Fig 1.

Schematic illustration of the CRISPR/Cas9^pyrR approach. (A) CRISPR/Cas9^pyrR involves the co-transfection of two plasmids: Plasmid 1 (p_gCD_goi) contains an expression cassette for SpCas9, a single-guide RNA (sgRNA) targeting a gene of interest (sgRNA_goi), and a donor template tailored to modify the gene of interest as desired. Plasmid 2 (p_gD-dhfr^pyrR) encodes a sgRNA targeting the pfdhfr-ts gene (sgRNA-dhfr) along with the dhfr^pyrR donor template that introduces four specific point mutations into the pfdhfr-ts coding region. These mutations result in a highly PYR-resistant PfDHFR-TS variant (PfDHFR-TS^pyrR; N51I, C59R, S108N, I164L) and are indicated by red asterisks. The central re-codonized region of the dhfr^pyrR donor sequence encompassing the four mutations is shaded in bright blue. The relative positions of the sgRNA target sites are indicated by vertical arrowheads. (B) Expected outcomes of transfection after the uptake of either plasmid 1 alone, plasmid 2 alone, or both plasmids together. Parasites that take up plasmid 1 alone will not survive PYR challenge because they express wild-type PfDHFR-TS. Parasites receiving plasmid 2 alone will also be sensitive to PYR because editing of the pfdhfr-ts gene using the dhfr^pyrR donor template is unlikely to occur in the absence of SpCas9 expression. Hence, only parasites receiving both plasmids can simultaneously edit both loci and survive in the presence of 500 nM PYR.