Functionally Tunable Star‐Shaped Multivalent crRNAs for Photocontrol CRISPR/Cas Editing

Abstract Clustered regularly interspaced shortpalindromic repeats/CRISPR‐associated (CRISPR/Cas)‐based genome editing has significantly advanced genetic engineering due to its precision, simplicity, and versatility. However, achieving precise spatial and temporal control remains challenging, restricting therapeutic and research applications. Herein, we introduce a novel class of star‐shaped, multivalent crRNAs engineered for precise spatiotemporal control of CRISPR/Cas9 and Cas12a editing systems. These crRNAs are synthesized via single‐site chemical modification and can be efficiently purified. By integrating distinct photo‐responsive chemical linkages, we achieved selective activation of crRNA activity upon irradiation with specific wavelengths, enabling orthogonal regulation of multiple genetic targets simultaneously. This method demonstrated robust OFF‐ON switching capabilities in vitro, characterized by minimal leakage and rapid activation. Importantly, the approach also proved highly effective for temporally controlled gene editing in mammalian cells in vivo, achieving considerable editing efficiency following brief photoactivation. Due to its target sequence‐independent, single‐site modification design, this strategy may serve as a universal solution for diverse CRISPR/Cas systems, eliminating cumbersome optimization processes. Future advancements incorporating long‐wavelength responsive and reversible linkers promise further enhancement of tissue penetration and control, significantly broadening the applicability and impact of this approach in biological research and therapeutic interventions.