2.2 Material Used for
Agroinfiltration of
N. benthamiana
1. Healthy 2–4-week-old N. benthamiana plants grown in a
greenhouse under 16/8-h light/dark conditions and 22
C
temperature.
2. Agrobacterium strain harboring the imaging expression construct (Fig. 2).
3. LB medium with 100 mg/L spectinomycin and 50 mg/L
rifampicin.
4. 100 mM Acetosyringone stock solution in ethanol, stored at
À20
C.
5. 0.5 M 2-(N-morpholino) ethanesulfonic acid buffer (MES-K),
adjusted to pH 5.6 with KOH.
6. Resuspension solution: 10 mM MgCl 2 , 10 mM MES-K.
7. Centrifuge for 50 mL tubes.
8. Spectrometer.
9. 5 mL Syringe.
2.3 Material
Necessary for
Microscopy of Plant
Leaves
1. Confocal laser scanning microscope (e.g., LSM780, Carl
Zeiss).
2. Phosphate buffer: 20 mM KH2PO4/Na2PO4, 0.01% Triton
X100, pH 7.0.
3. Caulking gun.
4. 50 mL Syringe.
5. 76 Â 26 mm Glass slides.
6. 40 Â 24 mm Coverslips.
7. Sticking tape.
8. Fixogum rubber cement, Marabu GmbH, Germany.
2.4 Material
Required for
Immunostaining
and FISH
1. Epifluorescence microscope (e.g., BX61, Olympus) equipped
with a camera (e.g., Orca ER, Hamamatsu).
2. Cytocentrifuge (Shandon CytoSpin3, see Note 2).
3. Cytology funnels for cytocentrifuge.
4. 5 mL Polystyrene round-bottom tube with cell strainer cap (see
Note 3).
5. Razor blade.
LB
Ubi4-P
SpdCas9
Pea3A-T
AtU26-P
sgRNA-Telomere
NLS
3xGFP/mRuby
RB
Fig. 2 Final CRISPR/dCas9 live-cell imaging construct. LB: left border, Ubi-4 p: ubiquitin promoter for
constitutive expression of Cas 9 protein, NLS: nuclear localization signal importing the expressed Cas9 protein
to nucleus, 3Â GFP/mRuby: fluorescent proteins, Pea3A T: terminator, AtU26-P: U6 promoter from A. thaliana
for expression of gRNA, sgRNA telomere: sgRNA-containing telomere-targeting protospacer
Live-Cell Imaging with a Telomere-Specific Guide RNA
347
Agroinfiltration of
N. benthamiana
1. Healthy 2–4-week-old N. benthamiana plants grown in a
greenhouse under 16/8-h light/dark conditions and 22
C
temperature.
2. Agrobacterium strain harboring the imaging expression construct (Fig. 2).
3. LB medium with 100 mg/L spectinomycin and 50 mg/L
rifampicin.
4. 100 mM Acetosyringone stock solution in ethanol, stored at
À20
C.
5. 0.5 M 2-(N-morpholino) ethanesulfonic acid buffer (MES-K),
adjusted to pH 5.6 with KOH.
6. Resuspension solution: 10 mM MgCl 2 , 10 mM MES-K.
7. Centrifuge for 50 mL tubes.
8. Spectrometer.
9. 5 mL Syringe.
2.3 Material
Necessary for
Microscopy of Plant
Leaves
1. Confocal laser scanning microscope (e.g., LSM780, Carl
Zeiss).
2. Phosphate buffer: 20 mM KH2PO4/Na2PO4, 0.01% Triton
X100, pH 7.0.
3. Caulking gun.
4. 50 mL Syringe.
5. 76 Â 26 mm Glass slides.
6. 40 Â 24 mm Coverslips.
7. Sticking tape.
8. Fixogum rubber cement, Marabu GmbH, Germany.
2.4 Material
Required for
Immunostaining
and FISH
1. Epifluorescence microscope (e.g., BX61, Olympus) equipped
with a camera (e.g., Orca ER, Hamamatsu).
2. Cytocentrifuge (Shandon CytoSpin3, see Note 2).
3. Cytology funnels for cytocentrifuge.
4. 5 mL Polystyrene round-bottom tube with cell strainer cap (see
Note 3).
5. Razor blade.
LB
Ubi4-P
SpdCas9
Pea3A-T
AtU26-P
sgRNA-Telomere
NLS
3xGFP/mRuby
RB
Fig. 2 Final CRISPR/dCas9 live-cell imaging construct. LB: left border, Ubi-4 p: ubiquitin promoter for
constitutive expression of Cas 9 protein, NLS: nuclear localization signal importing the expressed Cas9 protein
to nucleus, 3Â GFP/mRuby: fluorescent proteins, Pea3A T: terminator, AtU26-P: U6 promoter from A. thaliana
for expression of gRNA, sgRNA telomere: sgRNA-containing telomere-targeting protospacer
Live-Cell Imaging with a Telomere-Specific Guide RNA
347