recombination events between the genome and large donor fragments encoding the desired genetic change. As such, they involve
the individual cloning and optimization of each ~1–2 kb homologous repair template, or in the case of engineering gene deletions,
their manufacture by splicing overhang extension (SOE) PCR
[7, 8].
To facilitate the construction of isogenic strains in S. aureus, we
have recently developed a system for recombineering [9] and
CRISPR/Cas9-mediated counterselection [10] in that organism
[11]. Recombineering, which incorporates mutagenic oligonucleotides into a host genome through the action of bacteriophagederived single-stranded DNA (ssDNA) recombinases, allows
point mutations, variable-length deletions, and short insertions to
be precisely engineered [9, 12–14]. CRISPR/Cas9-mediated
endonuclease cleavage targeted to the wild-type allele subsequently
provides counterselection for the engineered change, even in the
absence of an externally selectable phenotype, by introducing
double-stranded DNA breaks into the genome of unedited cells
[15]. Advantageously, this strategy utilizes commercially synthesized synthetic DNA oligonucleotides as substrates for introducing
precise genomic modifications and for performing counterselection, making it possible to scalably, rapidly, and inexpensively engineer a range of genomic changes into laboratory or clinical
S. aureus strains by transforming them with effector plasmids and
oligonucleotides using electroporation [11]. Use of temperaturesensitive plasmid vectors ensures that exogenous genetic material
can be removed after brief passaging at elevated temperatures
[16, 17]. Although the protocols detailed here are specific for
S. aureus, the general principles discussed are broadly applicable
across the organisms for which recombineering and CRISPR/Cas9
counterselection systems have been established [13, 15, 18–21].
2 Materials
2.1 Plasmids
1. pCN-EF2132tet. The conditional recombineering vector,
pCN-EF2132tet [11], is available from Addgene
(ID 107191). This vector expresses recombinase EF2132
from Enterococcus faecalis, which we have found has high activity in S. aureus, under control of the strong, constitutive p23
promoter [22]. The plasmid is constructed on an Escherichia
coli–S. aureus shuttle vector [16]. It is maintained in E. coli at
37
under ampicillin selection (100 μg/mL). By virtue of its
T181cop-634ts origin of replication [16], the vector is temperature sensitive in S. aureus, where it must be maintained at
32
using chloramphenicol selection (10 μg/mL).
128
Kelsi Penewit and Stephen J. Salipante
the individual cloning and optimization of each ~1–2 kb homologous repair template, or in the case of engineering gene deletions,
their manufacture by splicing overhang extension (SOE) PCR
[7, 8].
To facilitate the construction of isogenic strains in S. aureus, we
have recently developed a system for recombineering [9] and
CRISPR/Cas9-mediated counterselection [10] in that organism
[11]. Recombineering, which incorporates mutagenic oligonucleotides into a host genome through the action of bacteriophagederived single-stranded DNA (ssDNA) recombinases, allows
point mutations, variable-length deletions, and short insertions to
be precisely engineered [9, 12–14]. CRISPR/Cas9-mediated
endonuclease cleavage targeted to the wild-type allele subsequently
provides counterselection for the engineered change, even in the
absence of an externally selectable phenotype, by introducing
double-stranded DNA breaks into the genome of unedited cells
[15]. Advantageously, this strategy utilizes commercially synthesized synthetic DNA oligonucleotides as substrates for introducing
precise genomic modifications and for performing counterselection, making it possible to scalably, rapidly, and inexpensively engineer a range of genomic changes into laboratory or clinical
S. aureus strains by transforming them with effector plasmids and
oligonucleotides using electroporation [11]. Use of temperaturesensitive plasmid vectors ensures that exogenous genetic material
can be removed after brief passaging at elevated temperatures
[16, 17]. Although the protocols detailed here are specific for
S. aureus, the general principles discussed are broadly applicable
across the organisms for which recombineering and CRISPR/Cas9
counterselection systems have been established [13, 15, 18–21].
2 Materials
2.1 Plasmids
1. pCN-EF2132tet. The conditional recombineering vector,
pCN-EF2132tet [11], is available from Addgene
(ID 107191). This vector expresses recombinase EF2132
from Enterococcus faecalis, which we have found has high activity in S. aureus, under control of the strong, constitutive p23
promoter [22]. The plasmid is constructed on an Escherichia
coli–S. aureus shuttle vector [16]. It is maintained in E. coli at
37
under ampicillin selection (100 μg/mL). By virtue of its
T181cop-634ts origin of replication [16], the vector is temperature sensitive in S. aureus, where it must be maintained at
32
using chloramphenicol selection (10 μg/mL).
128
Kelsi Penewit and Stephen J. Salipante