Construction of HygR Cassette for Cas9-Mediated Gene Deletion

A hygromycin B phosphotransferase expression cassette, referred to as here as HygR, was used as the selectable marker for Cas9-mediated gene deletion throughout this work. A 2,890-bp region, which spans 1,053 bp of the gpdA promoter, 1,020 bp of hygromycin B phosphotransferase (hph), and 817 bp of the trpC terminator, was PCR amplified from plasmid pUCGH (55 (link)) using the primers gpdA(p)-For and trpC(t)-Rev (Table 1). The resulting HygR cassette was cloned into pCR-Blunt II-TOPO using the Zero Blunt TOPO PCR cloning kit (Invitrogen) according to the manufacturer’s instructions. Positive clones were Sanger sequenced to confirm the absence of mutations, and the resulting plasmid was designated pCR-HygR. For generation of the repair templates needed for Cas9-mediated gene deletion, the HygR cassette was PCR amplified from plasmid pCR-HygR using either primer set 35 bp-pksP-HygR-F and 35 bp-pksP-HygR-R or primer set 50 bp-pksP-HygR-F and 50 bp-pksP-HygR-R. The resulting PCR fragments were purified using the GeneJET gel extraction kit (Thermo Scientific) and eluted using nuclease-free water. These purified PCR products were utilized as the completed repair templates and were composed of a 2,890-bp HygR cassette flanked by either 35 bp or 50 bp of microhomology regions targeting the pksP gene locus (Afu2g17600).