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

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, Invitrogen^TM, 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, Invitrogen^TM, 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.