Unlocking CRISPR-Cas9 editing for widely diverse Dictyostelid species

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

doi:10.1038/s44320-025-00180-8

Unlocking CRISPR-Cas9 editing for widely diverse Dictyostelid species

Mireia Garriga-Canut
, Nikki Cannon
, Matt Benton
, Andrea Zanon
, Samuel T. Horsfield
, Jacob Scheurich
, Kim Remans
, John Lees
, Alexandre Paix (Lead / Corresponding author)
, Jordi van Gestel (Lead / Corresponding author)

DArcy Thompson Unit

Research output: Contribution to journal › Article › peer-review

1 Citation (Scopus)

32 Downloads (Pure)

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.

Original language	English
Pages (from-to)	599-619
Number of pages	21
Journal	Molecular Systems Biology
Volume	22
Early online date	12 Jan 2026
DOIs	https://doi.org/10.1038/s44320-025-00180-8
Publication status	Published - 2 Apr 2026

Keywords

Cell Sorting
CRISPR-Cas9
Dictyostelids
Dictyostelium discoideum
Genome Editing

ASJC Scopus subject areas

Information Systems
General Immunology and Microbiology
General Biochemistry,Genetics and Molecular Biology
General Agricultural and Biological Sciences
Computational Theory and Mathematics
Applied Mathematics

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10.1038/s44320-025-00180-8Licence: CC BY

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Final published version, 4.03 MBLicence: CC BY

Cite this

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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.",

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Unlocking CRISPR-Cas9 editing for widely diverse Dictyostelid species

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Keywords

ASJC Scopus subject areas

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