Members of the amoebozoan genus Acanthamoeba are unicellular, heterotrophic protists that occupy a wide range of terrestrial and aquatic habitats. They are used widely to study fundamental cell biological processes such as cellular locomotion and phagocytosis, and they are of medical relevance as opportunistic pathogens. Acanthamoeba species also act as environmental hosts for disease-causing bacteria and for large DNA viruses. Despite its utility and biological importance, the repertoire of tools for precise genetic manipulation of Acanthamoeba has remained limited.
The preprint summarized here addresses that gap by reporting a genetic editing platform tailored to Acanthamoeba castellanii, strain Neff. The authors present this work as enabling further functional studies in cell and molecular biology, biochemistry and evolutionary biology using A. castellanii as a tractable model.
The study describes the development and implementation of CRISPR/Cas9-based methods for genetic engineering in A. castellanii (Neff). The report frames these methods as proof-of-principle tools aimed at establishing whether CRISPR-mediated editing can be achieved in this heterotrophic protist. Specific experimental details and protocols are reported in the preprint; the summary here restricts itself to the high-level outcomes described by the authors.
To demonstrate the feasibility of editing, the authors chose the myosin-II heavy chain gene as a target. Myosin-II is a functionally and cytoskeletally relevant gene whose perturbation can be assessed by multiple readouts. The preprint indicates that the myosin-II heavy chain locus was the focus of proof-of-principle editing experiments designed to test whether CRISPR/Cas9 can generate detectable modifications in A. castellanii.
Multiple lines of evidence for editing at the myosin-II heavy chain locus are reported. The authors detected editing outcomes using reverse transcriptase PCR, which can identify altered transcript products consistent with genomic modification. Microscopy was used as an orthogonal assay to visualize phenotypic consequences compatible with editing of a cytoskeletal gene. DNA sequencing of the target locus provided direct molecular evidence of modification and revealed sequence-level changes.
Sequencing data reported in the preprint identified unique alleles at the targeted locus, consistent with successful CRISPR/Cas9 activity and repair outcomes in the edited population. The authors characterize these findings as demonstrating that CRISPR can be employed to edit Acanthamoeba genes using a knock-in approach, although specific knock-in constructs and efficiencies are described in the full preprint.
The study emphasizes a knock-in strategy for introducing edits into the A. castellanii genome. Sequencing of the target region yielded distinct alleles, indicating diversity of repair outcomes following Cas9 cleavage. The presence of multiple unique alleles at the myosin-II locus supports the conclusion that genome editing occurred and that repair generated sequence variants rather than uniform changes across all alleles in the population.
The preprint presents these observations as a foundation for more refined genetic manipulations in Acanthamoeba, including targeted insertions or precise sequence modifications, but the report does not claim exhaustive optimization of editing efficiency or detailed comparative metrics versus other systems.
By establishing CRISPR/Cas9 editing in A. castellanii (Neff), the authors propose that this organism can be more broadly developed as a tractable experimental model. The availability of precise genetic tools will facilitate mechanistic studies of locomotion, phagocytosis and host–pathogen interactions, and will support biochemical and evolutionary investigations that require targeted gene perturbation.
The preprint frames the current work as an enabling step rather than a completed toolkit; additional technical development, optimization and community uptake will determine how widely these methods are applied in future Acanthamoeba research.
This work is presented as a bioRxiv preprint posted July 29, 2026, and has not been certified by peer review. Authors listed are Dudley Chung, Sari Matar and John M. Archibald (Dalhousie University). The authors declared no competing interests. Funding disclosed in the preprint includes support from the Gordon and Betty Moore Foundation (GBMF5782).
Notes: The summary above is constrained to information reported in the preprint. Specific experimental parameters, quantitative editing efficiencies, detailed methods and construct sequences are reported in the full preprint document and are not reproduced here.