5. Dilute the culture in a new T-25 flask to an OD 600 of 0.1–0.2
in 5 mL of the same media +100 ng/mL ATc (see Note 6). ATc
is used to induce the expression of Sth1 dCas9 and the sgRNA.
Grow in a T-25 flask as above for 2–3 generations (2–3 days) to
allow for the expression of CRISPRi components and transcriptional silencing of the target gene.
6. Transfer a culture volume containing 2 OD 600 units of cells (for
example, 2 mL of a culture at OD 600 of 1.0) or approximately
6 Â 10
8 cells to an equal volume of GTC buffer in a 15 mL
conical (see Note 8).
7. Pellet the cells by centrifugation at 4000 Â g for 10 min at 4
C.
8. Resuspend the pellet in 1 mL of TriZol with a p1000. Transfer
the pellet to a Lysing Matrix B tube.
9. Lyse the cells by bead beating at 6500 rpm in 3Â 1 min cycles,
incubating on ice for 1 min between each cycle.
10. Add 200 μL of chloroform to the lysed cells in TriZol. Vortex
for 5 s.
11. Allow the bead beating tubes to sit for 2 min to allow dissociation of nucleoprotein complexes (samples can be frozen at
À80
C prior to RNA isolation if desired).
12. Proceed with total RNA isolation and cDNA synthesis as per
kit instructions.
13. The dCas9-sgRNA complex functions as a roadblock for elongating RNA polymerase or a steric block when binding to the
target gene promoter. To quantify target gene knockdown,
quantify target gene mRNA levels downstream of the sgRNA
target sequence binding site (see Notes 9–11).
14. Set up qPCR reactions to determine transcript levels of the
target gene and a housekeeping gene (e.g., sigA). Use the
ΔΔC t method to determine the fold knockdown of your gene
of interest by comparing transcript levels of your target gene
relative to a housekeeping gene. The relevant comparisons can
be your M. tuberculosis CRISPRi strain grown in the presence
or absence of ATc, or the control M. tuberculosis CRISPRi
strain containing plJR965 grown in the presence of ATc.
3.8 Design of
CRISPRi-Resistant
Complementation
Constructs
To confirm that the phenotype seen in a M. tuberculosis CRISPRi
strain is due to the transcriptional knockdown of the targeted gene,
complementation with a CRISPRi-resistant allele of the target gene
is recommended (see Note 12). Since dCas9 is guided by sequence
complementarity of the target and sgRNA, a construct expressing a
CRISPRi-resistant allele of the targeted gene can be generated by
Gibson assembly to introduce silent mutations in the sgRNA binding site and/or the PAM.
Mycobacterial CRISPR Interference
355
in 5 mL of the same media +100 ng/mL ATc (see Note 6). ATc
is used to induce the expression of Sth1 dCas9 and the sgRNA.
Grow in a T-25 flask as above for 2–3 generations (2–3 days) to
allow for the expression of CRISPRi components and transcriptional silencing of the target gene.
6. Transfer a culture volume containing 2 OD 600 units of cells (for
example, 2 mL of a culture at OD 600 of 1.0) or approximately
6 Â 10
8 cells to an equal volume of GTC buffer in a 15 mL
conical (see Note 8).
7. Pellet the cells by centrifugation at 4000 Â g for 10 min at 4
C.
8. Resuspend the pellet in 1 mL of TriZol with a p1000. Transfer
the pellet to a Lysing Matrix B tube.
9. Lyse the cells by bead beating at 6500 rpm in 3Â 1 min cycles,
incubating on ice for 1 min between each cycle.
10. Add 200 μL of chloroform to the lysed cells in TriZol. Vortex
for 5 s.
11. Allow the bead beating tubes to sit for 2 min to allow dissociation of nucleoprotein complexes (samples can be frozen at
À80
C prior to RNA isolation if desired).
12. Proceed with total RNA isolation and cDNA synthesis as per
kit instructions.
13. The dCas9-sgRNA complex functions as a roadblock for elongating RNA polymerase or a steric block when binding to the
target gene promoter. To quantify target gene knockdown,
quantify target gene mRNA levels downstream of the sgRNA
target sequence binding site (see Notes 9–11).
14. Set up qPCR reactions to determine transcript levels of the
target gene and a housekeeping gene (e.g., sigA). Use the
ΔΔC t method to determine the fold knockdown of your gene
of interest by comparing transcript levels of your target gene
relative to a housekeeping gene. The relevant comparisons can
be your M. tuberculosis CRISPRi strain grown in the presence
or absence of ATc, or the control M. tuberculosis CRISPRi
strain containing plJR965 grown in the presence of ATc.
3.8 Design of
CRISPRi-Resistant
Complementation
Constructs
To confirm that the phenotype seen in a M. tuberculosis CRISPRi
strain is due to the transcriptional knockdown of the targeted gene,
complementation with a CRISPRi-resistant allele of the target gene
is recommended (see Note 12). Since dCas9 is guided by sequence
complementarity of the target and sgRNA, a construct expressing a
CRISPRi-resistant allele of the targeted gene can be generated by
Gibson assembly to introduce silent mutations in the sgRNA binding site and/or the PAM.
Mycobacterial CRISPR Interference
355