It is possible to overcome this problem using the catalytic null
mutant of the Cas9 RNA-guided nuclease (“dead” Cas9 or dCas9),
which induces gene silencing by steric hindrance of RNA polymerase, rather than disruption, thereby blocking transcription initiation (when dCas9 protein is bound to the promoter) or elongation
(when dCas9 is bound to the coding region) [13]. Based on our
previous results [12], when a single-guide RNA (sgRNA) is
designed to hybridize to the coding strand of the DNA, full gene
silencing is expected. We have created dCas9 and sgRNAexpressing plasmid and have successfully obtained gene silencing
using this novel tool.
2 Material
2.1 Bacterial Strains
and Media
1. Escherichia coli strain π1 [14].
2. Thymidine solution: 100 mM solution in water. Filter the
solution through a 0.22 filter for sterilization, and keep the
final solution refrigerated (4
C).
3. Spectinomycin: 100 mg/mL spectinomycin in water.
4. Sodium pyruvate: 10 mg/mL in water.
5. Luria-Bertani (LB) medium: 1% NaCl, 0.5% yeast extract, 1%
tryptone, pH to 7.0. Sterilize by autoclaving for 20 min at
121
C. For solid medium, include 1.5% bacteriological agar
before autoclaving.
6. L. biflexa serovar Patoc strain Patoc1 (see Note 1). Leptospiral
cells are grown in liquid EMJH medium with gentle shaking
(80–100 rpm) at 30
C or on solid medium at the same
temperature.
7. Genomic DNA from L. interrogans serovar Copenhageni strain
Fiocruz L1-130.
8. Liquid EMJH medium: Dissolve 2.3 g of Difco™ Leptospira
Medium Base EMJH (Becton Dickinson) in 900 mL Milli-Q
H 2 O, autoclave the solution (to avoid contamination), and
after cooling, add 100 mL Difco™ Leptospira Enrichment
EMJH (Becton Dickinson). Make sure the glass containers
are properly washed and sterile. The final medium can be stored
up to 2 months at 4
C. It is important to avoid excess handling
and always to check for possible contamination.
9. 2Â EMJH: Dissolve 0.46 g of Difco™ Leptospira Medium
Base EMJH (Becton Dickinson) in 80 mL of Milli-Q water,
and sterilize by autoclaving. After cooling, add 20 mL of
Difco™ Leptospira Enrichment EMJH (Becton Dickinson).
This can be stored up to 2 months at 4
C.
110
Luis Guilherme Virgı ´lio Fernandes and Ana Lucia Tabet Oller Nascimento
mutant of the Cas9 RNA-guided nuclease (“dead” Cas9 or dCas9),
which induces gene silencing by steric hindrance of RNA polymerase, rather than disruption, thereby blocking transcription initiation (when dCas9 protein is bound to the promoter) or elongation
(when dCas9 is bound to the coding region) [13]. Based on our
previous results [12], when a single-guide RNA (sgRNA) is
designed to hybridize to the coding strand of the DNA, full gene
silencing is expected. We have created dCas9 and sgRNAexpressing plasmid and have successfully obtained gene silencing
using this novel tool.
2 Material
2.1 Bacterial Strains
and Media
1. Escherichia coli strain π1 [14].
2. Thymidine solution: 100 mM solution in water. Filter the
solution through a 0.22 filter for sterilization, and keep the
final solution refrigerated (4
C).
3. Spectinomycin: 100 mg/mL spectinomycin in water.
4. Sodium pyruvate: 10 mg/mL in water.
5. Luria-Bertani (LB) medium: 1% NaCl, 0.5% yeast extract, 1%
tryptone, pH to 7.0. Sterilize by autoclaving for 20 min at
121
C. For solid medium, include 1.5% bacteriological agar
before autoclaving.
6. L. biflexa serovar Patoc strain Patoc1 (see Note 1). Leptospiral
cells are grown in liquid EMJH medium with gentle shaking
(80–100 rpm) at 30
C or on solid medium at the same
temperature.
7. Genomic DNA from L. interrogans serovar Copenhageni strain
Fiocruz L1-130.
8. Liquid EMJH medium: Dissolve 2.3 g of Difco™ Leptospira
Medium Base EMJH (Becton Dickinson) in 900 mL Milli-Q
H 2 O, autoclave the solution (to avoid contamination), and
after cooling, add 100 mL Difco™ Leptospira Enrichment
EMJH (Becton Dickinson). Make sure the glass containers
are properly washed and sterile. The final medium can be stored
up to 2 months at 4
C. It is important to avoid excess handling
and always to check for possible contamination.
9. 2Â EMJH: Dissolve 0.46 g of Difco™ Leptospira Medium
Base EMJH (Becton Dickinson) in 80 mL of Milli-Q water,
and sterilize by autoclaving. After cooling, add 20 mL of
Difco™ Leptospira Enrichment EMJH (Becton Dickinson).
This can be stored up to 2 months at 4
C.
110
Luis Guilherme Virgı ´lio Fernandes and Ana Lucia Tabet Oller Nascimento