利用CRISPRainbow在人体细胞中实时成像特定位点基因组

Thomas J. Versosky; Dilshodbek U. Nishonov; Li-Chun Tu

doi:10.21769/BioProtoc.5432

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Peer-reviewed

Real-Time Imaging of Specific Genomic Loci With CRISPR/dCas9 in Human Cells Using CRISPRainbow

利用CRISPRainbow在人体细胞中实时成像特定位点基因组

TV Thomas J. Versosky

DN Dilshodbek U. Nishonov

LT Li-Chun Tu email

发布: 2025年09月05日第15卷第17期 DOI: 10.21769/BioProtoc.5432 浏览次数: 3628

评审: David PaulAnonymous reviewer(s)

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The authors used this protocol in:

Cover of Nature Biotechnology, featuring study using the protocol.

May 2016

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Abstract

Proper genome organization is essential for genome function and stability. Disruptions to this organization can lead to detrimental effects and the transformation of cells into diseased states. Individual chromosomes and their subregions can move or rearrange during transcriptional activation, in response to DNA damage, and during terminal differentiation. Techniques such as fluorescence in situ hybridization (FISH) and chromosome conformation capture (e.g., 3C and Hi-C) have provided valuable insights into genome architecture. However, these techniques require cell fixation, limiting studies of the temporal evolution of chromatin organization in detail. Our understanding of the heterogeneity and dynamics of chromatin organization at the single-cell level is still emerging. To address this, clustered regularly interspaced short palindromic repeats (CRISPR)/dead Cas9 (dCas9) systems have been repurposed for precise live-cell imaging of genome dynamics. This protocol uses a system called CRISPRainbow, a powerful tool that allows simultaneous targeting of up to seven genomic loci and tracks their locations over time using spectrally distinct fluorescent markers to study real-time chromatin organization. Multiple single-guide RNA (sgRNA), carrying specific RNA aptamers for labeling, can be cloned into a single vector to improve transfection efficiency in human cells. The precise targeting of CRISPRainbow offers distinct advantages over previous techniques while also complementing them by validating findings in live cells.

Key features

• Simultaneous imaging of up to seven specific genomic loci in living cells.

• Multicolor imaging using a single CRISPR system from Streptococcus pyogenes.

• Signal amplification through targeting repetitive sequences.

• Targeting endogenous DNA without the need for foreign DNA insertion.

Keywords: CRISPRainbow

Live-cell imaging

Chromatin dynamics

Single-particle tracking

Fluorescence microscopy

Graphical overview

Background

CRISPR/Cas systems from Streptococcus pyogenes have been widely utilized in gene editing and silencing across various organisms owing to their ability to precisely target specific DNA sequences [1,2,3]. A particular adaptation has been the use of catalytically inactive Cas9 (dCas9), which binds DNA without introducing double-strand breaks. This enabled the repurposing of CRISPR/dCas9 into a powerful imaging platform for visualizing specific genomic loci and probing dynamic chromatin architecture in living cells [4,5,6]. Prior to the development of the CRISPR/dCas9-based imaging, studies of nuclear organization relied heavily on labor-intensive tools such as fluorescent transcription activator-like effector nucleases (TALENs) or endpoint techniques like fluorescence in situ hybridization (FISH), which are limited to fixed cells and provide only static snapshots of chromatin structure [7,8]. Although live-cell imaging using fluorescently tagged histone molecules (e.g., H2B-GFP) has provided valuable insights into global chromatin dynamics, their inability to resolve individual loci or local specific transcriptional states limits their utility in fine-scale studies of genome function. In contrast, systems such as the lac operon, which require insertion of foreign DNA elements, pose risks of artificial perturbation to the native chromatin environment. CRISPR-based imaging methods overcome many of these limitations. They allow for endogenous locus visualization without genomic modification, require minimal prior knowledge of genomic sequence context, and enable simultaneous tracking of multiple loci at the single-cell level. The ability to image chromatin structure in three dimensions and in real time opens the door to transformative insights into genome organization, transcriptional regulation, and epigenetic control—areas of immense relevance for understanding development, disease, and genome plasticity.

Labeling of genomic loci with the CRISPR/dCas9 system was initially achieved by fusing a fluorescent protein, such as EGFP, to the dCas9, allowing only one color per CRISPR system [4,5,6]. In our protocol, we engineered single-guide RNAs (sgRNAs) to carry RNA scaffolds that recruit fluorescent protein-fused RNA coat proteins for locus-specific labeling [3,9]. This method, known as CRISPRainbow, which uses spectrally separated fluorophores, facilitates multiplexed, multicolor imaging of distinct genomic regions in single cells by a single CRISPR system [9]. Such advancements greatly improve the resolution and scalability of CRISPR imaging and offer powerful complementary data when integrated with genome-wide mapping techniques like Hi-C. Altogether, CRISPR-based imaging offers unprecedented opportunities to investigate the spatial and temporal dynamics of the genome with high precision and minimal perturbation.

Materials and reagents

Biological materials

1. NEB^® Stable Competent E. coli (high efficiency) (New England Biolabs, catalog number: C3040H) or One Shot^TM TOP10 Chemically Competent E. coli (Thermo Fisher Scientific, catalog number: C404010), storage: -80 °C

2. One Shot^TMccdB Survival^TM 2 T1^R Competent Cells (Thermo Fisher Scientific, catalog number: A10460), storage: -80 °C

3. Human U2OS cells (ATCC, catalog number: HTB-96), storage: liquid nitrogen tank

Reagents

A. Annealing oligonucleotides

1. Custom forward and reverse oligonucleotides with standard desalting purification (Integrated DNA Technologies or similar vendors; see Section A for details), storage: -20 °C as a 100 μM stock solution. If oligos are ordered as powder, briefly spin the tubes in a minicentrifuge and dissolve the pellet in molecular biology–grade water to prepare a 100 μM solution

2. Corning^® molecular biology–grade water (Corning, catalog number: 46-000-Cl), storage: room temperature (RT)

3. Tris-HCl, 1 M, pH = 8.0, molecular biology grade, ultrapure (Thermo Fisher Scientific, catalog number: J22638-AP), storage: RT

4. Sodium chloride (NaCl) (Fisher Chemical, catalog number: S271-1), storage: RT

5. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E9884), storage RT

B. Cloning annealed oligos into vectors

1. CRISPR sgRNA vectors with stem loops: pLH-sgRNA1-2XMS2 (Addgene, ID: 75389), pLH-sgRNA1-2XPP7 (Addgene, ID: 75390), pLH-sgRNA1-2XboxB (Addgene, ID: 75391), pLH-sgRNA1-MS2-PP7 (Addgene, ID: 75392), pLH-sgRNA1-PP7-boxB (Addgene, ID: 75393), pLH-sgRNA1-boxB-MS2 (Addgene, ID: 75396), and pLH-sgRNA1-boxB-MS2-PP7 (Addgene, ID: 75397), storage: -20 °C

2. ATP, 100 mM (New England Biolabs, catalog number: P0756S), storage: -20 °C

3. BbsI-HF^® (New England Biolabs, catalog number: R3539L), storage: -20 °C

4. T7 DNA Ligase (New England Biolabs, catalog number: M0318L), storage: -20 °C

5. CutSmart^TM buffer, 10× (New England Biolabs, catalog number: B6004S), storage: -20 °C

6. Corning^® molecular biology–grade water (Corning, catalog number: 46-000-Cl), storage: RT

7. Zyppy^® Plasmid Miniprep kit (Zymo Research, catalog number: D4019), storage: RT (except for the neutralization buffer, storage: 4 °C)

C. LB-carbenicillin plates

1. LB broth with agar (Lennox) (Sigma-Aldrich, catalog number: L2897), storage: RT

2. Carbenicillin solution, 100 mg/mL (Teknova, catalog number: C2199), storage: -20 °C

D. LB-carbenicillin liquid media

1. LB broth (Lennox) (Sigma-Aldrich, catalog number: L7658), storage: RT

E. Restriction digest of individual sgRNA

1. NotI-HF^®, 20,000 U/mL (New England Biolabs, catalog number: R3189S), storage: -20 °C

2. EcoRI-HF^®, 20,000 U/mL (New England Biolabs, catalog number: R3101S), storage: -20 °C

3. CutSmart^TM buffer, 10× (New England Biolabs, catalog number: B6004S), storage: -20 °C

4. Corning^® molecular biology–grade water (Corning, catalog number: 46-000-Cl), storage: RT

F. 1% agarose gel

1. TAE buffer, tris-acetate-EDTA, 50× (Fisher Bioreagents^TM, catalog number: BP13321), storage: RT

2. Agarose (Sigma-Aldrich, catalog number: A9539), storage: RT

3. Ethidium bromide solution, 10 mg/mL (Sigma-Aldrich, catalog number: E1510), storage: RT

4. Gel loading dye, purple 6× (New England Biolabs, catalog number: B7024S), storage: -20 °C

5. 1 kb Plus DNA ladder, 500 μg/mL (New England Biolabs, catalog number: N3232L), storage: -20 °C

6. Monarch^® PCR & DNA Cleanup kit (5 μg) (New England Biolabs, catalog number: T1030L), storage: RT

G. PCR amplification of individual CRISPRainbow sgRNA inserts

1. PCR primers (Integrated DNA Technologies or other vendors that synthesize single-stranded DNA oligos at 25 nmol with standard desalting purification). Dissolve the primers using molecular biology–grade water into a 100 μM stock solution, storage: -20 °C

a. For template pLH-sgRNA1-2XMS2, the forward and reverse primers are:

Forward primer sgRNA-Ex-F1: AATAATggtctcaTGACccttcaccgagggcctatttcc

Reverse primer sgRNA-In-R1: AATAATggtctcATTGCTGCCATTTGTCTCGAGGTCGAGA

b. For template pLH-sgRNA1-2XPP7, the forward and reverse primers are:

Forward primer sgRNA-In-F2: AATAATggtctcaGCAAccttcaccgagggcctatttcc

Reverse primer sgRNA-In-R2: AATAATggtctcAGTTCTGCCATTTGTCTCGAGGTCGAGA

c. For template pLH-sgRNA1-2XboxB, the forward and reverse primers are:

Forward primer sgRNA-In-F3: AATAATggtctcaGAACccttcaccgagggcctatttcc

Reverse primer sgRNA-In-R3: AATAATggtctcAACTGTGCCATTTGTCTCGAGGTCGAGA

d. For template pLH-sgRNA1-MS2-PP7, the forward and reverse primers are:

Forward primer sgRNA-In-F4: AATAATggtctcaCAGTccttcaccgagggcctatttcc

Reverse primer sgRNA-In-R4: AATAATggtctcAGTGTTGCCATTTGTCTCGAGGTCGAGA

e. For template pLH-sgRNA1-PP7-boxB, the forward and reverse primers are:

Forward primer sgRNA-In-F5: AATAATggtctcaACACccttcaccgagggcctatttcc

Reverse primer sgRNA-In-R5: AATAATggtctcAGACATGCCATTTGTCTCGAGGTCGAGA

f. For template pLH-sgRNA1-boxB-MS2, the forward and reverse primers are:

Forward primer sgRNA-In-F6: AATAATggtctcaTGTCccttcaccgagggcctatttcc

Reverse primer sgRNA-Ex-R1: AATAATggtctcAGAGTTGCCATTTGTCTCGAGGTCGAGA

2. dNTP, 10 mM (New England Biolabs, catalog number: N0447S), storage: -20 °C

3. Q5^® Reaction Buffer pack, 5× (New England Biolabs, catalog number: B9027S), storage: -20 °C

4. Q5^® high-fidelity DNA polymerase, 2,000 U/mL (New England Biolabs, catalog number: M0491L), storage: -20 °C

5. Corning^® molecular biology–grade water (Corning, catalog number: 46-000-Cl), storage: RT

H. Assembling CRISPRainbow for the targets of interest

1. pCRISPRainbow-DONOR1 (Addgene, ID: 75398), storage: -20 °C

2. BsaI-HF^® v2, 20,000 U/mL (New England Biolabs, catalog number: R3733L), storage: -20 °C

3. T7 DNA ligase, 3,000,000 U/mL (New England Biolabs, catalog number: M0318L), storage: -20 °C

4. ATP, 100 mM (New England Biolabs, catalog number: P0756S), storage: -20 °C

5. CutSmart^TM buffer, 10× (New England Biolabs, catalog number: B6004S), storage: -20 °C

6. Corning^® molecular biology–grade water (Corning, catalog number: 46-000-Cl), storage: RT

I. Restriction digest of assembled CRISPRainbow

1. ApaLI, 10,000 U/mL (New England Biolabs, catalog number: R0507S), storage: -20 °C

2. CutSmart^TM buffer, 10× (New England Biolabs, catalog number: B6004S), storage: -20 °C

3. Corning^® molecular biology–grade water (Corning, catalog number: 46-000-Cl), storage: RT

J. Assembled CRISPRainbow amplification

1. ZymoPURE^TM II Plasmid Midiprep kit (Zymo Research, catalog number D4200), storage: RT (except for ZymoPURE^TM P1, storage: 4 °C)

2. Corning^® molecular biology–grade water (Corning, catalog number: 46-000-Cl), storage: RT

K. U2OS cell growth

1. Dulbecco’s modified Eagle medium (DMEM), 1× (Corning, catalog number: 10-013-CV), storage: 4 °C

2. Fetal bovine serum (FBS) (Gibco, catalog number: A5256701), storage: -20 °C

3. Penicillin/streptomycin (Pen/Strep, 100×), 10,000 U/mL (Gibco, catalog number: 15140122), storage: -20 °C

4. Dulbecco’s phosphate buffered saline (DPBS), 1× (Corning, catalog number: 21-030-CV), storage: 4 °C

5. TrypLE^TM Express enzyme, no phenol red, 1× (Gibco, catalog number: 12604013), storage: RT

L. Transfection

1. Opti-MEM^TM I reduced serum medium, 1× (Gibco, catalog number: 31985062), storage: 4 °C

2. Lipofectamine^® 2000 (Life Technologies, catalog number: 11668027), storage: 4 °C

3. Trypan Blue solution, 0.4% (Thermo Fisher Scientific, catalog number: 15250061), storage: RT

4. pHAGE-TO-dCas9 (Addgene, ID: 75381), storage: -20 °C

5. pHAGE-EFS-MCP-3XBFPnls (Addgene, ID: 75384), pHAGE-EFS-PCP-3XGFPnls (Addgene, ID: 75385), pHAGE-EFS-N22p-3XRFPnls (Addgene, ID: 75387), storage: -20 °C

M. Imaging

1. Olympus Type F immersion oil (Thorlabs, catalog number: MOIL-30), storage: RT

2. TetraSpeck^TM microspheres, 0.1 μm, fluorescent blue/green/orange/dark red (Life Technologies, catalog number: T7279), storage: 4 °C

Solutions

1. Annealing buffer (see Recipes)

2. 1× TAE buffer (see Recipes)

3. Medium for U2OS cells (see Recipes)

Recipes

1. Annealing buffer (25 mL)

Reagent	Final concentration	Quantity or Volume
1 M Tris-HCl, pH = 8.0	100 mM (v/v)	0.25 mL
5 M NaCl	50 mM (v/v)	0.25 mL
0.5 M EDTA, pH = 8.0	1 mM (v/v)	0.05 mL
ddH₂O	-	24.5 mL

2. 1× TAE buffer (1L)

Reagent	Final concentration	Quantity or Volume
50× TAE buffer	2% (v/v)	20.0 mL
ddH₂O	98% (v/v)	980.0 mL

3. Medium for U2OS cells (500 mL)

Reagent	Final concentration	Quantity or Volume
DMEM, 1×	89.1% (v/v)	450 mL
FBS	9.9% (v/v)	50 mL
Pen/Strep, 100×	1.0% (v/v)	5 mL

Laboratory supplies

1. TIPONE^® pipette tips in racks, 10 μL (USA Scientific, catalog number: 1111-3200)

2. TIPONE^® pipette tips in racks, 200 μL (USA Scientific, catalog number: 1110-1200)

3. TIPONE^® pipette tips in racks, 1,000 μL (USA Scientific, catalog number: 1111-2721)

4. Corning^® 100 mm × 20 mm style dish (Corning, catalog number: 430293)

5. 0.2 mL 8-strip PCR tube with flat cap, clear (GeneMate, catalog number: 490003-710)

6. DNA LoBind^® tubes, 1.5 mL (Eppendorf, catalog number: 022431021)

7. Fisherbrand^TM sterile polystyrene disposable serological pipettes with magnifier stripe, 5 mL (Thermo Fisher Scientific, catalog number: 13-678-11D)

8. Fisherbrand^TM sterile polystyrene disposable serological pipettes with magnifier stripe, 10 mL (Thermo Fisher Scientific, catalog number: 13-678-11E)

9. Fisherbrand^TM sterile polystyrene disposable serological pipettes with magnifier stripe, 50 mL (Thermo Fisher Scientific, catalog number: 13-678-11F)

10. VWR^® centrifuge tube, 15 mL (Avantor, catalog number: 525-1068)

11. VWR^® centrifuge tube, 50 mL (Avantor, catalog number: 525-1075)

12. Falcon^® 25 cm² rectangular canted neck cell culture flask with plug seal screw cap (Corning, catalog number: 353082)

13. 35 mm glass bottom dishes, No. 1.5 coverslips (MatTek, catalog number: P35G-1.5-10-C)

14. L-shaped cell spreader (Thermo Fisher Scientific, catalog number: 14665230)

15. Corning Falcon round-bottom polypropylene culture tubes (Thermo Fisher Scientific, catalog number: 352059)

Equipment

1. Eppendorf Research^®plus, 6-pack with Pipette Carousel 2 (Eppendorf, model: 3123000942)

2. Easypet 3 Motorized Pipette Controller (Eppendorf, model: 4430000018)

3. Mastercycler^® nexus GX2 (Eppendorf, model: 6336000023)

4. Enduro^TM UV Transilluminator (Labnet Internation, Inc., model: U1002-230V)

5. Enduro^TM Gel XL Electrophoresis System (Labnet International, Inc., model: E0160)

6. NewClassic MF Analytical Balance (Mettler Toledo, model: MS303S)

7. Synergy^® UV with integrated remote dispenser (Merck, model: 171-0774)

8. MaxQ^TM 6000 Incubated/Refrigerated Stackable Shakers (Thermo Fisher Scientific, model: SHKE6000-7)

9. NanoDrop^TM 2000c Spectrophotometer (Thermo Fisher Scientific, model: ND-2000C)

10. myECL Imager (Thermo Fisher Scientific, model: 62236X)

11. myFUGE^TM 12 Mini Centrifuge 100-240V (Benchmark, model: C1012)

12. Mini Vortexer (Thermo Fisher Scientific, model: 02215365)

13. CO₂ Incubator (Sanyo, model: MCO-18AIC)

14. Hermle Refrigerated Centrifuge (Labnet, model: Z 400 K)

15. Class II A2 Biological Safety Cabinet (Thermo Forma, model: 1284)

16. Centrifuge (Eppendorf, model: 5424 R)

17. Large centrifuge (Eppendorf, model: 5810 R)

18. QIAvac 24 Plus (Qiagen, model: 19413)

19. Phase Contrast Microscope (Nikon, model: Diaphot 200/300, for cell culture)

20. 4 °C refrigerator

21. -20 °C refrigerator

22. -80 °C freezer

23. Locator^TM Plus Rack and Box Systems (Thermo Fisher Scientific, model: Locator 8 Plus)

24. Countess^® II FL Automated Cell Counter (Invitrogen, discontinued) or Countess^® 3 FL Automated Cell Counter (Invitrogen, model: A49866)

25. Epifluorescence microscope (Leica, model: DMIRB, or similar) equipped with an EMCCD camera (Andor model: iXon-897D, or similar) and 100× apochromatic objective lens (oil immersion, Olympus, model: UPLXAPO100XO, NA 1.4 or higher)

Software and datasets

1. A plasmid Editor (ApE) (M. Wayne Davis, v3.1.7) (https://jorgensen.biology.utah.edu/wayned/ape/), free to use

2. Fiji (NIH, version 2.14.0 or newer) (https://imagej.net/software/fiji/downloads), free to use

3. MetaMorph (Molecular Devices) (https://www.moleculardevices.com/products/cellular-imaging-systems/high-content-analysis/metamorph-microscopy), requires a license (Leica Microsystems)

4. Camera registration codes are available upon request to Dr. David Grunwald at the University of Massachusetts Chan Medical School.

5. OriginLab Pro (OriginLab) (https://www.originlab.com/), requires a license

Procedure

English

中文翻译

文章信息

稿件历史记录

提交日期: May 20, 2025

接收日期: Jul 29, 2025

在线发布日期: Aug 11, 2025

出版日期: Sep 5, 2025

版权信息

如何引用

Versosky, T. J., Nishonov, D. U. and Tu, L. C. (2025). Real-Time Imaging of Specific Genomic Loci With CRISPR/dCas9 in Human Cells Using CRISPRainbow. Bio-protocol 15(17): e5432. DOI: 10.21769/BioProtoc.5432.