sterilized 50% (v/v) glycerol, 2.5 mL of filter sterilized 20%
(v/v) Tween80, and 100 mL of BBL Middlebrook OADC
Enrichment (BD).
4. 125 mL screw cap Erlenmeyer flask.
5. Sterile 10% glycerol.
2.6 Electroporation
of CRISPRi Constructs
into Electrocompetent
Mycobacteria
1. 2 mm electroporation cuvettes (Bio-Rad).
2. 7H10-OADC-Tween80 plates with 20 μg/mL kanamycin:
Dissolve 19 g Difco Middlebrook 7H10 Agar powder
(BD) in 887.5 mL deionized water and autoclave. Cool to
50
C and then aseptically add 4 mL of filter sterilized 50%
(v/v) glycerol, 2.5 mL of filter sterilized 20% (v/v) Tween80,
100 mL of BBL Middlebrook OADC Enrichment (BD), and
400 μL of a 50 mg/mL solution of kanamycin. Mix solution
and pour plates.
2.7 Quantification of
Transcriptional
Knockdown by qRTPCR
1. T-25 tissue culture flasks.
2. Anhydrotetracycline (ATc). A 10,000Â stock solution can be
made by dissolving ATc powder in methanol at a concentration
of 1 mg/mL.
3. Aluminum foil.
4. GTC Buffer: Dissolve 180 g guanidine thiocyanate, 1.5 g Nlauryl sarcosine, and 2.2 g sodium citrate in 297.9 mL water at
37
C. Add 2.1 mL of β-mercaptoethanol. Use within 48 h.
5. TRIzol Reagent.
6. Lysing Matrix B, 2 mL tube.
7. Chloroform.
8. RNA isolation kits (Zymo Direct-zol RNA Miniprep or
equivalent).
9. cDNA synthesis kits (Invitrogen SuperScript IV First-Strand
Synthesis System or equivalent).
10. Appropriate oligonucleotides for qPCR amplicon generation
and quantification.
2.8 Design of
CRISPRi-Resistant
Complementation
Constructs
1. Gibson assembly oligos. Used to PCR amplify and provide
DNA overhangs to clone the complementation gene of interest
into an appropriate expression vector.
2. Oligos to generate CRISPRi-resistance mutations. Designed to
include DNA overhangs for Gibson assembly and to introduce
synonymous mutations into the gene of interest that abrogate
dCas9-sgRNA binding.
3. Q5 High-Fidelity 2Â Master Mix (NEB) or equivalent. Used
to amplify DNA fragments for complementation constructs.
4. NEBuilder HiFi DNA Assembly Master Mix (NEB).
Mycobacterial CRISPR Interference
347
(v/v) Tween80, and 100 mL of BBL Middlebrook OADC
Enrichment (BD).
4. 125 mL screw cap Erlenmeyer flask.
5. Sterile 10% glycerol.
2.6 Electroporation
of CRISPRi Constructs
into Electrocompetent
Mycobacteria
1. 2 mm electroporation cuvettes (Bio-Rad).
2. 7H10-OADC-Tween80 plates with 20 μg/mL kanamycin:
Dissolve 19 g Difco Middlebrook 7H10 Agar powder
(BD) in 887.5 mL deionized water and autoclave. Cool to
50
C and then aseptically add 4 mL of filter sterilized 50%
(v/v) glycerol, 2.5 mL of filter sterilized 20% (v/v) Tween80,
100 mL of BBL Middlebrook OADC Enrichment (BD), and
400 μL of a 50 mg/mL solution of kanamycin. Mix solution
and pour plates.
2.7 Quantification of
Transcriptional
Knockdown by qRTPCR
1. T-25 tissue culture flasks.
2. Anhydrotetracycline (ATc). A 10,000Â stock solution can be
made by dissolving ATc powder in methanol at a concentration
of 1 mg/mL.
3. Aluminum foil.
4. GTC Buffer: Dissolve 180 g guanidine thiocyanate, 1.5 g Nlauryl sarcosine, and 2.2 g sodium citrate in 297.9 mL water at
37
C. Add 2.1 mL of β-mercaptoethanol. Use within 48 h.
5. TRIzol Reagent.
6. Lysing Matrix B, 2 mL tube.
7. Chloroform.
8. RNA isolation kits (Zymo Direct-zol RNA Miniprep or
equivalent).
9. cDNA synthesis kits (Invitrogen SuperScript IV First-Strand
Synthesis System or equivalent).
10. Appropriate oligonucleotides for qPCR amplicon generation
and quantification.
2.8 Design of
CRISPRi-Resistant
Complementation
Constructs
1. Gibson assembly oligos. Used to PCR amplify and provide
DNA overhangs to clone the complementation gene of interest
into an appropriate expression vector.
2. Oligos to generate CRISPRi-resistance mutations. Designed to
include DNA overhangs for Gibson assembly and to introduce
synonymous mutations into the gene of interest that abrogate
dCas9-sgRNA binding.
3. Q5 High-Fidelity 2Â Master Mix (NEB) or equivalent. Used
to amplify DNA fragments for complementation constructs.
4. NEBuilder HiFi DNA Assembly Master Mix (NEB).
Mycobacterial CRISPR Interference
347