2.9 Targeting
Multiple Genes for
Transcriptional
Silencing with CRISPRi
1. sgRNA oligos targeting genes of interest.
2. Q5 High-Fidelity 2Â Master Mix (NEB) or equivalent. Used
to amplify [promoter-sgRNA-terminator] cassette from individual CRISPRi plasmids.
3. 1 M DTT (Millipore Sigma).
4. 10 mM ATP (NEB).
5. SapI (10,000 U/μL; NEB).
6. 10Â CutSmart Buffer.
7. T4 DNA ligase (2,000,000 U/mL; NEB).
8. Oligos to amplify [promoter-sgRNA-terminator] cassette and
to add SapI digestion DNA overhangs for cloning into the
CRISPRi backbone.
9. LB agar.
10. Kanamycin.
11. Oligos to Sanger sequence Golden Gate assembled plasmids.
12. GG_seq_1: 5
0 -TGCGGCGCTTTTTTTTTTGAATTC-3
0 .
GG_seq_2: 5
0 -CTGCGTTATCCCCTGATTCTG-3
0 .
2.10 CRISPRi
Transcriptional
Interference in M.
smegmatis
1. M. smegmatis mc
2 155.
2. plJR962 (Addgene #115162): the CRISPRi backbone for
M. smegmatis (Fig. 2). This plasmid is identical to plJR965
except that it encodes a TetR expressed at higher levels than
plJR965 (unpublished data). Elevated TetR expression minimizes leaky dCas9 and sgRNA expression in the absence of ATc
but still allows for high-level target gene repression in
M. smegmatis.
3. ADC Enrichment: Dissolve 50 g of Bovine Serum Albumin
Fraction V, 20 g dextrose, and 8.5 g sodium chloride in
900 mL of deionized water, gently stirring and heating to
37
C. Once dissolved, remove from heat and add 30 mg of
catalase. Stir to dissolve, then supplement the volume to 1 L
with deionized water and filter sterilize.
4. 7H9-ADC-Tween80: Dissolve 4.7 g Difco Middlebrook 7H9
Broth powder (BD) in 893.5 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, and 100 mL of ADC Enrichment.
5. 7H10-ADC-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 ADC
348
Andrew I. Wong and Jeremy M. Rock
Multiple Genes for
Transcriptional
Silencing with CRISPRi
1. sgRNA oligos targeting genes of interest.
2. Q5 High-Fidelity 2Â Master Mix (NEB) or equivalent. Used
to amplify [promoter-sgRNA-terminator] cassette from individual CRISPRi plasmids.
3. 1 M DTT (Millipore Sigma).
4. 10 mM ATP (NEB).
5. SapI (10,000 U/μL; NEB).
6. 10Â CutSmart Buffer.
7. T4 DNA ligase (2,000,000 U/mL; NEB).
8. Oligos to amplify [promoter-sgRNA-terminator] cassette and
to add SapI digestion DNA overhangs for cloning into the
CRISPRi backbone.
9. LB agar.
10. Kanamycin.
11. Oligos to Sanger sequence Golden Gate assembled plasmids.
12. GG_seq_1: 5
0 -TGCGGCGCTTTTTTTTTTGAATTC-3
0 .
GG_seq_2: 5
0 -CTGCGTTATCCCCTGATTCTG-3
0 .
2.10 CRISPRi
Transcriptional
Interference in M.
smegmatis
1. M. smegmatis mc
2 155.
2. plJR962 (Addgene #115162): the CRISPRi backbone for
M. smegmatis (Fig. 2). This plasmid is identical to plJR965
except that it encodes a TetR expressed at higher levels than
plJR965 (unpublished data). Elevated TetR expression minimizes leaky dCas9 and sgRNA expression in the absence of ATc
but still allows for high-level target gene repression in
M. smegmatis.
3. ADC Enrichment: Dissolve 50 g of Bovine Serum Albumin
Fraction V, 20 g dextrose, and 8.5 g sodium chloride in
900 mL of deionized water, gently stirring and heating to
37
C. Once dissolved, remove from heat and add 30 mg of
catalase. Stir to dissolve, then supplement the volume to 1 L
with deionized water and filter sterilize.
4. 7H9-ADC-Tween80: Dissolve 4.7 g Difco Middlebrook 7H9
Broth powder (BD) in 893.5 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, and 100 mL of ADC Enrichment.
5. 7H10-ADC-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 ADC
348
Andrew I. Wong and Jeremy M. Rock