TBS-IGEPAL CA-630
    50 mM Tris (pH 7.5)
    150 mM NaCl
    0.1% IGEPAL CA-630
    For preparing 10 ml:
    9.99 ml TBS
    10 µl IGEPAL CA-630
  10. Elution buffer
    50 mM Tris (pH 7.5)
    150 mM NaCl
    0.1% IGEPAL CA-630
    500 µg/ml 3xFLAG peptide
    For preparing 500 µl:
    50 µl 5 mg/ml 3xFLAG peptide in TBS
    450 µl TBS-IGEPAL CA-630

Acknowledgments

This protocol was adapted from the previously published paper (Fujita et al., 2017a). We thank M. Yuno for technical assistance. This work was supported by Grant-in-Aid for Scientific Research (C) (#15K06895) (T.F.) and Grant-in-Aid for Scientific Research (B) (#15H04329) (T.F., H.F.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
T.F. and H.F. have patents on enChIP (“Method for isolating specific genomic region using molecule binding specifically to endogenous DNA sequence”; patent number: Japan 5,954,808; patent application number: WO2014/125668). T.F. and H.F. are founders of Epigeneron, LLC.

References

  1. Fujita, T. and Fujii, H. (2014a). Efficient isolation of specific genomic regions retaining molecular interactions by the iChIP system using recombinant exogenous DNA-binding proteins. BMC Mol Biol 15: 26.
  2. Fujita, T. and Fujii, H. (2014b). Identification of proteins interacting with genomic regions of interest in vivo using engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP). Bio Protoc 4(10): e1124.
  3. Fujita, T. and Fujii, H. (2015). Isolation of specific genomic regions and identification of associated molecules by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR. Methods Mol Biol 1288: 43-52.
  4. Fujita, T. and Fujii, H. (2016a). Biochemical analysis of genome functions using locus-specific chromatin immunoprecipitation technologies. Gene Regul Syst Bio 10(Suppl 1): 1-9.
  5. Fujita, T., Kitaura, F., Yuno, M., Suzuki, Y., Sugano, S. and Fujii, H. (2017a). Locus-specific ChIP combined with NGS analysis reveals genomic regulatory regions that physically interact with the Pax5 promoter in a chicken B cell line. DNA Res 24(5): 537-548.
  6. Fujita, T., Yuno, M. and Fujii, H. (2016b). Efficient sequence-specific isolation of DNA fragments and chromatin by in vitro enChIP technology using recombinant CRISPR ribonucleoproteins. Genes Cells 21(4): 370-377.
  7. Fujita, T., Yuno, M., Suzuki, Y., Sugano, S. and Fujii, H. (2017b). Identification of physical interactions between genomic regions by enChIP-Seq. Genes Cells 22(6): 506-520.

简介

我们开发了基因座特异性染色质免疫沉淀(基因座特异性芯片)技术,包括插入ChIP(iChIP)和工程DNA-结合分子介导ChIP(enChIP)。 基因座特异性ChIP是一种从细胞中分离感兴趣的基因组区域的方法,同时它还使用质谱(用于蛋白质)或下一代测序(用于RNA或DNA)鉴定什么与该区域结合,如Fujita等人 (2016a)。 最近,我们使用更新后的enChIP形式,结合NGS( in vitro enChIP-Seq),鉴定了与基因座物理相互作用的基因组区域(Fujimita et al。,2017a)。 在这里,我们描述了一个体外试验的方法,用于分离靶基因座以鉴定与基因座物理相互作用的基因组区域。
【背景】阐明基因组功能强调的分子机制需要鉴定与感兴趣的基因组区域相互作用的分子。为此,我们开发了由插入ChIP(iChIP)和工程化DNA结合分子介导的ChIP(enChIP)组成的基因座特异性染色质免疫沉淀技术(基因座特异性ChIP)技术(Fujita等人 ,2016a)。基因座特异性ChIP是从细胞生物化学分离感兴趣的基因组区域的方法。通过生物化学分析如质谱(MS)和下一代测序(NGS)鉴定与分离的基因组区域相互作用的分子。在iChIP中,外源DNA结合蛋白及其识别DNA序列用于“在细胞中”的基因座标记。在enChIP中,工程化的DNA结合分子,例如转录激活物样(TAL)蛋白和聚集的规则间隔短回文重复序列(CRISPR)被用于“细胞内”基因座标记。标记的基因座通过亲和纯化分离。我们最近开发了一种体外试剂盒,其中使用重组和/或合成的工程DNA结合分子在体外(在试管中)进行基因座标记(图1)(藤田和藤井,2014a和2016b)。在这里,我们描述了使用CRISPR核糖核蛋白(RNP)和NGS( in vitro enChIP-Seq)体外体外 enChIP的方案,用于鉴定物理相互作用的基因组区域感兴趣的位置(Fujita等人,2017a)。

“”src
图1.利用CRISPR RNP进行的体外试验图1.使用Creative Commons Attribution License转载的图1:Fujita et al。高效序列通过使用重组CRISPR核糖核蛋白的体外enChIP技术特异性分离DNA片段和染色质。基因细胞。 2016年21:370-377。

使用CRISPR RNP的体外 enChIP方案(图1)
1.将与表位 - 标签(例如,3xFLAG-dCas9)融合的核酸酶 - 死亡形式的Cas9(dCas9)制备为重组蛋白。识别感兴趣的基因组区域的DNA序列的引导RNA(gRNA)是化学产生的。至于gRNA,可以使用单个gRNA(sgRNA)或CRISPR RNA(crRNA)和反式激活crRNA(tracrRNA)的复合物。
2.如果需要,将要分析的细胞交联,并裂解,并使DNA片段化。
3.在试管中将3xFLAG-dCas9 / gRNA复合物与片段化的染色质DNA一起温育。结合3xFLAG-dCas9 / gRNA复合物的基因组DNA使用针对表位标签或dCas9本身的抗体进行亲和纯化。或者,可以使用与gRNA(例如,生物素)融合的标签来纯化复合物。分离的复合物保留与靶基因座相互作用的分子。
4.如果需要,反向交联,随后纯化DNA,RNA或蛋白质,可以鉴定和鉴定这些分子。例如,可以将NGS或MS组合来鉴定与靶基因座物理结合的基因组区域或蛋白质。

我们之前描述了一个详细的“in cell”enChIP协议,用于鉴定与Bio-protocol中感兴趣的位点相互作用的蛋白质(Fujita and Fujii,2014b)。在体外 enChIP中制备超声处理的染色质的程序与用于“in cell”enChIP的程序相同。另外,其他程序(例如,Dynabeads的准备)在它们之间是相似的。因此,我们推荐参考“in cell”enChIP协议(Fujita and Fujii,2014b)。

关键字:染色质免疫沉淀, ChIP, 基因座特异性ChIP, enChIP, 体外enChIP, 下一代测序, 体外enChIP-Seq

材料和试剂

  1. 1.5 ml离心管(SARSTEDT,目录号:72.690.001)
  2. 移液器吸头(DNase / RNase-free)
  3. DT40细胞(理化生物资源中心)(举例)
  4. 10μMcrRNA(FASMAC,稀释在无DNase / RNase的水中)(见步骤B1)
  5. 10μMtracrRNA(FASMAC,稀释在无DNase / RNase的水中)(见步骤B1)
  6. 3xFLAG-dCas9-D(Sysmex,ProCube)
  7. 37%的甲醛(NACALAI TESQUE,目录号:16223-55)
  8. 抗FLAG M2抗体(Sigma-Aldrich,目录号:F1804)
  9. 正常小鼠IgG(Santa Cruz Biotechnology,目录号:sc-2025)
  10. Dynabeads-Protein G(Thermo Fisher Scientific,Invitrogen TM,目录号:10004D)
  11. RNasin加RNA酶抑制剂(Promega,目录号:N261A)
  12. 10mg / ml RNA酶A(Sigma-Aldrich,目录号:R6513)
  13. 10%SDS溶液(NACALAI TESQUE,目录号:30562-04)
  14. 20mg / ml蛋白酶K(Roche Diagnostics,目录号:3115828001)
  15. ChIP DNA Clean and Concentrator(ZYMO RESEARCH,目录号:D5205)
  16. TruSeq ChIP样品制备试剂盒(Illumina,目录号:IP-202-1012)
  17. 氯化钾(KCl)(NACALAI TESQUE,目录号:28538-62)
  18. 二硫苏糖醇(DTT)(NACALAI TESQUE,目录号:14128-91)
  19. 1M HEPES(pH7.1-7.5)(NACALAI TESQUE,目录号:17557-94)
  20. 0.5M乙二胺四乙酸(EDTA)(pH8.0)(NACALAI TESQUE,目录号:06894-85)
  21. 1M氯化镁六水合物(MgCl 2•6H 2 O)(NACALAI TESQUE,目录号:20942-34)
  22. 完全,微量,不含EDTA的蛋白酶抑制剂(Roche Diagnostics,目录号:4693159001)
  23. UltraPure DNase / RNase-Free Distilled Water(DDW)(Thermo Fisher Scientific,Invitrogen TM,目录号:10977015)
  24. 10x磷酸盐缓冲盐水(PBS)(pH7.4)(NACALAI TESQUE,目录号:27575-31)
  25. 1x PBS(用蒸馏水10倍稀释10倍PBS)
  26. 吐温-20(Sigma-Aldrich,目录号:P5927)
    注:此产品已停产。
  27. BSA级分V(7.5%)(Thermo Fisher Scientific,目录号:15260037)
  28. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S9625)
  29. 1M Tris(pH8.0)(AppliChem,目录号:A4577)
  30. 1M Tris(pH 7.5)(AppliChem,目录号:A4263)
  31. 聚乙二醇单对异辛基苯基醚(Triton X-100)(NACALAI TESQUE,目录号:12967-45)
  32. IGEPAL CA-630(Sigma-Aldrich,目录号:I8896)
  33. 3xFLAG肽(Sigma-Aldrich,目录号:F4799)
  34. 1 M KCl(见食谱)
  35. 0.1 M DTT(见食谱)
  36. 体外CRISPR缓冲液(+ 0.5mM DTT)(见食谱)
  37. PBS-T(见食谱)
  38. PBS-T-BSA(见食谱)
  39. 5 M NaCl(见食谱)
  40. 改良低盐缓冲液(+ 0.04%SDS)(见食谱)
  41. TBS(见食谱)
  42. TBS-IGEPAL CA-630(见食谱)
  43. 洗脱缓冲液(见食谱)

设备

  1. 移液器
  2. 超声波干扰器(TOMY SEIKO,型号:UD-201)
  3. 座式加热器(Eppendorf,目录号:5355 000.046)
  4. 磁性支架(魔法捕手)(TOYOBO,目录号:MGS-101)
  5. 离心机(Eppendorf,目录号:5427 R)
  6. 涡旋混合器(Fisher Scientific,目录号:128101)
  7. 旋转器(AS ONE,产品目录号:TR-118)
  8. HiSeq 2500系统(Illumina,型号:HiSeq 2500系统)

软件

  1. Integrative Genomics Viewer, http://software.broadinstitute.org/software/igv/IGV

程序

  1. 超声处理染色质的制备
    1. 将细胞(例如2×10 7 DT40细胞,在培养基中的1%甲醛在37℃下5分钟)的甲醛交联和染色质的制备如所述进行先前(藤田和藤井,2014b步骤B和C)。
    2. 先前使用超声波干扰器UD-201(TOMY)超声处理制备的染色质(例如,输出:3,占空比:100%,时间:自由,10秒×15个循环)和Fujii,2014b),除了用800μl的体外CRISPR缓冲液(+ 0.5mM DTT)(参见食谱)代替MLB3外。
    3. 如前所述评估染色质片段化(平均片段长度约为2kbp)(藤田和藤井,2014b步骤E)。

  2. 制备gRNA
    1. 混合2μL10μMcrRNA和2μL10μMtracrRNA。在我们之前的研究(Fujita等人,2017a)中,使用crRNA(cggcaggcucgggugcgccuguuuuagagcuaugcuguuuug)和tracrRNA(aacagcauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaagaggcaccgagucggugcuuuuuuu)靶向鸡Pax5启动子区域;带下划线的序列对应于目标网站。
    2. 在100°C的环境中加热2分钟。孵育2分钟不会产生相当大的蒸发。加热后,将RNA从加热器中取出,自然冷却至室温。 RNA已准备好进行D6步。

  3. 与抗体缀合的Dynabeads(抗FLAG抗体或正常小鼠IgG)的制备
    1. 准备两个1.5毫升管;一个是抗FLAG抗体,另一个是正常小鼠IgG。在每个管中加入20μlDynabeads-Protein G的浆液(20μl×2管)。在这方面,我们通常使用20μl珠子浆液2μg抗体(见步骤C7)。在这个协议中,我们描述了用于NGS分析的一个样品的制备。在这方面,在步骤E1中使用正常的小鼠IgG进行预清除,在程序D中使用抗FLAG抗体。如果处理另一个样品(例如),则不用作为阴性对照的gRNA进行亲和纯化),您可以分别为其配制染色质和Dynabeads。
    2. 将磁力管置于冰上3分钟。弃去上清液。
      在Fujita和Fujii,2014b的图2中对使用磁性支架的详细说明进行了说明
    3. 用0.5ml的PBS-T重悬沉淀(参见食谱)。将磁力管置于冰上3分钟。
      弃去上清液
    4. 重复步骤F3(总共两次)。

    5. 在0.5毫升PBS-T-BSA中重悬沉淀(见食谱)。
    6. 向抗FLAG或正常小鼠IgG的珠子中加入2μg抗体。 4°C旋转过夜。至于与抗FLAG抗体结合的Dynabeads,进行程序D.对于与正常小鼠IgG结合的Dynabeads,进行下一步骤(C7)。
    7. 短暂离心(400 g x g,持续2-3秒)。将磁力管置于冰上3分钟。
      弃去上清液
    8. 用0.5ml的PBS-T重悬沉淀。反转数次并短暂离心(400g x g,持续2-3秒)。将磁力管置于冰上3分钟。
      弃去上清液
    9. 重复步骤C8,两次(总共三次)。与正常小鼠IgG结合的Dynabeads准备好了步骤E1。

  4. 制备与重组CRISPR RNP缀合的Dynabeads
    1. 向管中加入2μg重组的3xFLAG-dCas9-D,其中Dynabeads与0.5ml PBS-T-BSA中的抗-FLAG抗体缀合(步骤C6)。
      在4°C旋转2-3小时
    2. 短暂离心(400 g x g,持续2-3秒)。将磁力管置于冰上3分钟。
      弃去上清液
    3. 用0.5ml的PBS-T重悬沉淀。反转数次并短暂离心(400g x g,持续2-3秒)。将磁力管置于冰上3分钟。
      弃去上清液
    4. 重复步骤D3(总共两次)。
    5. 在0.5ml体外CRISPR缓冲液(+ 0.5mM DTT)中重悬沉淀。反转数次并短暂离心(400g x g,持续2-3秒)。将磁力管置于冰上3分钟。
      弃去上清液
    6. 加入100μl体外CRISPR缓冲液(+ 0.5mM DTT)和4μl程序B中制备的gRNA。
    7. 37°C孵育10分钟。与重组CRISPR RNP结合的Dynabeads已准备好进行步骤E3。

  5. 亲和纯化(染色质免疫沉淀)
    1. 取800μl碎片化的染色质(程序A),其对应于从2×10 7个细胞中提取的染色质,并加入到含有与正常小鼠IgG结合的Dynabeads的管中(步骤C9)。
      在4°C旋转1小时

    2. 将磁力架置于冰上3分钟
    3. 将澄清的上清液转移到管中,其中制备与重组CRISPR RNP缀合的Dynabeads(步骤D7)。加入RNase抑制剂(40U / ml,终浓度)。

    4. 在37°C孵育20分钟,650转/分钟
    5. 将磁力管置于冰上3分钟。
      弃去上清液
    6. 用1毫升改良低盐缓冲液(+ 0.04%SDS)洗珠(见食谱)。在4°C旋转10分钟。将磁力管置于冰上3分钟。
      弃去上清液
    7. 重复步骤E6,三次(共四次)。
    8. 加入1毫升TBS-IGEPAL CA-630(见食谱)。在4°C旋转10分钟。将磁力管置于冰上3分钟。
      弃去上清液
    9. 洗脱1:用60μl洗脱缓冲液重悬珠(参见食谱)。在37°C孵育20分钟,以850转/分钟的速度加热。将磁力管置于冰上3分钟。将上清转移到1.5毫升的管中。
    10. 洗脱2:用40μl洗脱缓冲液重悬珠。在37°C孵育5分钟,850转/分钟,将磁力管置于冰上3分钟。将上清液转移到1.5ml管中,收集60μl洗脱液(共100μl)。
    11. 加入4微升5 M NaCl(见食谱)。
      在65°C孵育过夜
    12. 加入2微升10毫克/毫升的核糖核酸酶A.在37°C孵育1小时。
    13. 加入5μl的10%SDS和5μl的20mg / ml蛋白酶K.在45℃孵育2小时。
    14. 用ChIP DNA Clean and Concentrator(Zymo Resarch)按照生产商的方案纯化DNA(例如,可以使用试剂盒中的0.6ml的ChIP DNA结合缓冲液和60μl的洗脱缓冲液)。

  6. NGS文库的制备和NGS分析
    1. 根据制造商的协议,使用TruSeq ChIP样品制备试剂盒(Illumina)从DNA样品制备NGS文库(步骤E14)。

    2. 使用HiSeq 2500系统(36 bp单端,20-50万读数)对DNA文库进行DNA测序。
    3. 您可以在合适的查看器中查看NGS数据(例如,Integrative Genomics Viewer, http://software.broadinstitute.org/software/igv/IGV )(图2和3)。


      图2.通过体外分离靶基因组区域 enChIP。 :一种。 gRNA的目标位置; B.在目标区域周围的NGS峰值图像。 NGS数据在IGV中可视化。图2是根据Creative Commons Attribution License转载的:Fujita et al。 Locus特异性ChIP结合NGS分析揭示了与Pax5启动子物理相互作用的基因组调控区鸡B细胞系。 DNA Res 。 2017,24:537-548。


      图3.鉴定与Pax5启动子区域相互作用的基因组区域。与Pax5启动子区域物理相互作用的基因组区域。 NGS数据在IGV中可视化。图3根据Creative Commons Attribution License转载:Fujita et al。 Locus特异性ChIP结合NGS分析揭示了与Pax5启动子物理相互作用的基因组调控区域鸡B细胞系。 DNA Res 。 2017,24:537-548。

数据分析

  1. 为了在IGV中可视化,将NGS数据(BAM文件)上传到IGV。加载参考基因组信息(例如,,galGal4)以映射NGS数据。
  2. 放大浏览器中目标基因组区域的位置以确认目标基因组区域的分离(图2B)。用阴性对照NGS数据( eg , in vitro enChIP-Seq without gRNA)检查NGS数据以判断目标基因组区域的信号是否不是噪音。
  3. 在全基因组范围内查看,您可以手动确定与目标基因组区域相互作用的基因组区域(图3)。在这方面,NGS数据中检测到的峰值区域,而不是阴性对照NGS数据可以被判断为相互作用的基因组区域。使用MACS2或其他软件(Fujita等人,2017b)可自动检测每个体外的窄峰enChIP-Seq数据集。

笔记

  1. 也可以将使用CRISPR的常规“in cell”enChIP与NGS(enChIP-Seq)相结合(Fujita等人,2017b)。在这种情况下,如前所述(Fujita和Fujii,2014b和2015),在细胞中表达CRISPR组分(例如,3xFLAG-dCas9和sgRNA),并进行enChIP以分离靶基因座。分离基因座后,如步骤E12-F3所示纯化和分析DNA。
  2. 在NGS分析之前,可以通过qPCR确认目标基因组区域的分离。为此目的,DNA样品(步骤E14)用对目标基因组区域特异的引物组进行qPCR。先前显示了一些例子(Fujita等人,2016b和2017a)。
  3. 可以列出脱靶结合位点作为相互作用的基因组区域的候选物。为了消除这种潜在的脱靶位点,有必要检查候选基因组区域的DNA序列是否不含与gRNA的靶序列相似的序列。
  4. 为了增加提取相互作用的基因组区域的可靠性,在两个或更多个gRNA对相同的基因组区域进行体外 enChIP将是有用的。在这方面,使用不同的gRNA(除了靶基因组区域)通过体外芯片通常检测到的基因组区域可以被判断为真正的相互作用的基因组区域。以前描述过细节(Fujita等人,2017b)

食谱

  1. 1 M KCl溶液(50毫升)
    3.73克KCl(分子量:74.55)
    DDW到50毫升
  2. 0.1 M DTT溶液(10 ml)(在-20°C储存)
    0.15克DTT(分子量:154.25)
    DDW至10毫升
  3. 体外CRISPR缓冲液(+ 0.5mM DTT)
    20 mM HEPES,pH 7.1-7.5
    150 mM KCl
    0.1mM EDTA
    10mM MgCl 2•/ 2 0.5 mM DTT
    1x蛋白酶抑制剂
    准备50毫升:
    1毫升1M HEPES(pH 7.1-7.5)
    7.5毫升1M KCl
    10μl0.5M EDTA
    500μl1M MgCl 2
    250μl0.1 M DTT(在使用前添加)
    5片cOmplete,迷你,无EDTA(使用前添加)
    41毫升DDW
  4. PBS-T(10毫升)
    PBS(pH7.4),0.01%Tween-20 10毫升PBS
    10μl10%Tween-20
  5. PBS-T-BSA
    PBS(pH 7.4)
    0.01%Tween-20
    0.1%BSA
    为了准备10毫升:
    10毫升PBS
    10μl10%Tween-20
    133μl7.5%BSA部分V
  6. 5 M NaCl溶液(50毫升)
    14.61克NaCl(分子量:58.44)
    DDW到50毫升
  7. 改良低盐缓冲液(+ 0.04%SDS)
    20mM Tris,pH8.0
    2 mM EDTA
    150 mM NaCl
    0.1%Triton X-100
    0.04%SDS
    1x蛋白酶抑制剂
    5U / ml RNA酶抑制剂
    准备50毫升:
    1毫升1M Tris(pH 8.0)
    200μl0.5M EDTA
    1.5毫升5M NaCl
    50μlTriton X-100
    200μl10%SDS(使用前添加)
    5片cOmplete-Mini(使用前添加)

    6.25μlRNasin plus(40 U / ml)(在使用前添加)
    47.05毫升DDW
  8. TBS
    50 mM Tris(pH 7.5)
    150 mM NaCl
    为了准备10毫升:
    500μl1 M Tris(pH 7.5)
    300μl5M NaCl
    9.2毫升DDW
  9. TBS-IGEPAL CA-630
    50 mM Tris(pH 7.5)
    150 mM NaCl
    0.1%IGEPAL CA-630
    为了准备10毫升:
    9.99毫升TBS
    10微升IGEPAL CA-630
  10. 洗脱缓冲液
    50 mM Tris(pH 7.5)
    150 mM NaCl
    0.1%IGEPAL CA-630
    500μg/ ml 3xFLAG肽
    准备500微升:
    在TBS中加入50μl5mg / ml 3xFLAG肽
    450μlTBS-IGEPAL CA-630

致谢

该协议是从以前发表的文章(藤田等人,2017a)改编的。我们感谢M. Yuno的技术援助。 (C)(#15K06895)(TF)和科学研究资助(B)(#15H04329)(TF,HF)日本的文化,体育和科技。
T.F.和H.F.在enChIP(“使用与内源DNA序列特异性结合的分子分离特定基因组区域的方法”,专利号:日本5,954,808,专利申请号WO2014 / 125668)上有专利。 T.F.和H.F.是Epigeneron,LLC的创始人。

参考

  1. Fujita,T。和Fujii,H。(2014a)。 使用重组外源性DNA结合蛋白,通过iChIP系统高效分离保留分子间相互作用的特定基因组区域。 BMC Mol Biol 15:26.
  2. Fujita,T。和Fujii,H。(2014b)。 使用工程化的DNA结合分子识别蛋白与体内感兴趣的基因组区域相互作用介导的染色质免疫沉淀(enChIP)。 Bio Protoc 4(10):e1124。
  3. Fujita,T。和Fujii,H。(2015)。 通过工程DNA结合分子介导的染色质免疫沉淀分离特定基因组区域和鉴定相关分子enChIP)。 Methods Mol Biol 1288:43-52。
  4. Fujita,T。和Fujii,H。(2016a)。 使用基因座特异性染色质免疫沉淀技术的基因组功能生化分析 基因Regul Syst Bio 10(Suppl 1):1-9。
  5. Fujita,T.,Kitaura,F.,Yuno,M.,Suzuki,Y.,Sugano,S.和Fujii,H。(2017a)。 基因座特异性ChIP结合NGS分析揭示了与Pax5发生物理相互作用的基因组调控区域在鸡B细胞系中的启动子。 DNA Res 24(5):537-548。
  6. Fujita,T.,Yuno,M.和Fujii,H。(2016b)。 通过体外体外有效的序列特异性分离DNA片段和染色质 enChIP技术使用重组CRISPR核糖核蛋白。基因细胞 21(4):370-377。
  7. Fujita,T.,Yuno,M.,Suzuki,Y.,Sugano,S.和Fujii,H。(2017b)。 通过enChIP-Seq鉴定基因组区域之间的物理相互作用 基因细胞 22(6):506-520。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Fujita, T. and Fujii, H. (2017). In vitro Engineered DNA-binding Molecule-mediated Chromatin Immunoprecipitation (in vitro enChIP) Using CRISPR Ribonucleoproteins in Combination with Next-generation Sequencing (in vitro enChIP-Seq) for the Identification of Chromosomal Interactions. Bio-protocol 7(22): e2612. DOI: 10.21769/BioProtoc.2612.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。