1. To disrupt CRISPRi targeting, introduce at least 2 mutations
in the target sequence and/or the PAM without affecting the
amino acid sequence of the ORF. Mutate the PAM to a
sequence not recognized by Sth1 dCas9 if possible, or to a
weaker PAM if the available silent mutations do not completely
inactivate the PAM (refer to Table 1). In the sgRNA target
sequence, silent mutations should be made in the seed
sequence, or the 8–9 most PAM-proximal nucleotides
[12, 24].
2. Use the NEBuilder tool (https://nebuilder.neb.com) to design
a Gibson assembly cloning scheme and oligos to amplify and
clone the targeted gene into the desired expression vector (see
Notes 13 and 14). To introduce the silent, CRISPRi-abrogating mutations, design an oligo containing the region of silent
mutations to be made, and 20 bp flanking upstream and downstream of the region to be mutated. Also design the reverse
complement of that oligo.
3. Using the PCR cloning scheme designed by NEBuilder, PCR
amplify the targeted gene in 2 fragments using the NEBuilderdesigned oligos and the oligos to introduce silent mutations
(see Note 15).
4. Use Gibson assembly to clone the CRISPRi-resistant expression construct into the desired expression vector. In parallel,
also clone a CRISPRi-sensitive (i.e., wild-type) allele into the
expression construct (see Note 16).
5. Sanger sequence a clone of each construct to confirm proper
assembly and/or introduction of the silent mutations.
6. Transform the CRISPRi-resistant and CRISPRi-sensitive constructs into the relevant electrocompetent cells of the
M. tuberculosis CRISPRi strain.
7. Perform phenotypic assays such as growth and survival assays
and microscopy to confirm complementation of the CRISPRi
knockdown phenotypes.
3.9 Targeting
Multiple Genes with
CRISPRi
To clone multiple sgRNAs into the same plasmid, the CRISPRi
backbones were designed with a SapI-based Golden Gate cloning
site. This site is placed downstream of the first sgRNA scaffold. In
principle, the SapI-based Golden Gate approach allows for the
cloning of an additional seven sgRNA cassettes in tandem into a
single CRISPRi backbone, which would in turn direct the expression of a total of eight sgRNAs from a single plasmid.
1. Clone all individual sgRNAs into the CRISPRi backbone (see
Subheadings 3.1–3.4 for detailed design and cloning
instructions).
2. Amplify each [promoter-sgRNA-terminator] cassette with primers that contain SapI-restriction sites as well as compatible
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Andrew I. Wong and Jeremy M. Rock
in the target sequence and/or the PAM without affecting the
amino acid sequence of the ORF. Mutate the PAM to a
sequence not recognized by Sth1 dCas9 if possible, or to a
weaker PAM if the available silent mutations do not completely
inactivate the PAM (refer to Table 1). In the sgRNA target
sequence, silent mutations should be made in the seed
sequence, or the 8–9 most PAM-proximal nucleotides
[12, 24].
2. Use the NEBuilder tool (https://nebuilder.neb.com) to design
a Gibson assembly cloning scheme and oligos to amplify and
clone the targeted gene into the desired expression vector (see
Notes 13 and 14). To introduce the silent, CRISPRi-abrogating mutations, design an oligo containing the region of silent
mutations to be made, and 20 bp flanking upstream and downstream of the region to be mutated. Also design the reverse
complement of that oligo.
3. Using the PCR cloning scheme designed by NEBuilder, PCR
amplify the targeted gene in 2 fragments using the NEBuilderdesigned oligos and the oligos to introduce silent mutations
(see Note 15).
4. Use Gibson assembly to clone the CRISPRi-resistant expression construct into the desired expression vector. In parallel,
also clone a CRISPRi-sensitive (i.e., wild-type) allele into the
expression construct (see Note 16).
5. Sanger sequence a clone of each construct to confirm proper
assembly and/or introduction of the silent mutations.
6. Transform the CRISPRi-resistant and CRISPRi-sensitive constructs into the relevant electrocompetent cells of the
M. tuberculosis CRISPRi strain.
7. Perform phenotypic assays such as growth and survival assays
and microscopy to confirm complementation of the CRISPRi
knockdown phenotypes.
3.9 Targeting
Multiple Genes with
CRISPRi
To clone multiple sgRNAs into the same plasmid, the CRISPRi
backbones were designed with a SapI-based Golden Gate cloning
site. This site is placed downstream of the first sgRNA scaffold. In
principle, the SapI-based Golden Gate approach allows for the
cloning of an additional seven sgRNA cassettes in tandem into a
single CRISPRi backbone, which would in turn direct the expression of a total of eight sgRNAs from a single plasmid.
1. Clone all individual sgRNAs into the CRISPRi backbone (see
Subheadings 3.1–3.4 for detailed design and cloning
instructions).
2. Amplify each [promoter-sgRNA-terminator] cassette with primers that contain SapI-restriction sites as well as compatible
356
Andrew I. Wong and Jeremy M. Rock