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<p>Gene manipulation is essential for understanding biological mechanisms, yet genetic modification in the social amoebas (Dictyostelia) has been largely limited to <i>Dictyostelium discoideum</i>. Here, we aimed to establish a CRISPR/Cas9-based genome-editing system applicable across the phylogenetic breadth of Dictyostelia, spanning Groups 1–4. Using an extrachromosomal CRISPR/Cas9 vector from <i>D. discoideum</i>, we disrupted <i>stlA</i> and <i>pkaC</i> in <i>Polysphondylium violaceum</i> and <i>pkaC</i> in two early-branching species, <i>Heterostelium pallidum</i> and <i>Cavenderia fasciculata</i>. In <i>D. discoideum</i>, co-introduction of donor oligos with the CRISPR vector enabled selection-free knockout generation of <i>pkaC</i> with 28.6% efficiency. In <i>H. pallidum</i>, where genome editing is typically inefficient, co-electroporation of donor oligos with the CRISPR/Cas9 vector followed by 4 days of drug selection increased the frequency of <i>pkaC</i> disruption from 0.9% to 8.3%. These results demonstrated that the <i>D. discoideum</i> CRISPR/Cas9 system can be extended across Dictyostelia, providing a versatile platform for comparative genetic and evolutionary developmental studies.</p>

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Genome editing across Dictyostelia species enables comparative functional genetics of social amoebas

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年2月5日

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  • Shuka Oishi,
  • Sousuke Doi,
  • Takumi Sekida,
  • Kensuke Yamashita,
  • Yoko Yamada,
  • Tetsuya Muramoto

摘要

Gene manipulation is essential for understanding biological mechanisms, yet genetic modification in the social amoebas (Dictyostelia) has been largely limited to Dictyostelium discoideum. Here, we aimed to establish a CRISPR/Cas9-based genome-editing system applicable across the phylogenetic breadth of Dictyostelia, spanning Groups 1–4. Using an extrachromosomal CRISPR/Cas9 vector from D. discoideum, we disrupted stlA and pkaC in Polysphondylium violaceum and pkaC in two early-branching species, Heterostelium pallidum and Cavenderia fasciculata. In D. discoideum, co-introduction of donor oligos with the CRISPR vector enabled selection-free knockout generation of pkaC with 28.6% efficiency. In H. pallidum, where genome editing is typically inefficient, co-electroporation of donor oligos with the CRISPR/Cas9 vector followed by 4 days of drug selection increased the frequency of pkaC disruption from 0.9% to 8.3%. These results demonstrated that the D. discoideum CRISPR/Cas9 system can be extended across Dictyostelia, providing a versatile platform for comparative genetic and evolutionary developmental studies.