ゼブラフィッシュにおける最適化CRISPR干渉を用いた系統的エンハンサーマッピングと機能解析

Noncoding cis-regulatory elements, particularly enhancers, are crucial for controlling gene expression. However, the in vivo use of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) interference (CRISPRi) to study enhancer function has been limited in zebrafish, which is widely used in early development and human disease research. Here, we optimized the CRISPRi system in zebrafish to achieve efficient suppression of tyr expression by fine-tuning component concentrations. Applying this optimized system, we functionally annotated distal enhancers of globin genes. Using Hi-C and histone modification assays, we systematically mapped 434 enhancer-promoter (EP) interactions across the genome. Among these EP loops, CRISPRi perturbation identified previously unreported enhancers with regulatory strengths surpassing known elements, demonstrated by disrupted phenotypes in fin and blood cell development. Additionally, several unreported EP loops were validated, underscoring the robustness of our integrated approach. This study not only provides an optimized CRISPRi system for zebrafish but also introduces a powerful platform that integrates computational and experimental strategies for advancing cis-regulatory element annotation in vertebrate gene regulation.