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

The study examined various approaches to enhance CRISPR/Cas9 genome editing in Toxoplasma gondii, a parasitic organism. Researchers explored different promoters, gRNA scaffolds, and a novel tRNA-based polycistronic system to improve editing efficiency and reduce costs. Four constructs were employed to compare the U6 and H1 promoters, along with standard and modified gRNA scaffolds.

The U6-driven constructs exhibited robust gRNA-targeted Cas9 activity, while the H1-driven constructs did not generate any observable phenotypes. By modifying the gRNA scaffold with base changes and insertions, the researchers observed an increase in knockout efficiency compared to the standard scaffold. To streamline dual gRNA-based methods, a tRNA-based polycistronic system was introduced, enabling simultaneous targeting of two independent loci.

In this proof-of-concept study, U6-driven polycistronic constructs efficiently knocked out the SAG1 locus and conferred resistance to 5-fluorodeoxyuridine. Conversely, H1-driven constructs showed position-dependent activity, with only the third gRNA proving functional. The findings demonstrate that a U6-driven, tRNA-based polycistronic gRNA expression system facilitates reliable multiplexed genome editing in T. gondii, with the less potent H1 promoter being inadequate for consistent multi-gRNA activity. This optimized system offers a scalable framework for dual-gRNA CRISPR strategies in this organism.