(g) Plate aliquots of the cell suspension onto LB containing
10 μg/mL chloramphenicol. We recommend plating
three aliquots of 250 μL each, and one aliquot of 100 μL.
(h) Incubate plates up to 48 h at 32
C, until colonies appear.
5. Once a transformant is isolated, prepare electrocompetent cells
of the conditionally recombinogenic S. aureus strain (see Subheading 3.3). This batch of cells can be used for many different
genome editing experiments.
3.6 Preparation
of Counterselection
Vector
This step targets Cas9 counterselection to a specific site in the
genome through the cloning of targeting oligos into the sgRNA
element of pCAS9counter. The protocol will result in a large quantity of digested pCAS9counter that can be stored frozen and used
to construct many different targeting constructs.
3.6.1 Plasmid Digestion
and Size Selection
1. Extract plasmid from the E. coli strain carrying pCAS9counter
using a commercial plasmid extraction kit. At least 1ug of
plasmid is recommended.
2. Prepare restriction digest of plasmid as follows:
1 μg pCAS9counter.
5 μL 10Â Cutsmart buffer.
1 μL BsaI-HF.
Water to 50 μL.
3. Incubate reaction at 37
C for 1 h, followed by a 65
C heat
inactivation for 10 min.
4. Add 1 μL CIP and incubate at 37
C for 30 min.
5. Perform gel purification of digested plasmid to exclude uncut
vector using a 0.8% agarose gel. The digested plasmid will
migrate just higher than 10 kb.
3.6.2 Insertion
of Targeting
Oligonucleotides
1. Prepare a reaction to phosphorylate and subsequently anneal
the two targeting oligos (designed in Subheading 3.1) as
follows:
(a) 0.5 μL oligo 1 (100 μM)
(b) 0.5 μL oligo 2 (100 μM)
(c) μL T4 Ligase Buffer,
(d) 0.5 μL T4 PNK
(e) 21 μL H 2 O.
2. Using a thermocycler, incubate reaction at 37
for 40 min, then
95
for 5 min, then gradually cool to 20
over 42 min.
3. Prepare ligation reaction to insert annealed targeting oligos
into pCAS9counter as follows:
(a) 2.9 μL annealed targeting oligo, diluted 1:50 in water.
Genome Editing in Staphylococcus aureus by Conditional. . .
137
10 μg/mL chloramphenicol. We recommend plating
three aliquots of 250 μL each, and one aliquot of 100 μL.
(h) Incubate plates up to 48 h at 32
C, until colonies appear.
5. Once a transformant is isolated, prepare electrocompetent cells
of the conditionally recombinogenic S. aureus strain (see Subheading 3.3). This batch of cells can be used for many different
genome editing experiments.
3.6 Preparation
of Counterselection
Vector
This step targets Cas9 counterselection to a specific site in the
genome through the cloning of targeting oligos into the sgRNA
element of pCAS9counter. The protocol will result in a large quantity of digested pCAS9counter that can be stored frozen and used
to construct many different targeting constructs.
3.6.1 Plasmid Digestion
and Size Selection
1. Extract plasmid from the E. coli strain carrying pCAS9counter
using a commercial plasmid extraction kit. At least 1ug of
plasmid is recommended.
2. Prepare restriction digest of plasmid as follows:
1 μg pCAS9counter.
5 μL 10Â Cutsmart buffer.
1 μL BsaI-HF.
Water to 50 μL.
3. Incubate reaction at 37
C for 1 h, followed by a 65
C heat
inactivation for 10 min.
4. Add 1 μL CIP and incubate at 37
C for 30 min.
5. Perform gel purification of digested plasmid to exclude uncut
vector using a 0.8% agarose gel. The digested plasmid will
migrate just higher than 10 kb.
3.6.2 Insertion
of Targeting
Oligonucleotides
1. Prepare a reaction to phosphorylate and subsequently anneal
the two targeting oligos (designed in Subheading 3.1) as
follows:
(a) 0.5 μL oligo 1 (100 μM)
(b) 0.5 μL oligo 2 (100 μM)
(c) μL T4 Ligase Buffer,
(d) 0.5 μL T4 PNK
(e) 21 μL H 2 O.
2. Using a thermocycler, incubate reaction at 37
for 40 min, then
95
for 5 min, then gradually cool to 20
over 42 min.
3. Prepare ligation reaction to insert annealed targeting oligos
into pCAS9counter as follows:
(a) 2.9 μL annealed targeting oligo, diluted 1:50 in water.
Genome Editing in Staphylococcus aureus by Conditional. . .
137