Abstract

Members of the amoebozoan genus Acanthamoeba are unicellular heterotrophic protists that inhabit a wide range of terrestrial and aquatic environments. They are opportunistic pathogens known to host disease-causing bacteria as well large DNA viruses. While Acanthamoeba is commonly used to study cellular locomotion, phagocytosis, and disease, genetic tools for precise editing of the Acanthamoeba genome are still in their infancy. Here we have developed CRISPR/Cas9 tools for genetically engineering the model acanthamoebid Acanthamoeba castellanii strain Neff. In proof-of-principle experiments, we targeted the myosin-II heavy chain gene. Evidence for editing was found using reverse transcriptase PCR, microscopy, and DNA sequencing, which also identified unique alleles for the target locus. Collectively, we demonstrate that CRISPR can be employed to edit Acanthamoeba genes using a knock-in approach. This work serves as a foundation to further develop A. castellanii as a model system with which to study diverse questions in cell and molecular biology, biochemistry and evolution.