Genetic Manipulation of Group A Streptococcus—Gene Deletion by Allelic Replacement

Genetic manipulation of Streptococcus pyogenes (Group A Streptococcus, GAS) has historically been a challenging process, with considerable variation in efficiency between different strains. Here, we outline an optimized, rapid method for creating markerless isogenic mutations that combines Gibson assembly cloning with a new temperature-sensitive plasmid, pLZts. This method is highly efficient and reduces the time needed to create GAS mutants to ~2–3 weeks, with the ability to prepare multiple mutants simultaneously.