Using Polycistronic gRNAs and a tRNA Cleavage System in CRISPR-Based Genome Editing of Toxoplasma gondii

Abstract CRISPR/Cas9 is widely used for genetic manipulation in Toxoplasma gondii , but optimizing gRNA expression and multiplexed editing remains challenging. Here, we evaluated alternative promoters, gRNA scaffolds, and a tRNA-based polycistronic system to improve editing efficiency and reduce synthetic DNA costs. Four constructs targeting the SAG1 locus were used to compare the U6 and H1 promoters, as well as standard and modified gRNA scaffolds. U6-driven constructs supported robust gRNA-targeted Cas9 activity, whereas H1-driven constructs failed to produce detectable phenotypes. The alternative gRNA scaffold, modified by a number of base changes and insertions, increased knockout efficiency compared to the standard scaffold. To simplify dual gRNA-based methods, we establish a tRNA-based polycistronic system to simultaneously target two independent loci. Targeting the SAG1 and UPRT locus for proof-of-concept, all U6-driven polycistronic constructs conferred efficient SAG1 knockout and 5-fluorodeoxyuridine resistance, whereas H1-driven constructs exhibited position-dependent activity, with only the 3′ gRNA being functional. These results demonstrate that U6-driven, tRNA-based polycistronic gRNA expression enables reliable multiplexed genome editing in T. gondii , with the minimal H1 promoter being insufficient for consistent multi-gRNA activity. This optimized system provides a scalable framework for dual-gRNA CRISPR strategies in this organism.