Endogenous tagging and generation of knockouts in <i>T</i>. <i>gondii</i>.

<p><b>A</b>. Schematic of the CRISPR/Cas9 tagging system. Tagging plasmids were generated with various tags (green box) flanked by common ends (red and black boxes) and including a common stop codon (gray box) followed by the <i>HXGPRT</i> 3’ UTR (yellow box) and the selectable marker HXGPRT. Amplification of this central region with primers that contained short homology regions HR1 (purple box) and HR2 (blue box) together with the common flanks (red and black boxes) generated products for gene-specific tagging. Co-transfection of these amplicons with a CRISPR/Cas9 plasmid bearing the gene-specific single guide RNA (sgRNA3’) was used to add an epitope tag (green box) at the C-terminus of the endogenous locus. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006379#ppat.1006379.s006" target="_blank">S1 Fig</a> for more details. <b>B</b>. Localization of CaM1, CaM2 and CaM3 containing C-terminal 6HA tags. Detected with mouse anti-HA (green) and rabbit anti-GAP45 (red). Scale bar, 2 μM. <b>C</b>. Schematic of the double CRISPR/Cas9 gRNA system used for generation of clean knockouts using two sgRNAs matching the 5’ and 3’ ends of the coding sequence. The entire coding sequence was replaced by the DHFR marker flanked by short homology regions (HR3, red; HR2, blue). Primers (p) used for diagnostic PCR. <b>D</b>. Diagnostic PCR of knockouts compared to the parental ku80^KO line. <i>CDPK1</i>, PCR control. <b>E.</b> Plaque numbers formed by the knockouts compared to the parental ku80^KO line. ns, not significant, analyzed by one-way ANOVA.