Validation of site-specific genome integration of integration plasmids for S. pyogenes SF370 using whole-genome nanopore sequencing
As part of the newly developed genetic toolbox for Streptococcus pyogenes SF370, we designed site-specific integrative plasmids that insert into the streptococcal genome based on homologous recombination or by a mechanism facilitated by site-specific integrases. The plasmids pSpy0C2, pSpy0C4, pSpy0K6 and p7INT.1 were transformed into S. pyogenes SF370 and successful integration was verified by PCR and Sanger sequencing of the integration site. To validate the site-specific integration and rule out potential off-target effects, such as insertions, deletions, single nucleotide polymorphisms or translocations, we performed whole-genome nanopore sequencing of the strains (two isolated single clones each).