Base editing-mediated precision genome engineering in Toxoplasma gondii

Functional genomics in Toxoplasma gondii ( T. gondii ), an obligate intracellular parasite of global medical importance, primarily relies on CRISPR-Cas9-mediated gene disruption. However, conventional CRISPR-Cas9 editing relies on generating double-strand breaks (DSBs), which are repaired either by error-prone non-homologous end joining or by homology-directed repair requiring donor templates and time-consuming clone isolation. Here, we established the first two base editor platforms in T. gondii : adenine base editors (ABEs) and cytosine base editors (CBEs), which enable precise point mutations via direct base conversion without DSBs or donor templates. We successfully employed ABE and CBE to introduce high-efficiency A > G and C > T conversions at defined genomic loci, respectively. By targeting the critical virulence gene ROP18 , we demonstrate that ABE-mediated i-Silence (start codon disruption) and CBE-mediated i-STOP (premature stop codon introduction) strategies can efficiently generate functional T. gondii null mutants. The resulting base-edited parasite strains exhibited a complete loss of ROP18 protein expression and showed attenuated virulence and proliferation phenotypes equivalent to those of a conventional ROP18 knockout strain, as validated in both cellular and mouse models. We then conducted a genome-wide analysis predicting that the i-Silence and i-STOP strategies enable targeted inactivation of nearly all genes in the T. gondii genome (>99.6% coverage). Since our approach does not require DSBs, it enables researchers to achieve efficient, precise nucleotide substitutions without the risk of chromosomal rearrangements. We propose base editing as a powerful genetic tool in T. gondii , paving the way for high-resolution functional pathogenesis studies and the further development of genetically stable attenuated vaccines.