Efficient Genome Engineering of <i>Toxoplasma gondii</i> Using CRISPR/Cas9

<div><p><i>Toxoplasma gondii</i> is a parasite of humans and animals, and a model for other apicomplexans including <i>Plasmodium spp.</i>, the causative agents of malaria. Despite many advances, manipulating the <i>T. gondii</i> genome remains labor intensive, and is often restricted to lab-adapted strains or lines carrying mutations that enable selection. Here, we use the RNA-guided Cas9 nuclease to efficiently generate knockouts without selection, and to introduce point mutations and epitope tags into the <i>T. gondii</i> genome. These methods will streamline the functional analysis of parasite genes and enable high-throughput engineering of their genomes.</p>