Implementation of a Mycobacterial CRISPRi Platform in Mycobacterium Abscessus and Demonstration of the Essentiality of FtsZ

Overview

Journal Tuberculosis (Edinb)

Specialties Infectious Diseases
Pulmonary Medicine

Date 2022 Dec 10

PMID 36495774

Authors

Rashmi Gupta

Kyle H Rohde

Affiliations

Soon will be listed here.

Abstract

Mycobacterium abscessus (Mab) is a highly drug-resistant non-tuberculous mycobacterial species that causes debilitating TB-like pulmonary infections. The lack of genetic tools has hampered characterization of its extensive repertoire of virulence factors, antimicrobial resistance mechanisms, and drug targets. In this study, we evaluated the performance of a mycobacterial single plasmid CRISPRi-dCas9 system optimized for M. tuberculosis and M. smegmatis for inducible gene silencing in Mab. The efficacy of CRISPRi-mediated repression of two antibiotic resistance genes (bla, whiB7) and two putative essential genes (ftsZtopA) was determined by measuring mRNA transcript levels and phenotypic outcomes. While our results support the utility of this mycobacterial CRISPRi dCas9 single-plasmid platform for inducible silencing of specific target genes in Mab, they also highlighted several caveats and nuances that may warrant species-specific optimization for Mab. We observed overall lower levels of gene repression in Mab including variable silencing of different target genes despite use of PAMs of similar predicted strength. In addition, leaky gene repression in the absence of inducer was noted for some genes but not others. Nonetheless, using CRISPRi we demonstrated the silencing of multiple target genes and validated ftsZ as an essential gene and promising drug target for the first time.

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