appropriate sticky ends. This can be accomplished by modifying the desired guide RNA sequence as follows in order to
separately design the two strands of that element as individual
targeting oligonucleotides:
(a) 5
0 -AGCTC–guide RNA sequence–G
0 .
(b) 5
0 -AAAAC–reverse complement of the guide RNA
sequence–G
0 .
3. Procure each strand of the targeting oligonucleotides of the
required length as standard, salt-purified ssDNA oligonucleotides from Integrated DNA Technologies (IDT), or another
commercial supplier. If large numbers of targeting oligos are
required, they may be synthesized most inexpensively in a
plate-based format.
3.2 Design
of Recombineering
Oligonucleotides
Recombineering oligonucleotides encode the mutagenic change of
interest and are incorporated into the genome through the action
of EF2132 recombinase. As recombineering oligonucleotides must
be relatively short, they are not suitable for engineering the insertion of entire genes, but can be used to effectively manufacture
gene deletions of even relatively large size, single or multiple nucleotide substitutions, and short indels.
3.2.1 General
Considerations
1. For optimal recombineering efficiencies in S. aureus, we have
found that recombineering oligos should be 90 bp in length
and carry a series of four phosphorothioate bonds at the 5
0 end
in order to inhibit exonuclease digestion [11, 27]. Efforts
should be taken to avoid designing recombineering oligonucleotides containing strong secondary structures [28].
2. In E. coli, recombineering oligonucleotides are preferentially
incorporated into the lagging strand during DNA replication
[14]. In our experiments in S. aureus, we have found that only
some oligonucleotides demonstrate preferential incorporation
on one strand or the other; nevertheless, both a primary
recombineering oligonucleotide and its reverse complement
may be designed and empirically tested.
3. Recombineering oligonucleotides are most inexpensively generated as “ultramers” (IDT) with standard salt purification,
manufactured on the smallest possible scale (4 nM). If large
numbers of recombineering oligonucleotides are required,
they may be ordered most inexpensively in a plate-based synthesis format.
3.2.2 Gene Deletions
For gene deletions, we recommend generating in-frame deletions
to escape potential polar effects.
1. The junction of the gene deletion should be placed as centrally
as possible within the oligonucleotide: as a starting point, the
132
Kelsi Penewit and Stephen J. Salipante
separately design the two strands of that element as individual
targeting oligonucleotides:
(a) 5
0 -AGCTC–guide RNA sequence–G
0 .
(b) 5
0 -AAAAC–reverse complement of the guide RNA
sequence–G
0 .
3. Procure each strand of the targeting oligonucleotides of the
required length as standard, salt-purified ssDNA oligonucleotides from Integrated DNA Technologies (IDT), or another
commercial supplier. If large numbers of targeting oligos are
required, they may be synthesized most inexpensively in a
plate-based format.
3.2 Design
of Recombineering
Oligonucleotides
Recombineering oligonucleotides encode the mutagenic change of
interest and are incorporated into the genome through the action
of EF2132 recombinase. As recombineering oligonucleotides must
be relatively short, they are not suitable for engineering the insertion of entire genes, but can be used to effectively manufacture
gene deletions of even relatively large size, single or multiple nucleotide substitutions, and short indels.
3.2.1 General
Considerations
1. For optimal recombineering efficiencies in S. aureus, we have
found that recombineering oligos should be 90 bp in length
and carry a series of four phosphorothioate bonds at the 5
0 end
in order to inhibit exonuclease digestion [11, 27]. Efforts
should be taken to avoid designing recombineering oligonucleotides containing strong secondary structures [28].
2. In E. coli, recombineering oligonucleotides are preferentially
incorporated into the lagging strand during DNA replication
[14]. In our experiments in S. aureus, we have found that only
some oligonucleotides demonstrate preferential incorporation
on one strand or the other; nevertheless, both a primary
recombineering oligonucleotide and its reverse complement
may be designed and empirically tested.
3. Recombineering oligonucleotides are most inexpensively generated as “ultramers” (IDT) with standard salt purification,
manufactured on the smallest possible scale (4 nM). If large
numbers of recombineering oligonucleotides are required,
they may be ordered most inexpensively in a plate-based synthesis format.
3.2.2 Gene Deletions
For gene deletions, we recommend generating in-frame deletions
to escape potential polar effects.
1. The junction of the gene deletion should be placed as centrally
as possible within the oligonucleotide: as a starting point, the
132
Kelsi Penewit and Stephen J. Salipante