Abstract
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.
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All data are presented in the manuscript or the supplementary materials. The plasmids and cell lines generated in this study are available from NBRP Nenkin.
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Acknowledgements
We thank Dr. Takaaki B. Narita for valuable advice on the stlA gene of P. violaceum, and are grateful to Mr. Yuichiro Ishiyama, Mr. Takanori Ogasawara, and Ms. Mio Ito for their assistance with cloning parts of the CRISPR/Cas9 vectors. We also acknowledge the National BioResource Project (NBRP) Nenkin for supplying the dictyostelid species used in this work. Paperpal and ChatGPT were used solely to improve the English language during the revision process.
Funding
This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI (23K05785 to T.M.) and by a JSPS Research Fellowship for Young Scientists (DC1; 23KJ1977 to K.Y.).
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S.O. performed most of the experiments. S.D. and T.S. optimized CRISPR/Cas9 conditions in non-model Dictyostelia. K.Y. and Y.Y. contributed to improving cell culture and selection procedures. T.M. conceived and designed the study, and supervised the project. T.M. wrote the manuscript with contributions from K.Y. and S.O. All authors discussed the results and agreed to the published version of the manuscript.
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Oishi, S., Doi, S., Sekida, T. et al. Genome editing across Dictyostelia species enables comparative functional genetics of social amoebas. Sci Rep (2026). https://doi.org/10.1038/s41598-026-38605-5
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DOI: https://doi.org/10.1038/s41598-026-38605-5
