All-in-one, Cas13d-based cell-specific gene knockdown system for zebrafish
Heo, D.; Call, C. L.; Chen, J.; Freeman, M. R.; Monk, K. R.
Show abstract
Cell type-specific genetic manipulation is essential for dissecting neural circuits and glial biology in vivo; however, methods for achieving and validating cell-specific gene knockdown in zebrafish remain limited. Here, we introduce a CRISPR-Cas13d-based mRNA knockdown platform for zebrafish that leverages RNA targeting, rather than DNA editing, to enable efficient and flexible perturbation of gene function in defined cell types of interest. We engineered an all-in-one vector system for cell type-specific RfxCas13d expression and ubiquitous expression of crispr RNAs (crRNAs) and validated its utility across the major classes of CNS glia: astrocytes, oligodendrocytes, and microglia. Cas13d-mediated knockdown consistently achieved target mRNA depletion and produced heritable reproducible morphological and functional phenotypes, demonstrating the robustness of the approach. This approach complements permanent genome-editing methods such as CRISPR-Cas9 by providing a reliable, efficient, and scalable strategy for RNA-level perturbation. Our Cas13d toolkit expands the repertoire of zebrafish genetic technologies, offering a powerful resource for in vivo cell type-specific studies to uncover cell-autonomous mechanisms and possibility for cell type-specific genetic screens in development and disease.
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