manipulation of M. tuberculosis genes, be they essential or nonessential for in vitro growth. The efficient cellular and tissue penetration of doxycycline [18] should allow CRISPRi-mediated control
of the M. tuberculosis transcriptome in numerous experimental
settings, including axenic in vitro culture, ex vivo M. tuberculosisinfected macrophages, and in vivo animal infection models. An
additional advantage of the Sth1 dCas9 CRISPRi system is that
the magnitude of target gene silencing is tunable, either by varying
targeted PAM “strength” [9] or by varying the length of the
sgRNA targeting sequence [10]. This allows for rheostat-like control of target gene production spanning two orders of magnitude
[9, 10]. Tunability enables the hypomorphic or partial silencing of
target gene production to create an allelic series, thereby enabling
the study of interactions (chemical and genetic) between in vitro
essential genes [9, 19]. Lastly, CRISPRi is scalable. With advances
in array-based synthesis, generating large pools of unique sgRNA
targeting sequences is fast and inexpensive. Altogether, these
unique features set the stage to develop CRISPRi as a powerful
new genetic method in M. tuberculosis. In the next sections, we
discuss the application and limitations of this approach for targeted
gene silencing in M. tuberculosis.
2 Materials
2.1 Design of
CRISPRi sgRNA
Targeting Sequence
Oligonucleotides
(sgRNA Oligos)
1. sgRNA oligos: There will be two oligos for each sgRNA to be
cloned. Resuspend each oligo in deionized water to a concentration of 100 μM.
2.2 Annealing sgRNA
Oligos for Ligation into
the CRISPRi Plasmid
Backbone
1. PCR tubes.
2. Oligos (top and bottom).
3. Oligo Annealing Buffer: 50 mM Tris pH 7.5, 50 mM NaCl,
1 mM EDTA.
2.3 BsmBI-Digestion
of the CRISPRi Plasmid
Backbone
1. BsmBI-v2 (10,000 U/mL; NEB).
2. NEBuffer 3.1 (supplied with BsmBI-v2).
3. plJR965 (Addgene #115163): the CRISPRi backbone for
M. tuberculosis. This plasmid contains an Sth1 dCas9 allele, an
sgRNA scaffold, a Tet repressor (TetR) codon optimized for
expression in M. tuberculosis, an L5 integrase, a kanR gene that
confers kanamycin resistance, and an E. coli origin of replication
(Fig. 2). dCas9 and the sgRNA are expressed from a TetRregulated promoter that is induced in the presence of anhydrotetracycline (ATc) or doxycycline. The plasmid integrates into
Mycobacterial CRISPR Interference
345
of the M. tuberculosis transcriptome in numerous experimental
settings, including axenic in vitro culture, ex vivo M. tuberculosisinfected macrophages, and in vivo animal infection models. An
additional advantage of the Sth1 dCas9 CRISPRi system is that
the magnitude of target gene silencing is tunable, either by varying
targeted PAM “strength” [9] or by varying the length of the
sgRNA targeting sequence [10]. This allows for rheostat-like control of target gene production spanning two orders of magnitude
[9, 10]. Tunability enables the hypomorphic or partial silencing of
target gene production to create an allelic series, thereby enabling
the study of interactions (chemical and genetic) between in vitro
essential genes [9, 19]. Lastly, CRISPRi is scalable. With advances
in array-based synthesis, generating large pools of unique sgRNA
targeting sequences is fast and inexpensive. Altogether, these
unique features set the stage to develop CRISPRi as a powerful
new genetic method in M. tuberculosis. In the next sections, we
discuss the application and limitations of this approach for targeted
gene silencing in M. tuberculosis.
2 Materials
2.1 Design of
CRISPRi sgRNA
Targeting Sequence
Oligonucleotides
(sgRNA Oligos)
1. sgRNA oligos: There will be two oligos for each sgRNA to be
cloned. Resuspend each oligo in deionized water to a concentration of 100 μM.
2.2 Annealing sgRNA
Oligos for Ligation into
the CRISPRi Plasmid
Backbone
1. PCR tubes.
2. Oligos (top and bottom).
3. Oligo Annealing Buffer: 50 mM Tris pH 7.5, 50 mM NaCl,
1 mM EDTA.
2.3 BsmBI-Digestion
of the CRISPRi Plasmid
Backbone
1. BsmBI-v2 (10,000 U/mL; NEB).
2. NEBuffer 3.1 (supplied with BsmBI-v2).
3. plJR965 (Addgene #115163): the CRISPRi backbone for
M. tuberculosis. This plasmid contains an Sth1 dCas9 allele, an
sgRNA scaffold, a Tet repressor (TetR) codon optimized for
expression in M. tuberculosis, an L5 integrase, a kanR gene that
confers kanamycin resistance, and an E. coli origin of replication
(Fig. 2). dCas9 and the sgRNA are expressed from a TetRregulated promoter that is induced in the presence of anhydrotetracycline (ATc) or doxycycline. The plasmid integrates into
Mycobacterial CRISPR Interference
345