3.4 Ligation of the
Annealed Oligos into
the CRISPRi Backbone
The annealed sgRNA oligos will be cloned into the CRISPRi
plasmid backbone using ligation of complementary “sticky end”
overhangs.
1. Each oligo pair will be ligated into the CRISPRi plasmid in
separate ligation reactions. A control ligation with digested
vector in the absence of oligos should also be prepared to
confirm the complete digestion of the CRISPRi backbone. To
prepare a master mix for the ligation reactions, multiply the
number of reactions by the following required volumes of each
reagent and aliquot the reagents into a master mix tube:
0.25 μL vector (BsmBI-digested, gel-purified; approximately
9 ng, see Subheading 3.3).
0.5 μL 10Â T4 DNA ligase buffer.
0.25 μL T4 DNA ligase (2,000,000 U/mL).
3.5 μL deionized water.
2. Transfer 4.5 μL of the master mix to a tube with 0.5 μL
annealed oligos (see Subheading 3.2).
3. Incubate at room temperature for 30 min—overnight.
4. Transform 0.5 μL of the ligation reaction into 5 μL of chemically competent E. coli cells.
5. Add 50 μL of LB broth to the transformation and recover for
1 h at 37
C.
6. Plate the transformation on LB with 50 μg/mL kanamycin to
select for kanamycin-resistant clones.
7. Miniprep a single clone from each reaction and validate cloning
by Sanger sequencing with oligo:
5
0 -TTCCTGTGAAGAGCCATTGATAATG-3
0 .
In our experience, sgRNA cloning in E. coli with this protocol
typically yields a >90% success rate.
3.5 Preparation of
Electrocompetent
Mycobacteria
1. Start a culture of M. tuberculosis in 5 mL of 7H9-OADCTween80 at an OD 600 of 0.025 in a T-25 flask. Grow in
stationary conditions at 37
C with 5% CO 2 for approximately
3–4 days to saturation (OD 600 ~ 1.0).
2. Expand the culture in a 125 mL screw cap Erlenmeyer flask by
transferring 3 mL of the starter culture to 22 mL 7H9-OADCTween80. Grow for at least 3 generations (approximately
3 days) to an OD 600 of 0.8–1.0 at 37
C with shaking.
3. Transfer the culture to a 50 mL conical tube.
4. Centrifuge the cells in the 50 mL conical tube at 4000 Â g for
10 min.
5. Discard the supernatant, and gently resuspend the pelleted cells
in 1 mL 10% glycerol with a p1000 pipette.
Mycobacterial CRISPR Interference
353
Annealed Oligos into
the CRISPRi Backbone
The annealed sgRNA oligos will be cloned into the CRISPRi
plasmid backbone using ligation of complementary “sticky end”
overhangs.
1. Each oligo pair will be ligated into the CRISPRi plasmid in
separate ligation reactions. A control ligation with digested
vector in the absence of oligos should also be prepared to
confirm the complete digestion of the CRISPRi backbone. To
prepare a master mix for the ligation reactions, multiply the
number of reactions by the following required volumes of each
reagent and aliquot the reagents into a master mix tube:
0.25 μL vector (BsmBI-digested, gel-purified; approximately
9 ng, see Subheading 3.3).
0.5 μL 10Â T4 DNA ligase buffer.
0.25 μL T4 DNA ligase (2,000,000 U/mL).
3.5 μL deionized water.
2. Transfer 4.5 μL of the master mix to a tube with 0.5 μL
annealed oligos (see Subheading 3.2).
3. Incubate at room temperature for 30 min—overnight.
4. Transform 0.5 μL of the ligation reaction into 5 μL of chemically competent E. coli cells.
5. Add 50 μL of LB broth to the transformation and recover for
1 h at 37
C.
6. Plate the transformation on LB with 50 μg/mL kanamycin to
select for kanamycin-resistant clones.
7. Miniprep a single clone from each reaction and validate cloning
by Sanger sequencing with oligo:
5
0 -TTCCTGTGAAGAGCCATTGATAATG-3
0 .
In our experience, sgRNA cloning in E. coli with this protocol
typically yields a >90% success rate.
3.5 Preparation of
Electrocompetent
Mycobacteria
1. Start a culture of M. tuberculosis in 5 mL of 7H9-OADCTween80 at an OD 600 of 0.025 in a T-25 flask. Grow in
stationary conditions at 37
C with 5% CO 2 for approximately
3–4 days to saturation (OD 600 ~ 1.0).
2. Expand the culture in a 125 mL screw cap Erlenmeyer flask by
transferring 3 mL of the starter culture to 22 mL 7H9-OADCTween80. Grow for at least 3 generations (approximately
3 days) to an OD 600 of 0.8–1.0 at 37
C with shaking.
3. Transfer the culture to a 50 mL conical tube.
4. Centrifuge the cells in the 50 mL conical tube at 4000 Â g for
10 min.
5. Discard the supernatant, and gently resuspend the pelleted cells
in 1 mL 10% glycerol with a p1000 pipette.
Mycobacterial CRISPR Interference
353