7. Add 50 μL of LB broth to the transformation and recover for
1 h at 37
C.
8. Plate the transformation on LB with 50 μg/mL kanamycin to
select for kanamycin-resistant clones.
9. Miniprep and validate cloning by Sanger sequencing with the
following primers:
GG_seq_1: 5
0 -TGCGGCGCTTTTTTTTTTGAATTC-3
0 .
GG_seq_2: 5
0 -CTGCGTTATCCCCTGATTCTG-3
0 .
In larger arrays of multiple sgRNA cassettes, the oligos used to
clone each sgRNA target sequence, as described in Subheading
3.1, can be used in Sanger sequencing for cloning validation.
3.10 CRISPRi
Transcriptional
Interference in M.
smegmatis
1. All culturing of M. smegmatis is done with 7H9-ADCTween80 for liquid media or 7H10-ADC-Tween80 for solid
media (see Note 20).
2. To prepare competent cells, grow M. smegmatis overnight to
saturation in 5 mL of 7H9-ADC-Tween80 in a 30 mL inkwell
shaking at 37
C. In a 125 mL inkwell, dilute the overnight
culture to an OD 600 of 0.01–0.02 in a total volume of 25 mL of
the same media. Grow for 16 h to an OD 600 of ~1.0. Prepare
electrocompetent cells as described for M. tuberculosis (see Subheading 3.5).
3. Electroporations can be performed in M. smegmatis as in
M. tuberculosis (see Subheading 3.6). Transformations should
be recovered in 7H9-ADC-Tween80 for at least 3 h prior to
plating on 7H10-ADC-Tween80 with 20 μg/mL kanamycin.
4. The level of target-gene transcriptional repression can be quantified similarly in M. smegmatis as in M. tuberculosis. Grow an
M. smegmatis CRISPRi strain to saturation (OD 600 ~ 1.0)
shaking at 37
C in 7H9-ADC-Tween80 with 20 μg/mL
kanamycin in a 30 mL inkwell.
5. Dilute the culture in at least 5 mL 7H9-ADC-Tween80 with
20 μg/mL kanamycin in a new 30 mL inkwell for at least
3 generations (9 h) at 37
C to log phase (OD 600 0.4–0.8).
6. Dilute the culture in at least 5 mL 7H9-ADC-Tween80 in a
new 30 mL inkwell with 20 μg/mL kanamycin and 100 ng/
mL ATc to an OD 600 of 0.1.
7. Grow for at least 3 generations at 37
C in shaking conditions
to allow for the expression of CRISPRi components and to
allow for dCas9:sgRNA-mediated transcriptional interference
at the gene of interest.
8. Harvest cells for RNA extraction and cDNA preparation as
with M. tuberculosis RNA samples.
Mycobacterial CRISPR Interference
359
1 h at 37
C.
8. Plate the transformation on LB with 50 μg/mL kanamycin to
select for kanamycin-resistant clones.
9. Miniprep and validate cloning by Sanger sequencing with the
following primers:
GG_seq_1: 5
0 -TGCGGCGCTTTTTTTTTTGAATTC-3
0 .
GG_seq_2: 5
0 -CTGCGTTATCCCCTGATTCTG-3
0 .
In larger arrays of multiple sgRNA cassettes, the oligos used to
clone each sgRNA target sequence, as described in Subheading
3.1, can be used in Sanger sequencing for cloning validation.
3.10 CRISPRi
Transcriptional
Interference in M.
smegmatis
1. All culturing of M. smegmatis is done with 7H9-ADCTween80 for liquid media or 7H10-ADC-Tween80 for solid
media (see Note 20).
2. To prepare competent cells, grow M. smegmatis overnight to
saturation in 5 mL of 7H9-ADC-Tween80 in a 30 mL inkwell
shaking at 37
C. In a 125 mL inkwell, dilute the overnight
culture to an OD 600 of 0.01–0.02 in a total volume of 25 mL of
the same media. Grow for 16 h to an OD 600 of ~1.0. Prepare
electrocompetent cells as described for M. tuberculosis (see Subheading 3.5).
3. Electroporations can be performed in M. smegmatis as in
M. tuberculosis (see Subheading 3.6). Transformations should
be recovered in 7H9-ADC-Tween80 for at least 3 h prior to
plating on 7H10-ADC-Tween80 with 20 μg/mL kanamycin.
4. The level of target-gene transcriptional repression can be quantified similarly in M. smegmatis as in M. tuberculosis. Grow an
M. smegmatis CRISPRi strain to saturation (OD 600 ~ 1.0)
shaking at 37
C in 7H9-ADC-Tween80 with 20 μg/mL
kanamycin in a 30 mL inkwell.
5. Dilute the culture in at least 5 mL 7H9-ADC-Tween80 with
20 μg/mL kanamycin in a new 30 mL inkwell for at least
3 generations (9 h) at 37
C to log phase (OD 600 0.4–0.8).
6. Dilute the culture in at least 5 mL 7H9-ADC-Tween80 in a
new 30 mL inkwell with 20 μg/mL kanamycin and 100 ng/
mL ATc to an OD 600 of 0.1.
7. Grow for at least 3 generations at 37
C in shaking conditions
to allow for the expression of CRISPRi components and to
allow for dCas9:sgRNA-mediated transcriptional interference
at the gene of interest.
8. Harvest cells for RNA extraction and cDNA preparation as
with M. tuberculosis RNA samples.
Mycobacterial CRISPR Interference
359