Application of CRISPR/Cas۹ in the Functional Genomics of Toxoplasma gondii: From Targeted Editing to Genome-Wide, Context-Dependent and In Vivo Screens

CRISPR/Cas۹ has transformed functional genomics in Toxoplasma gondii by enabling targeted genome editing and scalable genetic screening. This review examines the progression from individual gene disruption to genome-wide fitness profiling, conditional and phenotypic screens, synthetic-lethal analyses, and in vivo studies. Early work established efficient editing across parasite strains and demonstrated that pooled libraries could identify genes required for growth and invasion. Subsequent screens revealed genetic determinants of host-cell entry and egress, resistance to interferon-γ-mediated restriction, oxidative-stress tolerance, nutrient acquisition, lipid homeostasis, and virulence. Recent studies have extended screening to acute animal infection and linked genetic perturbation with imaging, transcriptomics, proteomics, and chromatin profiling. These advances show that gene function cannot always be reduced to a single essential or nonessential designation: the measured effect of a perturbation depends on parasite strain, host-cell type, immune state, nutrient availability, developmental stage, and experimental design. Interpretation also requires attention to guide efficiency, library coverage, population bottlenecks, genetic redundancy, and interactions among parasites that may allow diffusible products to rescue neighboring mutants. Accordingly, high-confidence discoveries require validation with independent perturbations, complementation where feasible, and orthogonal molecular or phenotypic assays. This review synthesizes the strengths and limitations of current CRISPR/Cas۹ strategies and considers how conditional screening, single-cell measurements, quantitative imaging, and multi-omic integration may improve mechanistic resolution. Together, these approaches offer a framework for identifying context-dependent vulnerabilities in T. gondii and prioritizing candidates for further therapeutic investigation.

کلیدواژه ها:

CRISPR/Cas۹ ، Toxoplasma gondii ، functional genomics ، genome-wide screening ، parasite fitness ، virulence ، host–parasite interaction