transduction system described by Bardarov [7] has been used in
numerous studies over the last 17 years. More recent improvements
in this system have involved increasing the efficiency of constructing allelic exchange substrates and introducing the capability of
making unmarked knockouts [8].
The introduction of recombineering technology for the manipulation of mycobacterial chromosomes by van Kessel and Hatfull in
2007 allowed for efficient targeting of genes using simpler allelicexchange substrates (linear dsDNA generated by plasmid digestion,
or overlapping PCRs) [9, 10]. This methodology, which employs
the phage Che9 RecET recombination system, has been used by
many investigators over the last decade [10–14]. It was most
recently instrumental in the construction of a large library of
M. tuberculosis strains where nearly every essential gene was tagged
with a degradation epitope, allowing for the generation of a hypomorphic library [15]. The RecET system has also been employed to
increase the frequency of recombination in the specialized transduction protocol of mycobacterial gene replacement discussed
above [16].
Just as significant, but perhaps less utilized, has been the use of
the Che9 phage RecT annealase to promote oligo-mediated recombineering in M. tuberculosis, first described in 2008 [17]. In the
most routine use of this method, an electroporated oligonucleotide
(oligo) carrying a single nucleotide polymorphism (SNP) is bound
by RecT in vivo. RecT subsequently anneals the oligo to the
lagging-strand template of the replication fork where it gets
incorporated into the bacterial chromosome. This method is most
suited for the verification of SNPs suspected of being associated
with spontaneous drug-resistance, as was done by Ioerger et al.
[18], or for the transfer of SNPs from clinical isolates to laboratory
strains of mycobacteria to test their relevance to a specific disease
phenotype. A likely mechanism of oligo-recombineering is shown
in Fig. 1a.
The widespread use of oligo-mediated recombineering in
M. tuberculosis is generally lacking though, principally because of
the low frequency of recombinant formation (~1:1000), and
because of the inability to select for incorporation of the oligo
after transfer to the chromosome. Oligos containing large insertions, like those corresponding to drug-resistant markers, are not
suitable for recombineering. While there are schemes where an
oligo can be selected biologically, or be genetically designed to be
selected, these strategies are not generally useful for transfer of
SNPs of unknown function. Promising avenues for increasing the
efficiency of finding SNP transfers involve CRISPR-associated
recombineering schemes, where Cas9 or Cas12a proteins are utilized to remove cells from a population that have not taken up the
SNP, as has been reported in E. coli [19] and other species. While a
Cas12a-assisted scheme has been reported for M. smegmatis [20],
302
Kenan C. Murphy
numerous studies over the last 17 years. More recent improvements
in this system have involved increasing the efficiency of constructing allelic exchange substrates and introducing the capability of
making unmarked knockouts [8].
The introduction of recombineering technology for the manipulation of mycobacterial chromosomes by van Kessel and Hatfull in
2007 allowed for efficient targeting of genes using simpler allelicexchange substrates (linear dsDNA generated by plasmid digestion,
or overlapping PCRs) [9, 10]. This methodology, which employs
the phage Che9 RecET recombination system, has been used by
many investigators over the last decade [10–14]. It was most
recently instrumental in the construction of a large library of
M. tuberculosis strains where nearly every essential gene was tagged
with a degradation epitope, allowing for the generation of a hypomorphic library [15]. The RecET system has also been employed to
increase the frequency of recombination in the specialized transduction protocol of mycobacterial gene replacement discussed
above [16].
Just as significant, but perhaps less utilized, has been the use of
the Che9 phage RecT annealase to promote oligo-mediated recombineering in M. tuberculosis, first described in 2008 [17]. In the
most routine use of this method, an electroporated oligonucleotide
(oligo) carrying a single nucleotide polymorphism (SNP) is bound
by RecT in vivo. RecT subsequently anneals the oligo to the
lagging-strand template of the replication fork where it gets
incorporated into the bacterial chromosome. This method is most
suited for the verification of SNPs suspected of being associated
with spontaneous drug-resistance, as was done by Ioerger et al.
[18], or for the transfer of SNPs from clinical isolates to laboratory
strains of mycobacteria to test their relevance to a specific disease
phenotype. A likely mechanism of oligo-recombineering is shown
in Fig. 1a.
The widespread use of oligo-mediated recombineering in
M. tuberculosis is generally lacking though, principally because of
the low frequency of recombinant formation (~1:1000), and
because of the inability to select for incorporation of the oligo
after transfer to the chromosome. Oligos containing large insertions, like those corresponding to drug-resistant markers, are not
suitable for recombineering. While there are schemes where an
oligo can be selected biologically, or be genetically designed to be
selected, these strategies are not generally useful for transfer of
SNPs of unknown function. Promising avenues for increasing the
efficiency of finding SNP transfers involve CRISPR-associated
recombineering schemes, where Cas9 or Cas12a proteins are utilized to remove cells from a population that have not taken up the
SNP, as has been reported in E. coli [19] and other species. While a
Cas12a-assisted scheme has been reported for M. smegmatis [20],
302
Kenan C. Murphy