Additional file 2 of Downregulation of FeSOD-A expression in Leishmania infantum alters trivalent antimony and miltefosine susceptibility

Additional file 2: Figure S1. First attempt to knock out FeSOD-A using the CRISPR/Cas9 system. For this attempt, parasites bearing the pLPhygCas9 plasmid were transfected with the pSPneoHHsgRNAaH, containing sgRNA_144 or sgRNA_282, and with their respective donor DNA containing stop codons. Two other transfections were performed to provide more donor DNA for mutant parasites. The knockout was evaluated by PCR, using genomic DNA of wild-type parasites, of parasites expressing Cas9 and of the mutant parasites (after one, two or three transfections with the donor DNA). The correct integration of the stop codons was evaluated by PCR by annealing a primer within the stop codon sequence and another primer within the FeSOD-A sequence. Two different guides were evaluated, a sgRNA144 and b sgRNA282. c After three transfections, the FeSOD-A protein levels were evaluated by western blot. MW: Molecular Weight Standard; bp, base pairs; NC: negative control; WT: wild-type; KDa, kilodalton.