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Re-sensitization of Mycobacterium smegmatis to Rifampicin Using CRISPR Interference Demonstrates Its Utility for the Study of Non-essential Drug Resistance Traits

Kendall, Sharon L.; Faulkner, Valwynne; Cox, Adrienne Adele; Goh, Shan; van Bohemen, Annelies; Gibson, Amanda J.; Liebster, Oliver; Wren, Brendan W.; Willcocks, Sam

Authors

Sharon L. Kendall

Valwynne Faulkner

Adrienne Adele Cox

Shan Goh

Annelies van Bohemen

Amanda J. Gibson

Oliver Liebster

Brendan W. Wren

Sam Willcocks



Contributors

Sharon Kendall
Project Leader

Abstract

A greater understanding of the genes involved in antibiotic resistance in Mycobacterium tuberculosis (Mtb) is necessary for the design of improved therapies. Clustered regularly interspaced short palindromic repeat interference (CRISPRi) has been previously utilized in mycobacteria to identify novel drug targets by the demonstration of gene essentiality. The work presented here shows that it can also be usefully applied to the study of non-essential genes involved in antibiotic resistance. The expression of an ADP-ribosyltransferase (Arr) involved in rifampicin resistance in Mycobacterium smegmatis was silenced using CRISPRi and the impact on rifampicin susceptibility was measured. Gene silencing resulted in a decrease in the minimum inhibitory concentration (MIC) similar to that previously reported in an arr deletion mutant. There is contradictory evidence for the toxicity of Streptococcus pyogenes dCas9 (dCas9Spy) in the literature. In this study the expression of dCas9Spy in M. smegmatis showed no impact on viability. Silencing was achieved with concentrations of the aTc inducer lower than previously described and with shorter induction times. Finally, designing small guide RNAs (sgRNAs) that target transcription initiation, or the early stages of elongation had the most impact on rifampicin susceptibility. This study demonstrates that CRISPRi based gene silencing can be as impactful as gene deletion for the study of non-essential genes and further contributes to the knowledge on the design and induction of sgRNAs for CRISPRi. This approach can be applied to other non-essential antimicrobial resistance genes such as drug efflux pumps.

Citation

Kendall, S. L., Faulkner, V., Cox, A. A., Goh, S., van Bohemen, A., Gibson, A. J., …Willcocks, S. (in press). Re-sensitization of Mycobacterium smegmatis to Rifampicin Using CRISPR Interference Demonstrates Its Utility for the Study of Non-essential Drug Resistance Traits. Frontiers in Microbiology, 11, https://doi.org/10.3389/fmicb.2020.619427

Journal Article Type Article
Acceptance Date Dec 21, 2020
Online Publication Date Feb 1, 2021
Deposit Date Feb 22, 2021
Publicly Available Date Mar 28, 2024
Journal Frontiers in Microbiology
Publisher Frontiers Media
Peer Reviewed Peer Reviewed
Volume 11
DOI https://doi.org/10.3389/fmicb.2020.619427
Keywords Microbiology (medical); Microbiology
Public URL https://rvc-repository.worktribe.com/output/1443473

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