4. Linearize the gRNA expression vector by digestion with BbsI
and purify the linearized vector. Adjust the concentration to
5 ng/μL (see Note 10) (Fig. 3a and b).
5. For the subsequent ligation, mix 2 μL of the linearized gRNA
expression vector with 3 μL of the annealed oligonucleotides
and proceed according to the manufacturer’s protocol of the
respective T4 DNA ligase (see Note 11).
6. Transform 5 μL of the ligation in 100 μL competent DH5α
cells via the heat-shock method, plate 100 μL on LB medium
with 100 mg/L ampicillin, and grow overnight at 37
C.
7. Verify successful transformation via colony PCR, inoculate a
single colony in 5 mL LB medium with 100 mg/L ampicillin,
and grow overnight at 37
C.
8. Isolate the plasmid DNA and verify successful cloning via
sequencing. Adjust the concentration to 100 ng/μL.
9. Digest the specified gRNA expression vector with MluI (for
pEn-Sa-Chimera) or AvrII (for pChimera) and purify the
excised gRNA expression cassette.
10. Linearize the dCas9-eGFP/mRuby2 expression vectors via
digestion with MluI (for SaCas9) or AvrII (for SpCas9) and
purify the linearized vector.
11. Ligate the specified gRNA expression cassette into the linearized dCas9-eGFP/mRuby2 expression vector according to the
manufacturer’s protocol of the respective T4 DNA ligase.
5’-GAAGTAGTGATTGggGTCTTCGAGAAGACCTGTTTTA-3’
3’-CTTCATCACTAACccCAGAAGCTCTTCTGGACAAAAT-5’
5’-GAAGTAGTGATTGggGTCTTCGAGAAGACCTGTTTTA-3’
3’-CTTCATCACTAACccCAGAAGCTCTTCTGGACAAAAT-5’
BbsI
BbsI
a
b
c
5’-ATTGNNNNNNNNNNNNNNNNNNNN-3’
3’-NNNNNNNNNNNNNNNNNNNNCAAA- 5’
20 bp protospacer
digesƟon
Ligate with annealed protospacer
Fig. 3 Ligation of a telomere protospacer into the sgRNA expression vector. (a)
BbsI restriction site in pChimera/pEn-Sa-Chimera vectors. (b) BbsI-linearized
sgRNA expression vector DNA has TAAC and GTTT sticky ends. (c) Protospacer
sequence which carries ATTG and CAAA sticky ends that are complementary to
the sticky ends of the linearized sgRNA expression vector
Live-Cell Imaging with a Telomere-Specific Guide RNA
349
and purify the linearized vector. Adjust the concentration to
5 ng/μL (see Note 10) (Fig. 3a and b).
5. For the subsequent ligation, mix 2 μL of the linearized gRNA
expression vector with 3 μL of the annealed oligonucleotides
and proceed according to the manufacturer’s protocol of the
respective T4 DNA ligase (see Note 11).
6. Transform 5 μL of the ligation in 100 μL competent DH5α
cells via the heat-shock method, plate 100 μL on LB medium
with 100 mg/L ampicillin, and grow overnight at 37
C.
7. Verify successful transformation via colony PCR, inoculate a
single colony in 5 mL LB medium with 100 mg/L ampicillin,
and grow overnight at 37
C.
8. Isolate the plasmid DNA and verify successful cloning via
sequencing. Adjust the concentration to 100 ng/μL.
9. Digest the specified gRNA expression vector with MluI (for
pEn-Sa-Chimera) or AvrII (for pChimera) and purify the
excised gRNA expression cassette.
10. Linearize the dCas9-eGFP/mRuby2 expression vectors via
digestion with MluI (for SaCas9) or AvrII (for SpCas9) and
purify the linearized vector.
11. Ligate the specified gRNA expression cassette into the linearized dCas9-eGFP/mRuby2 expression vector according to the
manufacturer’s protocol of the respective T4 DNA ligase.
5’-GAAGTAGTGATTGggGTCTTCGAGAAGACCTGTTTTA-3’
3’-CTTCATCACTAACccCAGAAGCTCTTCTGGACAAAAT-5’
5’-GAAGTAGTGATTGggGTCTTCGAGAAGACCTGTTTTA-3’
3’-CTTCATCACTAACccCAGAAGCTCTTCTGGACAAAAT-5’
BbsI
BbsI
a
b
c
5’-ATTGNNNNNNNNNNNNNNNNNNNN-3’
3’-NNNNNNNNNNNNNNNNNNNNCAAA- 5’
20 bp protospacer
digesƟon
Ligate with annealed protospacer
Fig. 3 Ligation of a telomere protospacer into the sgRNA expression vector. (a)
BbsI restriction site in pChimera/pEn-Sa-Chimera vectors. (b) BbsI-linearized
sgRNA expression vector DNA has TAAC and GTTT sticky ends. (c) Protospacer
sequence which carries ATTG and CAAA sticky ends that are complementary to
the sticky ends of the linearized sgRNA expression vector
Live-Cell Imaging with a Telomere-Specific Guide RNA
349