CRISPR/Cas9-Engineered Fluorescent Reporter Lines of Babesia duncani

CRISPR-Cas9 technology is a valuable tool for gene editing in various apicomplexan parasites, including Plasmodium falciparum, the causative agent of human malaria. However, there is a dearth of efficient CRISPR-based genetic modification tools for Babesia duncani, a related apicomplexan parasite responsible for human babesiosis, an emerging tick-borne disease. Such tools could play a pivotal role in probing gene function and identifying new targets for drug and vaccine development. The availability of an established in vitro culture system for B. duncani and a published genome offers a promising opportunity to develop an efficient gene editing system in this parasite. We leveraged advances in genome modification protocols for bovine Babesia parasites and P. falciparum to create B. duncani lines that stably express GFP or mCherry for various downstream applications, including compound screening. To achieve this, we developed a Cas9/gRNA expression plasmid that is codon-optimized for Babesia parasites. This plasmid expresses Cas9 from Streptococcus pyogenes and a guide RNA scaffold into which target-specific gRNAs can be cloned. To examine the practical utility of this Cas9/gRNA expression vector, gRNAs specific for the parasite apical membrane antigen 1 (AMA-1) or thioredoxin peroxidase 1 (TPX-1) were cloned into this vector. The resulting plasmid and a donor DNA plasmid expressing either mCherry-tagged AMA-1 or GFP-tagged TPX-1 were co-transfected into B. duncani parasites by electroporation. B. duncani parasites expressing GFP or mCherry were observed 14 days after the transfection, with 100% editing efficiency. These results highlight the practical utility of this gene editing system. This work will contribute towards the identification of novel targets for drug and vaccine development using reverse genetics, and a better understanding of the biology of Babesia parasites that affect humans.