telomeres in living plant cells has been accomplished in transiently
transformed Nicotiana benthamiana and N. tabacum plants
[20, 21]. For N. benthamiana, dCas9 from two different bacterial
sources, Streptococcus pyogenes and Staphylococcus aureus, was
employed. Accordingly, both orthologs of Cas9 could visualize
telomeric regions with equal efficiency [20].
In this chapter, the protocol of using different dCas9 orthologs
in combination with target-specific gRNA for live-cell imaging of
telomeres in N. benthamiana leaves is explained in detail. To start
live-cell imaging of telomeres with CRISPR/Cas, pChimera and
pEn-Sa-Chimera vectors are used to express gRNA from S. pyogenes
and S. aureus, respectively. The dCas9-eGFP/mRuby2 vector is
used for the expression of dCas9 from S. pyogenes (pCAS9-TP-SpdCAS9-eGFP/mRuby2) or S. aureus (pCAS9-TP-Sa-dCAS9eGFP/mRuby2) fused with a fluorescent protein, either GFP or
mRuby2 (see Note 1).
The protocol starts by designing the protospacers for labeling
of telomers and annealing them to the pChimera vectors. Next, the
gRNA from pChimera vectors is cloned next to dCas9-eGFP/
mRuby2 with the help of conventional cloning. The resulting
vector is agro-infiltrated into N. benthamiana leaves for the visualization of telomeres by confocal laser scanning microscopy. Subsequently, the specificity of observed signals is confirmed by
immunostaining and FISH.
2 Materials
2.1 Material Used to
Prepare CRISPR/Cas9
Constructs
1. gRNA expression vectors (here pChimera and pEn-Sa-Chimera) which express gRNA from S. pyogenes and S. aureus,
respectively (see Note 1).
2. dCas9-eGFP/mRuby2 expression vectors (see Note 1):
(a) pCAS9-TP-Sp-dCAS9-eGFP/mRuby2, referred to as
pSpCas in this chapter.
(b) pCAS9-TP-Sa-dCAS9-eGFP/mRuby2, referred to as
pSaCas in this chapter.
3. E. coli NEB5α, DH5α derivative.
4. LB medium pH 7: 10 g Tryptone, 10 g sodium chloride
(NaCl), 5 g yeast extract dissolved in 1 /L distilled water,
supplement with 100 mg/L ampicillin or 100 mg/L
spectinomycin.
5. Restriction enzymes BbsI, MluI, AvrII, and respective restriction enzyme buffers.
6. T4 DNA ligase and T4 DNA ligase buffer.
7. Plasmid DNA isolation kit.
8. PCR purification kit.
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transformed Nicotiana benthamiana and N. tabacum plants
[20, 21]. For N. benthamiana, dCas9 from two different bacterial
sources, Streptococcus pyogenes and Staphylococcus aureus, was
employed. Accordingly, both orthologs of Cas9 could visualize
telomeric regions with equal efficiency [20].
In this chapter, the protocol of using different dCas9 orthologs
in combination with target-specific gRNA for live-cell imaging of
telomeres in N. benthamiana leaves is explained in detail. To start
live-cell imaging of telomeres with CRISPR/Cas, pChimera and
pEn-Sa-Chimera vectors are used to express gRNA from S. pyogenes
and S. aureus, respectively. The dCas9-eGFP/mRuby2 vector is
used for the expression of dCas9 from S. pyogenes (pCAS9-TP-SpdCAS9-eGFP/mRuby2) or S. aureus (pCAS9-TP-Sa-dCAS9eGFP/mRuby2) fused with a fluorescent protein, either GFP or
mRuby2 (see Note 1).
The protocol starts by designing the protospacers for labeling
of telomers and annealing them to the pChimera vectors. Next, the
gRNA from pChimera vectors is cloned next to dCas9-eGFP/
mRuby2 with the help of conventional cloning. The resulting
vector is agro-infiltrated into N. benthamiana leaves for the visualization of telomeres by confocal laser scanning microscopy. Subsequently, the specificity of observed signals is confirmed by
immunostaining and FISH.
2 Materials
2.1 Material Used to
Prepare CRISPR/Cas9
Constructs
1. gRNA expression vectors (here pChimera and pEn-Sa-Chimera) which express gRNA from S. pyogenes and S. aureus,
respectively (see Note 1).
2. dCas9-eGFP/mRuby2 expression vectors (see Note 1):
(a) pCAS9-TP-Sp-dCAS9-eGFP/mRuby2, referred to as
pSpCas in this chapter.
(b) pCAS9-TP-Sa-dCAS9-eGFP/mRuby2, referred to as
pSaCas in this chapter.
3. E. coli NEB5α, DH5α derivative.
4. LB medium pH 7: 10 g Tryptone, 10 g sodium chloride
(NaCl), 5 g yeast extract dissolved in 1 /L distilled water,
supplement with 100 mg/L ampicillin or 100 mg/L
spectinomycin.
5. Restriction enzymes BbsI, MluI, AvrII, and respective restriction enzyme buffers.
6. T4 DNA ligase and T4 DNA ligase buffer.
7. Plasmid DNA isolation kit.
8. PCR purification kit.
346
Solmaz Khosravi et al.