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.

Keywords: CRISPR/Cas; homologous recombination; mutagenesis; non-tuberculosis mycobacteria (NTMs); recombineering; transposon mutagenesis.

FIGURE 1

Graphical representation of recombineering with ds and ssDNA and ORBIT. dsDNA substrates (left) usually contain an antibiotic resistance marker for selection, while ssDNA substrates (middle) carry only the desired mutation flanked by short regions (25–50 bp) of homology. In ORBIT (right), the ssDNA substrate contains a Bxb1 integration site flanked by short regions (25–50 bp) of homology. The gp60 exonuclease (RecE homolog) generates single stranded overhangs from the dsDNA fragments, while gp61 (RecT homolog) promotes annealing of ssDNA to homologous regions in the chromosome. Recombineering is more efficient when oligonucleotides are targeted to the lagging strand (indicated in diagram). In ORBIT, Bxb1 facilitates concomitant integration of the payload plasmid. Images adapted from Servier Medical Art by Servier which is licensed under a Creative Commons Attribution 3.0 Unported License (https://smart.servier.com/).

FIGURE 2

Graphical representation of CRISPR/Cas mutagenesis. Synthetic sgRNAs (black) direct binding of the Cas nuclease (pink) to a specific gene through Watson-Crick base paring between the sgRNA and the target sequence. Initial binding of the sgRNA-Cas complex is dependent on the recognition of an adjacent PAM sequence (red). Nuclease cleavage results in a double strand break in the chromosome, which is repaired either through homologous recombination, in the presence of a homologous substrate, or non-homologous end joining. The error-prone nature of NHEJ it results in random mutations at the site of repair. Phage proteins gp60 and gp61 are proposed to function in DSB repair with ssDNA and dsDNA substrates (CRISPR/Cas-mediated recombineering).