6. 4% (vol/vol) Paraformaldehyde solution (see Note 4).
7. LB01 lysis buffer: 15 mM Tris, 2 mM Na 2 EDTA, 0.5 mM
spermine tetrahydrochloride, 80 mM KCL, 20 mM NaCl,
15 mM β-mercaptoethanol, 0.1% (vol/vol) Triton X-100.
Adjust pH to 7.5 with 1 M NaOH. Filtrate the buffer through
0.22 μm filter to sterilize it for longer storage.
8. 4% (vol/vol) Bovine serum albumin (BSA) solution.
9. PBS pH 7.4: 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO4,
1.8 mM KH 2 PO 4 .
10. 4
0 ,6-Diamidino-2
0 -phenylindole dihydrochloride (DAPI) solution (see Note 5).
11. VECTASHIELD antifade mounting medium (Vector Laboratories) (see Note 6).
12. Ethanol solutions: 70, 90, 100%.
13. Fixation solution: (1:3) glacial acetic acid: 100% Ethanol.
14. Denaturation solution: 0.2 M NaOH in 70% ethanol.
15. FISH hybridization solution: 50% (vol/vol) Formamide, 10%
(vol/vol) dextran sulfate in 2Â SSC.
16. 2Â Saline sodium citrate (SSC): 0.30 M Sodium citrate,
0.030 M NaCl, pH 7.0.
17. GFP antibody (directly labeled GFP mouse antibody Dylight
488; Rockland).
18. 5
0 -Cy5-labeled
oligonucleotide
probe
(5
0 -Cy5GGGTTTAGGGTTTAGGGTTT-3
0 ).
19. Sucrose buffer: 100 mM Tris, 50 mM KCl, 2 mM MgCl2,
0.05% Tween, 5% sucrose.
3 Methods
3.1 Preparation of
the Protospacer and
Cloning of the CRISPR/
Cas9-gRNA Vectors
1. Select a 20 nt protospacer for the telomere sequence upstream
of 5
0 -NGG-3
0 and 5
0 -NNGRRT-3
0 PAM sequences for Cas9
from S. pyogenesis and S. aureus, respectively (see Note 7).
2. Synthesize the protospacer as oligonucleotides with appropriate overhangs for cloning into the gRNA expression vector
(5
0 -ATTG-protospacer-3
0 and 5
0 -AAAC-rev-com_protospacer-3
0 ) (see Note 8) (Fig. 3c).
3. Mix the oligonucleotides with a final concentration of 2 μM
each in a final volume of 50 μL and anneal them by incubation
at 95
C for 5 min and subsequent incubation at room temperature for 20 min (see Note 9).
348
Solmaz Khosravi et al.
7. LB01 lysis buffer: 15 mM Tris, 2 mM Na 2 EDTA, 0.5 mM
spermine tetrahydrochloride, 80 mM KCL, 20 mM NaCl,
15 mM β-mercaptoethanol, 0.1% (vol/vol) Triton X-100.
Adjust pH to 7.5 with 1 M NaOH. Filtrate the buffer through
0.22 μm filter to sterilize it for longer storage.
8. 4% (vol/vol) Bovine serum albumin (BSA) solution.
9. PBS pH 7.4: 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO4,
1.8 mM KH 2 PO 4 .
10. 4
0 ,6-Diamidino-2
0 -phenylindole dihydrochloride (DAPI) solution (see Note 5).
11. VECTASHIELD antifade mounting medium (Vector Laboratories) (see Note 6).
12. Ethanol solutions: 70, 90, 100%.
13. Fixation solution: (1:3) glacial acetic acid: 100% Ethanol.
14. Denaturation solution: 0.2 M NaOH in 70% ethanol.
15. FISH hybridization solution: 50% (vol/vol) Formamide, 10%
(vol/vol) dextran sulfate in 2Â SSC.
16. 2Â Saline sodium citrate (SSC): 0.30 M Sodium citrate,
0.030 M NaCl, pH 7.0.
17. GFP antibody (directly labeled GFP mouse antibody Dylight
488; Rockland).
18. 5
0 -Cy5-labeled
oligonucleotide
probe
(5
0 -Cy5GGGTTTAGGGTTTAGGGTTT-3
0 ).
19. Sucrose buffer: 100 mM Tris, 50 mM KCl, 2 mM MgCl2,
0.05% Tween, 5% sucrose.
3 Methods
3.1 Preparation of
the Protospacer and
Cloning of the CRISPR/
Cas9-gRNA Vectors
1. Select a 20 nt protospacer for the telomere sequence upstream
of 5
0 -NGG-3
0 and 5
0 -NNGRRT-3
0 PAM sequences for Cas9
from S. pyogenesis and S. aureus, respectively (see Note 7).
2. Synthesize the protospacer as oligonucleotides with appropriate overhangs for cloning into the gRNA expression vector
(5
0 -ATTG-protospacer-3
0 and 5
0 -AAAC-rev-com_protospacer-3
0 ) (see Note 8) (Fig. 3c).
3. Mix the oligonucleotides with a final concentration of 2 μM
each in a final volume of 50 μL and anneal them by incubation
at 95
C for 5 min and subsequent incubation at room temperature for 20 min (see Note 9).
348
Solmaz Khosravi et al.