initial 45 bp and the terminal 45 bp of a gene may be joined to
generate a central, in frame gene deletion.
2. We have observed a negative correlation between the size of the
engineered deletion and the efficiency by which it can be generated [11, 28], so the 5
0 and 3
0 sites of the fusion can be
moved more internally for engineering deletions in particularly
long genes, if necessary.
3. The gene deletion must excise the PAM site that is targeted by
the counterselection oligonucleotide.
3.2.3 Point Mutations
and Small Indels
For engineering point mutations or small indels, separate design
considerations are required.
1. The desired change or changes should be placed as centrally as
possible within the oligo. At a minimum, mismatches should be
placed 15 bp or farther from the ends of the oligo
sequence [29].
2. Measures must be taken to bypass repair of the engineered
changes through the bacterial mismatch repair (MMR) system
[29]. This may be accomplished through a number of design
features in the recombineering oligonucleotide.
(a) Single base C:C mismatches (i.e., G>C transversions)
cannot be efficiently repaired by MMR and can be introduced as singular changes [29].
(b) Alteration of four or more consecutive bases (base substitutions or indels) produce lesions which are too large to
be corrected by MMR, and can be introduced without
additional alterations [29].
(c) Four or more wobble positions in adjacent codons can
similarly be changed to produce large lesions which do not
alter protein coding sequence [29]. This strategy is the
most facile for many applications, as it provides the greatest flexibility in design. The desired mutation may either
be adjoining or contained within the altered wobble bases.
(d) Alternatively, an MMR-deficient, mutS deletion strain
may be generated from the strain of interest and can be
used to incorporate single nucleotide changes in the
absence of additional mutations (silent or otherwise)
encoded by the recombineering oligonucleotide
[11]. However, MMR deficiency results in strains with
elevated rates of spontaneous mutation [11].
3. If the engineered change does not directly ablate the targeted
PAM, separate mutations must be encoded to destroy the PAM
site in engineered cells. These mutations are optimally silent so
as not to introduce undesired amino acid substitutions with the
desired, engineered change. In some cases, introducing silent
Genome Editing in Staphylococcus aureus by Conditional. . .
133
generate a central, in frame gene deletion.
2. We have observed a negative correlation between the size of the
engineered deletion and the efficiency by which it can be generated [11, 28], so the 5
0 and 3
0 sites of the fusion can be
moved more internally for engineering deletions in particularly
long genes, if necessary.
3. The gene deletion must excise the PAM site that is targeted by
the counterselection oligonucleotide.
3.2.3 Point Mutations
and Small Indels
For engineering point mutations or small indels, separate design
considerations are required.
1. The desired change or changes should be placed as centrally as
possible within the oligo. At a minimum, mismatches should be
placed 15 bp or farther from the ends of the oligo
sequence [29].
2. Measures must be taken to bypass repair of the engineered
changes through the bacterial mismatch repair (MMR) system
[29]. This may be accomplished through a number of design
features in the recombineering oligonucleotide.
(a) Single base C:C mismatches (i.e., G>C transversions)
cannot be efficiently repaired by MMR and can be introduced as singular changes [29].
(b) Alteration of four or more consecutive bases (base substitutions or indels) produce lesions which are too large to
be corrected by MMR, and can be introduced without
additional alterations [29].
(c) Four or more wobble positions in adjacent codons can
similarly be changed to produce large lesions which do not
alter protein coding sequence [29]. This strategy is the
most facile for many applications, as it provides the greatest flexibility in design. The desired mutation may either
be adjoining or contained within the altered wobble bases.
(d) Alternatively, an MMR-deficient, mutS deletion strain
may be generated from the strain of interest and can be
used to incorporate single nucleotide changes in the
absence of additional mutations (silent or otherwise)
encoded by the recombineering oligonucleotide
[11]. However, MMR deficiency results in strains with
elevated rates of spontaneous mutation [11].
3. If the engineered change does not directly ablate the targeted
PAM, separate mutations must be encoded to destroy the PAM
site in engineered cells. These mutations are optimally silent so
as not to introduce undesired amino acid substitutions with the
desired, engineered change. In some cases, introducing silent
Genome Editing in Staphylococcus aureus by Conditional. . .
133