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<p><i>Dictyostelids</i> 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 <i>Dictyostelids</i> has focused on only a single species, <i>Dictyostelium discoideum</i>, 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 <i>Dictyostelid</i> 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 <i>Dictyostelid</i> species, including those that have never been genome edited before. Our methods therefore open the door to performing broad-scale genetic interrogations across the <i>Dictyostelids</i>.</p>

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Unlocking CRISPR-Cas9 editing for widely diverse Dictyostelid species

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2026年1月12日

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  • Mireia Garriga-Canut,
  • Nikki Cannon,
  • Matt Benton,
  • Andrea Zanon,
  • Samuel T Horsfield,
  • Jacob Scheurich,
  • Kim Remans,
  • John Lees,
  • Alexandre Paix,
  • Jordi van Gestel

摘要

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.