Unlocking CRISPR-Cas9 editing for widely diverse Dictyostelid species

Abstract Dictyostelids are a species-rich clade of cellular slime molds that are widely found in soils and have been studied for over a century. Due to a lack of genome editing methods, most molecular research in Dictyostelids has focused on only a single species, Dictyostelium discoideum , which has severely limited broad-scale comparative analyses. Here, we introduce the first CRISPR-Cas9 editing approach that is cloning-free, selection-free, highly efficient, and effective across Dictyostelid species that diverged millions of years ago. Depending on the CRISPR-Cas9 target site, our editing approach generates knock-out efficiencies of up to 90% and knock-in efficiencies of up to 50% without a selective marker. We show that mutants can be isolated as soon as one day post-transfection, vastly outpacing existing methods for generating knock-outs, fusion proteins, and expression reporters. Leveraging single-cell sorting and fluorescent microscopy, we could readily apply our CRISPR-Cas9 editing approach to phylogenetically distant Dictyostelid species, including those that have never been genome edited before. Our methods therefore open the door to performing broad-scale genetic interrogations across the Dictyostelids .