Chapter 16
CRISPR Interference (CRISPRi) for Targeted Gene Silencing
in Mycobacteria
Andrew I. Wong and Jeremy M. Rock
Abstract
The genetic basis for Mycobacterium tuberculosis pathogenesis is incompletely understood. One reason for
this knowledge gap is the relative difficulty of genetic manipulation of M. tuberculosis. To close this gap, we
recently developed a robust CRISPR interference (CRISPRi) platform for programmable gene silencing in
mycobacteria. In this chapter, we: (1) discuss some of the advantages and disadvantages of CRISPRi relative
to more traditional genetic approaches; and (2) provide a protocol for the application of CRISPRi to reduce
transcription of target genes in mycobacteria.
Key words CRISPR, CRISPRi, CRISPR interference, Gene silencing, Gene knockdown, Transcriptional silencing
1 Introduction
The past 20 years have seen dramatic improvements in our ability to
genetically manipulate Mycobacterium tuberculosis. These improvements have advanced our understanding of this pathogen. While
powerful, each genetic method has advantages and disadvantages.
For example, current genetic approaches in M. tuberculosis include
promoter replacement and inducible protein degradation systems
that allow the regulation of target protein levels over two orders of
magnitude [1–3], but can take months to target a single gene.
Similarly, several methodologies [4, 5] now allow for facile gene
deletion in M. tuberculosis, but these methods are necessarily
restricted to the analysis of genes nonessential for in vitro growth
and remain slow to implement. To increase throughput, Kenan
Murphy and colleagues developed ORBIT [6], a method that
dramatically improves the efficiency of M. tuberculosis genetic engineering. While it is a major advance, this method in its present form
does not yet scale to parallelized genome-wide genetic manipulation. To work at scale, Transposon-sequencing (TnSeq) allows the
simultaneous assessment of hundreds of thousands of loss-ofTanya Parish and Anuradha Kumar (eds.), Mycobacteria Protocols, Methods in Molecular Biology, vol. 2314,
https://doi.org/10.1007/978-1-0716-1460-0_16, © Springer Science+Business Media, LLC, part of Springer Nature 2021
343
CRISPR Interference (CRISPRi) for Targeted Gene Silencing
in Mycobacteria
Andrew I. Wong and Jeremy M. Rock
Abstract
The genetic basis for Mycobacterium tuberculosis pathogenesis is incompletely understood. One reason for
this knowledge gap is the relative difficulty of genetic manipulation of M. tuberculosis. To close this gap, we
recently developed a robust CRISPR interference (CRISPRi) platform for programmable gene silencing in
mycobacteria. In this chapter, we: (1) discuss some of the advantages and disadvantages of CRISPRi relative
to more traditional genetic approaches; and (2) provide a protocol for the application of CRISPRi to reduce
transcription of target genes in mycobacteria.
Key words CRISPR, CRISPRi, CRISPR interference, Gene silencing, Gene knockdown, Transcriptional silencing
1 Introduction
The past 20 years have seen dramatic improvements in our ability to
genetically manipulate Mycobacterium tuberculosis. These improvements have advanced our understanding of this pathogen. While
powerful, each genetic method has advantages and disadvantages.
For example, current genetic approaches in M. tuberculosis include
promoter replacement and inducible protein degradation systems
that allow the regulation of target protein levels over two orders of
magnitude [1–3], but can take months to target a single gene.
Similarly, several methodologies [4, 5] now allow for facile gene
deletion in M. tuberculosis, but these methods are necessarily
restricted to the analysis of genes nonessential for in vitro growth
and remain slow to implement. To increase throughput, Kenan
Murphy and colleagues developed ORBIT [6], a method that
dramatically improves the efficiency of M. tuberculosis genetic engineering. While it is a major advance, this method in its present form
does not yet scale to parallelized genome-wide genetic manipulation. To work at scale, Transposon-sequencing (TnSeq) allows the
simultaneous assessment of hundreds of thousands of loss-ofTanya Parish and Anuradha Kumar (eds.), Mycobacteria Protocols, Methods in Molecular Biology, vol. 2314,
https://doi.org/10.1007/978-1-0716-1460-0_16, © Springer Science+Business Media, LLC, part of Springer Nature 2021
343