Editing the Constructs
In order to further modify an existing construct without having to recreate the whole assembly de novo, we have developed a multisite-directed mutagenesis approach based on homologous recombination that results in high efficiencies even when applied to relatively large episomes. The strategy is summarized in Figure 2A.
Plasmids of up to 14 kbp are subjected to a 12−20-min methylation reaction followed by 12 to 18 cycles of either a single multiplex or three independent PCR reactions. The mutation site in each oligonucleotide must be flanked with at least 10 unchanged nucleotides at both sides. After amplification, an aliquot of the reaction is subjected to a 15-min pulse of recombination activity, followed by transformation into One Shot® MAX Efficiency® DH5α™-T1R cells, where the methylated template is degraded by the mcrBC restriction system.
The high mutagenesis efficiencies observed (between 70% and 95%) significantly reduce the number of clones needed to be validated compared to other methodologies (Figure 2B). The approach also works with sites in close proximity (Figure 2B) and with oligonucleotides harboring up to 12 degenerated nucleotides for single-site and 3 degenerated nucleotides for multisite mutagenesis (not shown).