Versatile CRISPR‐Cas Tools for Gene Regulation in Zebrafish via an Enhanced Q Binary System

ABSTRACT CRISPR‐Cas systems revolutionize gene regulation across diverse organisms, including zebrafish. However, most zebrafish studies still rely on transient delivery of CRISPR components, with limited use of transgenic models, primarily restricted to Cas9‐mediated knockouts. This limitation arises from challenges in achieving sustained, tissue‐specific, and efficient expression of transgenic CRISPR effectors. To address these challenges, we introduce CRISPR‐Q, a transgenic system that combines the QFvpr/QUAS binary expression platform with CRISPR‐Cas technologies. CRISPR‐Q overcomes the drawbacks of transient mRNA or protein delivery and circumvents the toxicity and transgene silencing issues associated with other binary systems, such as Gal4/UAS. The system enables robust and spatiotemporal expression of CasRx or dCas9vpr, allowing precise transcript knockdown (CRISPR‐Q KD ) or gene activation (CRISPR‐Qa). Using CRISPR‐Q KD , we achieve effective knockdown of smn1 and simultaneous knockdown of tardbp and tardbpl , modeling spinal muscular atrophy and amyotrophic lateral sclerosis, respectively. CRISPR‐Qa activates endogenous lin28a and sox9b , demonstrating its functional versatility. We further validate CRISPR‐Q's tissue‐specific applicability in heart‐specific transgenic zebrafish. Together, CRISPR‐Q represents a robust and versatile platform for studying gene function and modeling human diseases in zebrafish, with broad potential for adaptation in other model organisms.