CRISPR-Cas Systems in Protozoan Parasites: A Biological-Fit Decision Framework for Cas9/Cas12a Editing and dCas-Based Regulation

CRISPR-Cas systems have transformed functional genomics in protozoan parasites; however, their genome engineering outcomes are strongly influenced by parasite-specific biological and genetic contexts. This review focuses on experimentally established CRISPR applications in representative protozoan parasite systems and examines how DNA repair pathways, delivery architectures, the biological consequences of DNA double-strand breaks (DSBs), and the cellular burden associated with Cas expression collectively shape genome-editing outcomes. We summarize recent advances in Cas9- and Cas12a-mediated genome editing, as well as dCas-based transcriptional regulation (CRISPRi/a), in protozoan parasites. Available evidence indicates that no universally optimal CRISPR platform exists across protozoan systems. Instead, CRISPR strategy selection should be guided by experimental objectives, target-gene properties, and species-specific biological characteristics. Cas9- and Cas12a-based systems remain advantageous for targeted gene disruption and endogenous tagging, whereas DSB-independent regulatory approaches such as dCas-mediated CRISPRi/a may provide biologically less disruptive alternatives in parasites with high sensitivity to DNA damage. Emerging RNA-targeting, precision-editing, and compact RNA-guided effectors may further expand the protozoan genetic engineering toolbox, although most remain at an early stage of development. Overall, CRISPR applications in protozoan parasites are transitioning from a tool-centered paradigm toward a biologically informed and decision-oriented framework for protozoan genome engineering.