Efficient CRISPR/Cas9-mediated Gene Disruption in the Tetraploid Protist

CRISPR/Cas9-mediated genome editing has become an extremely powerful technique used to modify gene expression in many organisms, including parasitic protists. , a protist parasite that infects approximately 280 million people around the world each year, has been eluding the use of CRISPR/Cas9 to generate knockout cell lines due to its tetraploid genome. In this work, we show the ability of the assembled CRISPR/Cas9 components to successfully edit the genome of . The cell line that stably expresses Cas9 in both nuclei of showed effective recombination of the cassette containing the transcription units for the gRNA and the resistance marker. This highly efficient process led to the removal of all gene copies at once for three independent experimental genes, , and The method was also applicable to incomplete disruption of the essential gene, as evidenced by significantly reduced expression of Finally, testing the efficiency of Cas9-induced recombination revealed that homologous arms as short as 150 bp can be sufficient to establish a complete knockout cell line in .