Topology‐Engineered Guide RNAs for Programmable Control of CRISPR/Cas Activity

Abstract CRISPR/Cas systems have transformed genome editing, yet achieving precise temporal and conditional control remains challenging. Traditional strategies involving linear guide RNAs (gRNAs) modified with multiple chemical groups throughout their strands often face limitations such as heterogeneous reaction outcomes, irreversibility, and variable editing efficiencies. To overcome these issues, topology‐engineered guide RNAs (TE‐gRNAs) have emerged, featuring defined structural architectures including polymeric, circular, and dendrimer‐like topologies that enable precise spatial control, reversibility, and programmable activation of CRISPR activity. By selectively incorporating physical or chemically responsive linkers and stimuli‐sensitive groups at specific sites, TE‐gRNAs facilitate dynamic and conditional genome editing that can be activated or deactivated with external triggers such as light or chemical signals. These engineered RNA structures significantly improve synthesis feasibility, stability, reduce off‐target effects, and provide unprecedented control over gene editing processes. Recent advancements in TE‐gRNAs demonstrate their broad applicability in synthetic biology, functional genomics, and therapeutic interventions, highlighting their potential to achieve precise spatiotemporal modulation of CRISPR systems. This review summarizes the current strategies, benefits, and challenges associated with TE‐gRNAs, and discusses future directions for enhancing their performance and utility in complex genome editing applications.