Genetic Manipulation of Non-tuberculosis Mycobacteria

Abstract

Non-tuberculosis mycobacteria (NTMs) comprise a large group of organisms that are phenotypically diverse. Analysis of the growing number of completed NTM genomes has revealed both significant intra-genus genetic diversity, and a high percentage of predicted genes that appear to be unique to this group. Most NTMs have not been studied, however, the rise in NTM infections in several countries has prompted increasing interest in these organisms. Mycobacterial research has recently benefitted from the development of new genetic tools and a growing number of studies describing the genetic manipulation of NTMs have now been reported. In this review, we discuss the use of both site-specific and random mutagenesis tools in NTMs, highlighting the challenges that exist in applying these techniques to this diverse group of organisms.

A dual-plasmid CRISPR/Cas9-based method for rapid and efficient genetic disruption in Mycobacterium abscessus

“…In turn, this enables the screening of only small numbers of clones to obtain the desired mutants. Indeed, we report an efficiency rate of 1.1%–9.3% using this two-plasmid system for the disruption of a single gene, which is at least 10 2 –10 4 more efficient than rates reported for homologous recombination ( 9 – 12 ). Compared to traditional approaches where success is limited due to the occurrence of spontaneous background resistance and low efficiency of homologous recombination, the high frameshift frequency suggests that CRISPR/Cas9 is an important addition to the toolbox for genetic manipulation in M. abscessus .…”

“…Current methods for genetically engineering M. abscessus have been largely limited to suboptimal and tedious recombination-based methods due to the high rates of spontaneous background antibiotic resistance. These methods are inefficient, necessitating the selection of numerous colonies to identify the correct mutants, or ineffective, where the desired clone cannot be obtained ( 9 – 12 ). Meanwhile, CRISPR/Cas9 has revolutionized the field of genome editing but has only recently been adapted for use in mycobacteria for this purpose ( 19 – 21 ).…”

“…While transposon-sequencing is a viable approach for genome-wide studies, homologous recombination-based methods have been the primary tool for producing genetic deletion mutants in many bacterial species including M. abscessus ( 9 – 14 ). In mycobacteria, homologous recombination using allelic exchange mutagenesis or recombineering has been used extensively to generate gene deletions, although the efficiency is suboptimal with the frequency of obtaining a desired mutation ranging from 10 –6 to 10 –4 ( 11 , 12 ). Moreover, in M. abscessus , high rates of spontaneous background resistance to the antibiotic selection marker, in combination with the low success rates of homologous recombination, result in a cumbersome process requiring the screening of a lot of clones to find the rare, desired correct mutant ( 9 – 12 ).…”

“…In mycobacteria, homologous recombination using allelic exchange mutagenesis or recombineering has been used extensively to generate gene deletions, although the efficiency is suboptimal with the frequency of obtaining a desired mutation ranging from 10 –6 to 10 –4 ( 11 , 12 ). Moreover, in M. abscessus , high rates of spontaneous background resistance to the antibiotic selection marker, in combination with the low success rates of homologous recombination, result in a cumbersome process requiring the screening of a lot of clones to find the rare, desired correct mutant ( 9 – 12 ). These technical challenges have motivated the development of improvements or alternate methods.…”

Silencing essential gene expression in Mycobacterium abscessus during infection

“…Gene manipulation has been widely developed during these last two decades, mainly in M. tuberculosis and M. smegmatis , through the generation of knockout mutants and the development of regulated expression systems, which have been instrumental to our understanding of pathogenic mechanisms and to demonstrate the essentiality of genes encoding drug targets. However, functional genomics tools are limited in M. abscessus , with mutagenesis approaches developed in other species often proving to be challenging to adopt in M. abscessus ( 17 , 18 ). Despite these difficulties, recent studies have reported the successful use of CRISPR/Cas-based systems to inducibly silence the expression of targeted genes in M. abscessus ( 19 ).…”