37
C for 1 min and 16
C for 1 min, then heat at 60
C for
5 min.
19. When cloning multiple sgRNA cassettes into a single Golden
Gate site (i.e., such that a single plasmid will express more than
2 sgRNAs), it may be necessary to increase the volumes of
competent E. coli cells used to increase cloning yield.
20. The methods described in this protocol are likely applicable to
other mycobacterial species not discussed here, including
M. abscessus, M. avium complex, M. marinum, and others.
References
1. Kim J-H, Wei J-R, Wallach JB et al (2011)
Protein inactivation in mycobacteria by controlled proteolysis and its application to deplete
the beta subunit of RNA polymerase. Nucleic
Acids Res 39:2210–2220. https://doi.org/10.
1093/nar/gkq1149
2. Ehrt S, Guo XV, Hickey CM et al (2005)
Controlling gene expression in mycobacteria
with anhydrotetracycline and Tet repressor.
Nucleic Acids Res 33:e21. https://doi.org/
10.1093/nar/gni013
3. Wei J-R, Krishnamoorthy V, Murphy K et al
(2011) Depletion of antibiotic targets has
widely varying effects on growth. Proc Natl
Acad Sci U S A 108:4176–4181. https://doi.
org/10.1073/pnas.1018301108
4. van Kessel JC, Hatfull GF (2007) Recombineering in Mycobacterium tuberculosis. Nat
Methods 4:147–152. https://doi.org/10.
1038/nmeth996
5. Yan M-Y, Li S-S, Ding X-Y et al (2020) A
CRISPR-assisted nonhomologous end-joining
strategy for efficient genome editing in Mycobacterium
tuberculosis.
MBio
11:
e02364–e02319. https://doi.org/10.1128/
mBio.02364-19
6. Murphy KC, Nelson SJ, Nambi S, et al (2018)
ORBIT: a new paradigm for genetic engineering of mycobacterial chromosomes bioRxiv
249292. https://doi.org/10.1101/249292
7. Zhang YJ, Ioerger TR, Huttenhower C et al
(2012) Global assessment of genomic regions
required for growth in Mycobacterium tuberculosis. PLoS Pathog 8:e1002946. https://doi.
org/10.1371/journal.ppat.1002946
8. Griffin JE, Gawronski JD, DeJesus MA et al
(2011) High-resolution phenotypic profiling
defines genes essential for mycobacterial
growth and cholesterol catabolism. PLoS
Pathog 7:e1002251. https://doi.org/10.
1371/journal.ppat.1002251
9. Rock JM, Hopkins FF, Chavez A et al (2017)
Programmable transcriptional repression in
mycobacteria using an orthogonal CRISPR
interference platform. Nat Microbiol 2:1–9.
https://doi.org/10.1038/nmicrobiol.2016.
274
10. Qi LS, Larson MH, Gilbert LA et al (2013)
Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene
expression. Cell 152:1173–1183. https://doi.
org/10.1016/j.cell.2013.02.022
11. Bikard D, Jiang W, Samai P et al (2013) Programmable repression and activation of bacterial gene expression using an engineered
CRISPR-Cas system. Nucleic Acids Res
41:7429–7437.
https://doi.org/10.1093/
nar/gkt520
12. Jinek M, Chylinski K, Fonfara I et al (2012) A
programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science
337:816–821.
https://doi.org/10.
1126/science.1225829
13. Horvath P, Romero DA, Cou ˆte ´-Monvoisin AC
et al (2008) Diversity, activity, and evolution of
CRISPR loci in Streptococcus thermophilus. J
Bacteriol 190:1401–1412. https://doi.org/
10.1128/JB.01415-07
14. Deveau H, Barrangou R, Garneau JE et al
(2008) Phage response to CRISPR-encoded
resistance in Streptococcus thermophilus. J
Bacteriol 190:1390–1400. https://doi.org/
10.1128/JB.01412-07
15. Mojica FJM, Dı ´ez-Villasen ˜or C, Garcı ´aMartı ´nez J, Almendros C (2009) Short motif
sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology 155:733–740. https://doi.org/10.1099/
mic.0.023960-0
16. Anders C, Niewoehner O, Duerst A, Jinek M
(2014) Structural basis of PAM-dependent target DNA recognition by the Cas9
Mycobacterial CRISPR Interference
363
C for 1 min and 16
C for 1 min, then heat at 60
C for
5 min.
19. When cloning multiple sgRNA cassettes into a single Golden
Gate site (i.e., such that a single plasmid will express more than
2 sgRNAs), it may be necessary to increase the volumes of
competent E. coli cells used to increase cloning yield.
20. The methods described in this protocol are likely applicable to
other mycobacterial species not discussed here, including
M. abscessus, M. avium complex, M. marinum, and others.
References
1. Kim J-H, Wei J-R, Wallach JB et al (2011)
Protein inactivation in mycobacteria by controlled proteolysis and its application to deplete
the beta subunit of RNA polymerase. Nucleic
Acids Res 39:2210–2220. https://doi.org/10.
1093/nar/gkq1149
2. Ehrt S, Guo XV, Hickey CM et al (2005)
Controlling gene expression in mycobacteria
with anhydrotetracycline and Tet repressor.
Nucleic Acids Res 33:e21. https://doi.org/
10.1093/nar/gni013
3. Wei J-R, Krishnamoorthy V, Murphy K et al
(2011) Depletion of antibiotic targets has
widely varying effects on growth. Proc Natl
Acad Sci U S A 108:4176–4181. https://doi.
org/10.1073/pnas.1018301108
4. van Kessel JC, Hatfull GF (2007) Recombineering in Mycobacterium tuberculosis. Nat
Methods 4:147–152. https://doi.org/10.
1038/nmeth996
5. Yan M-Y, Li S-S, Ding X-Y et al (2020) A
CRISPR-assisted nonhomologous end-joining
strategy for efficient genome editing in Mycobacterium
tuberculosis.
MBio
11:
e02364–e02319. https://doi.org/10.1128/
mBio.02364-19
6. Murphy KC, Nelson SJ, Nambi S, et al (2018)
ORBIT: a new paradigm for genetic engineering of mycobacterial chromosomes bioRxiv
249292. https://doi.org/10.1101/249292
7. Zhang YJ, Ioerger TR, Huttenhower C et al
(2012) Global assessment of genomic regions
required for growth in Mycobacterium tuberculosis. PLoS Pathog 8:e1002946. https://doi.
org/10.1371/journal.ppat.1002946
8. Griffin JE, Gawronski JD, DeJesus MA et al
(2011) High-resolution phenotypic profiling
defines genes essential for mycobacterial
growth and cholesterol catabolism. PLoS
Pathog 7:e1002251. https://doi.org/10.
1371/journal.ppat.1002251
9. Rock JM, Hopkins FF, Chavez A et al (2017)
Programmable transcriptional repression in
mycobacteria using an orthogonal CRISPR
interference platform. Nat Microbiol 2:1–9.
https://doi.org/10.1038/nmicrobiol.2016.
274
10. Qi LS, Larson MH, Gilbert LA et al (2013)
Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene
expression. Cell 152:1173–1183. https://doi.
org/10.1016/j.cell.2013.02.022
11. Bikard D, Jiang W, Samai P et al (2013) Programmable repression and activation of bacterial gene expression using an engineered
CRISPR-Cas system. Nucleic Acids Res
41:7429–7437.
https://doi.org/10.1093/
nar/gkt520
12. Jinek M, Chylinski K, Fonfara I et al (2012) A
programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science
337:816–821.
https://doi.org/10.
1126/science.1225829
13. Horvath P, Romero DA, Cou ˆte ´-Monvoisin AC
et al (2008) Diversity, activity, and evolution of
CRISPR loci in Streptococcus thermophilus. J
Bacteriol 190:1401–1412. https://doi.org/
10.1128/JB.01415-07
14. Deveau H, Barrangou R, Garneau JE et al
(2008) Phage response to CRISPR-encoded
resistance in Streptococcus thermophilus. J
Bacteriol 190:1390–1400. https://doi.org/
10.1128/JB.01412-07
15. Mojica FJM, Dı ´ez-Villasen ˜or C, Garcı ´aMartı ´nez J, Almendros C (2009) Short motif
sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology 155:733–740. https://doi.org/10.1099/
mic.0.023960-0
16. Anders C, Niewoehner O, Duerst A, Jinek M
(2014) Structural basis of PAM-dependent target DNA recognition by the Cas9
Mycobacterial CRISPR Interference
363