2. pCAS9counter. The conditional counterselection vector,
pCAS9counter [11], is available from Addgene (ID 107192).
This vector expresses self-sufficient small guide RNA (sgRNA)
[23] and Cas9 derived from Streptococcus pyogenes SF370
[10]. The specificity of Cas9 for introducing double stranded
DNA breaks is determined through the sequence of targeting
DNA oligonucleotides cloned into the sgRNA (see Subheading
3.1): in its native state, the sgRNA element contains a nonfunctional ~100 bp spacer, which enables screening for successfully
cloned targeting oligonucleotides using fragment length analysis PCR. The plasmid contains a temperature sensitive E194ts
S. aureus origin of replication [17] (which is compatible with
T181cop-634ts). It is maintained in E. coli at 37
under ampicillin selection (100 μg/mL), and must be maintained in
S. aureus at 32
using erythromycin selection (10 μg/mL).
2.2 Bacterial Strains
2.2.1 S. aureus Strains
With successful preparation of electrocompetent cells and plasmid
preconditioning through the appropriate E. coli plasmid artificial
modification strain (see Subheading 2.2.2), we have found that we
are able to modify the genomes of laboratory strains or primary
clinical isolates from a wide variety of genetic backgrounds and
lineages, although the efficiency of genome editing appears to be
strain specific [11].
2.2.2 E. coli Plasmid
Artificial Modification
Strains
A number of transgenic E. coli strains have been constructed which
are able to modify the methylation profiles of plasmids prior to their
introduction into the S. aureus strain of interest, which is necessary
to bypass the restriction barrier in transferring DNA between those
species. Some of the most useful plasmid artificial modification
strains are summarized below:
1. DC10B. This is a transgenic, cytosine methylase-negative E. coli
strain which enables bypass of the S. aureus type IV restriction
system [7]. The strain is useful in conditioning plasmids for
introduction into many S. aureus backgrounds, including those
for which the clonal complex designation is unusual or is not
known, but transformation efficiencies are generally not as high
as can be obtained when using a plasmid artificial modification
strain which actively mimics the methylation profile of a destination S. aureus strain.
2. SA08B. SA08B is a derivative of DC10B which has been further
modified to actively mimic the methylation profile of S. aureus
clonal complex 8/sequence type 8 (i.e., CC8/ST8), which
consequently enables bypass of the S. aureus type III restriction
system [24]. We have found that use of this E. coli strain can
condition plasmids for their introduction into the majority of
S. aureus laboratory and clinical isolates, which are largely
derived from CC8 or CC8-restriction compatible groups.
Genome Editing in Staphylococcus aureus by Conditional. . .
129
pCAS9counter [11], is available from Addgene (ID 107192).
This vector expresses self-sufficient small guide RNA (sgRNA)
[23] and Cas9 derived from Streptococcus pyogenes SF370
[10]. The specificity of Cas9 for introducing double stranded
DNA breaks is determined through the sequence of targeting
DNA oligonucleotides cloned into the sgRNA (see Subheading
3.1): in its native state, the sgRNA element contains a nonfunctional ~100 bp spacer, which enables screening for successfully
cloned targeting oligonucleotides using fragment length analysis PCR. The plasmid contains a temperature sensitive E194ts
S. aureus origin of replication [17] (which is compatible with
T181cop-634ts). It is maintained in E. coli at 37
under ampicillin selection (100 μg/mL), and must be maintained in
S. aureus at 32
using erythromycin selection (10 μg/mL).
2.2 Bacterial Strains
2.2.1 S. aureus Strains
With successful preparation of electrocompetent cells and plasmid
preconditioning through the appropriate E. coli plasmid artificial
modification strain (see Subheading 2.2.2), we have found that we
are able to modify the genomes of laboratory strains or primary
clinical isolates from a wide variety of genetic backgrounds and
lineages, although the efficiency of genome editing appears to be
strain specific [11].
2.2.2 E. coli Plasmid
Artificial Modification
Strains
A number of transgenic E. coli strains have been constructed which
are able to modify the methylation profiles of plasmids prior to their
introduction into the S. aureus strain of interest, which is necessary
to bypass the restriction barrier in transferring DNA between those
species. Some of the most useful plasmid artificial modification
strains are summarized below:
1. DC10B. This is a transgenic, cytosine methylase-negative E. coli
strain which enables bypass of the S. aureus type IV restriction
system [7]. The strain is useful in conditioning plasmids for
introduction into many S. aureus backgrounds, including those
for which the clonal complex designation is unusual or is not
known, but transformation efficiencies are generally not as high
as can be obtained when using a plasmid artificial modification
strain which actively mimics the methylation profile of a destination S. aureus strain.
2. SA08B. SA08B is a derivative of DC10B which has been further
modified to actively mimic the methylation profile of S. aureus
clonal complex 8/sequence type 8 (i.e., CC8/ST8), which
consequently enables bypass of the S. aureus type III restriction
system [24]. We have found that use of this E. coli strain can
condition plasmids for their introduction into the majority of
S. aureus laboratory and clinical isolates, which are largely
derived from CC8 or CC8-restriction compatible groups.
Genome Editing in Staphylococcus aureus by Conditional. . .
129