Blastocrithidia nonstop is a protist with a highly unusual nuclear genetic code, in which all three standard stop codons are reassigned to encode amino acids, with UAA also serving as a sole termination codon. In this study, we demonstrate that this parasitic flagellate is amenable to genetic manipulation, enabling gene ablation and protein tagging. Using preassembled Cas9 ribonucleoprotein complexes, we successfully disrupted and tagged the non-essential gene encoding catalase. These advances establish this single-celled eukaryote as a model organism for investigating the malleability and evolution of the genetic code in eukaryotes.

Department of Parasitology Faculty of Science Charles University Praha Czechia

Division of Infectious Diseases Department of Medicine University of Alberta Edmonton Alberta Canada

Faculty of Science University of South Bohemia České Budějovice Czechia

Institute of Parasitology Biology Centre Czech Academy of Sciences České Budějovice Czechia

Life Science Research Centre Faculty of Science University of Ostrava Ostrava Czechia

Zoological Institute Russian Academy of Sciences St Petersburg Russia

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