An orthogonal Cro/OR3 binary system for targeted gene expression

Precise spatiotemporal control of gene expression is essential for dissecting complex biological systems. Here, we introduce a phage-derived Cro/OR3 binary system for transcriptional activation that functions robustly in zebrafish and human cells. A Cro repressor fused to the VP16 activation domain efficiently drives expression of OR3 operator-linked genes, and its activity can be targeted to specific cell types using enhancer-trap and promoter-controlled zebrafish lines. As a proof of concept, we manipulate glutamate transporter Glast/Slc1a3b-expressing cells in the central nervous system by Cro/OR3-mediated expression of a human ataxia-associated, uptake-deficient Glast/Slc1a3b variant, causing spastic, uncoordinated movement and axial deformation. Concurrent Gal4/UAS-mediated visualization reveals substantially disrupted positioning of motor neurons, highlighting their selective sensitivity to extracellular glutamate homeostasis during spinal circuit assembly. Together, these results establish the Cro/OR3 system as an orthogonal platform for targeted gene expression, enabling precise mechanistic dissection of how specific cell types and their interactions underlie diverse biological processes.