A comprehensive review of CRISPR-Cas9-mediated genome editing in Leishmania strains: methodologies, applications,...

Genome editing employing CRISPR-Cas9 has rapidly transformed experimental research in Leishmania, providing opportunities to investigate the genetic factors responsible for parasite survival, response to drugs and pathogenic traits. This review provides a comprehensive synthesis of CRISPR-based systems implemented across Leishmania species, spanning Cas9-mediated gene deletion, precise genome editing, endogenous locus tagging and pooled screening strategies. Furthermore, we highlight the emergence of Cas variants and next-generation CRISPR systems which expand the range of targetable genomic regions, improve editing precision and reduce the need for generation of double-strand DNA breaks (DSBs). Particular emphasis is placed on conditional and inducible genome-editing platforms, cytosine base-editing technologies, and recently developed CRISPR-based approaches such as prime editing, CRISPR activation/interference and Cas12-associated implementations. This review also discusses the principal biological and technical constraints influencing CRISPR-based studies in Leishmania, including genome plasticity, multicopy gene families, required genes, guide RNA design limitations and off-target considerations. Notably, the review also addresses CRISPR-Cas implementations in sand-fly vector biology, drawing on a foundational study in Phlebotomus papatasi. Through systematic compilation of published studies into comparative tables, we evaluate the strengths, limitations, experimental utility, delivery strategies, experimental workflows and representative applications of major CRISPR platforms. Together, these advances highlight the transition of CRISPR-Cas systems from proof-of-concept tools to versatile platforms for functional genomics, target validation and translational research in Leishmania, while offering a consolidated guide for selecting suitable CRISPR-Cas technologies and underscoring important considerations for their continued development in leishmaniasis research.