7. Transformations of CRISPRi plasmids encoding sgRNAs that
achieve strong inhibition of in vitro essential genes plated in the
presence of ATc should result in growth inhibition. It is normal
for some “escaper” colonies to arise in the presence of ATc at a
low frequency (~1:500 to 1:1000 transformants,
unpublished data).
8. GTC buffer contains: guanidine thiocyanate to lyse cells and
inhibit RNases; N-lauryl sarcosine to inhibit new rounds of
transcription initiation during cell lysis; and sodium citrate to
chelate divalent cations that can contribute to RNA hydrolysis.
9. Another method for assessing CRISPRi target gene knockdown is by monitoring the targeted gene’s protein levels by
western blot. However, this is not as broadly applicable given
the paucity of antibodies to endogenous M. tuberculosis
proteins.
10. We use the PrimerQuest tool from IDT (https://www.idtdna.
com/PrimerQuest/Home/Index) to design 2–3 qPCR
primer pairs for the target gene of interest. Each qPCR primer
pair is then validated for efficiency, specificity, and the linear
range of amplification using standard qPCR approaches. A
single amplification product is confirmed for each validated
qPCR primer pair by inspection of the amplification melt
curve.
11. Due to the common occurrence of promoters in the
M. tuberculosis genome [25, 26], including within ORFs and
in the sense and antisense directions, exercise caution when
interpreting the magnitude of target knockdown by qRT-PCR.
For instance, CRISPRi knockdown of some target genes may
appear ineffective by qRT-PCR. One possible explanation for
this lack of measured knockdown is the presence of multiple
promoters driving transcription through the target gene, not
all of which are disrupted by the chosen sgRNA. In such
instances, consult published transcription start site (TSS) annotations in the M. tuberculosis genome to determine the location
and direction of TSSs proximal to the gene of interest. Design
and validate qPCR amplicons that avoid quantifying interfering transcripts and/or use strand-specific qPCR to measure
direction-specific transcription.
12. It is important to consider the operon structure of genes
targeted with CRISPRi. CRISPRi targeting of a gene in an
operon results in transcriptional knockdown of the targeted
gene and all downstream genes in the operon. This polar effect
is due to the inhibition of RNA polymerase elongation beyond
the site of dCas9 occupancy, as defined by the sgRNA targeting
sequence. Hence, genes downstream of the sgRNA targeting
sequence
will
also
experience
CRISPRi-mediated
Mycobacterial CRISPR Interference
361
achieve strong inhibition of in vitro essential genes plated in the
presence of ATc should result in growth inhibition. It is normal
for some “escaper” colonies to arise in the presence of ATc at a
low frequency (~1:500 to 1:1000 transformants,
unpublished data).
8. GTC buffer contains: guanidine thiocyanate to lyse cells and
inhibit RNases; N-lauryl sarcosine to inhibit new rounds of
transcription initiation during cell lysis; and sodium citrate to
chelate divalent cations that can contribute to RNA hydrolysis.
9. Another method for assessing CRISPRi target gene knockdown is by monitoring the targeted gene’s protein levels by
western blot. However, this is not as broadly applicable given
the paucity of antibodies to endogenous M. tuberculosis
proteins.
10. We use the PrimerQuest tool from IDT (https://www.idtdna.
com/PrimerQuest/Home/Index) to design 2–3 qPCR
primer pairs for the target gene of interest. Each qPCR primer
pair is then validated for efficiency, specificity, and the linear
range of amplification using standard qPCR approaches. A
single amplification product is confirmed for each validated
qPCR primer pair by inspection of the amplification melt
curve.
11. Due to the common occurrence of promoters in the
M. tuberculosis genome [25, 26], including within ORFs and
in the sense and antisense directions, exercise caution when
interpreting the magnitude of target knockdown by qRT-PCR.
For instance, CRISPRi knockdown of some target genes may
appear ineffective by qRT-PCR. One possible explanation for
this lack of measured knockdown is the presence of multiple
promoters driving transcription through the target gene, not
all of which are disrupted by the chosen sgRNA. In such
instances, consult published transcription start site (TSS) annotations in the M. tuberculosis genome to determine the location
and direction of TSSs proximal to the gene of interest. Design
and validate qPCR amplicons that avoid quantifying interfering transcripts and/or use strand-specific qPCR to measure
direction-specific transcription.
12. It is important to consider the operon structure of genes
targeted with CRISPRi. CRISPRi targeting of a gene in an
operon results in transcriptional knockdown of the targeted
gene and all downstream genes in the operon. This polar effect
is due to the inhibition of RNA polymerase elongation beyond
the site of dCas9 occupancy, as defined by the sgRNA targeting
sequence. Hence, genes downstream of the sgRNA targeting
sequence
will
also
experience
CRISPRi-mediated
Mycobacterial CRISPR Interference
361