Erythromycin (stock at 50 mg/mL, prepared in 100% ethanol).
Rifampin (stock at 50 mg/mL, prepared in 100% methanol).
3 Methods
3.1 Design
of Counterselection
Targeting
Oligonucleotides
The specificity of Cas9 counterselection is determined by cloning a
20 bp guide RNA, encoded by synthetic targeting oligonucleotides, into the sgRNA element of pCAS9counter. This element
targets Cas9 to a specific protospacer adjacent motif, or PAM,
sequence (5
0 -NGG-3
0 ) and enables its subsequent endonuclease
activity.
1. Guide RNAs can be designed by copying the sequence of your
gene of interest into a program such as CRISPRscan (http://
www.crisprscan.org/) [26], which optimizes the design of targeting oligonucleotides for their activity based on empirical
model. If using CRISPRscan, select the “No Search” option
for predicting off-target effects, and “Cas9 – NGG” as the
enzyme. A list of possible targeting oligonucleotides will be
displayed, along with their accompanying “CRISPRscan
score,” a rating of how likely the oligo will be at promoting
Cas9-mediated cutting: higher scoring oligos are most likely to
have high activity. The sequence of designed guide RNAs are
displayed in capital letters within the larger oligonucleotide
construct. Other, equivalent programs may also be used for
this purpose.
(a) If the objective is to engineer a gene deletion, any PAM
internal to the site of deletion may be selected, as successful genome editing events will eliminate the PAM site and
consequently eliminate Cas9-mediated DNA cleavage in
the engineered strain.
(b) If the objective is to engineer a point mutation, the targeted PAM site must be near the desired edit (at most,
~50 bp distant). In general, selecting a PAM closest to the
engineered edit is desirable: in ideal cases, the intended
mutation will overlap the targeted PAM. However, silent
mutations to eliminate the PAM and escape counterselection must ideally avoid altering the protein sequence of
the gene so that a more distal PAM which can be ablated
through silent mutation may be favored over a PAM more
proximal to the desired edit but which necessitates
removal by a nonsynonymous amino acid change (see
Subheading 3.2).
2. To be cloned into pCAS9counter, the guide RNA must be
reconstituted as double stranded DNA (dsDNA) with
Genome Editing in Staphylococcus aureus by Conditional. . .
131
Rifampin (stock at 50 mg/mL, prepared in 100% methanol).
3 Methods
3.1 Design
of Counterselection
Targeting
Oligonucleotides
The specificity of Cas9 counterselection is determined by cloning a
20 bp guide RNA, encoded by synthetic targeting oligonucleotides, into the sgRNA element of pCAS9counter. This element
targets Cas9 to a specific protospacer adjacent motif, or PAM,
sequence (5
0 -NGG-3
0 ) and enables its subsequent endonuclease
activity.
1. Guide RNAs can be designed by copying the sequence of your
gene of interest into a program such as CRISPRscan (http://
www.crisprscan.org/) [26], which optimizes the design of targeting oligonucleotides for their activity based on empirical
model. If using CRISPRscan, select the “No Search” option
for predicting off-target effects, and “Cas9 – NGG” as the
enzyme. A list of possible targeting oligonucleotides will be
displayed, along with their accompanying “CRISPRscan
score,” a rating of how likely the oligo will be at promoting
Cas9-mediated cutting: higher scoring oligos are most likely to
have high activity. The sequence of designed guide RNAs are
displayed in capital letters within the larger oligonucleotide
construct. Other, equivalent programs may also be used for
this purpose.
(a) If the objective is to engineer a gene deletion, any PAM
internal to the site of deletion may be selected, as successful genome editing events will eliminate the PAM site and
consequently eliminate Cas9-mediated DNA cleavage in
the engineered strain.
(b) If the objective is to engineer a point mutation, the targeted PAM site must be near the desired edit (at most,
~50 bp distant). In general, selecting a PAM closest to the
engineered edit is desirable: in ideal cases, the intended
mutation will overlap the targeted PAM. However, silent
mutations to eliminate the PAM and escape counterselection must ideally avoid altering the protein sequence of
the gene so that a more distal PAM which can be ablated
through silent mutation may be favored over a PAM more
proximal to the desired edit but which necessitates
removal by a nonsynonymous amino acid change (see
Subheading 3.2).
2. To be cloned into pCAS9counter, the guide RNA must be
reconstituted as double stranded DNA (dsDNA) with
Genome Editing in Staphylococcus aureus by Conditional. . .
131