A Conformational Restriction Strategy for the Control of CRISPR/Cas Gene Editing with Photoactivatable Guide RNAs.

The CRISPR/Cas system is one of the most powerful tools for gene editing. However, approaches for precise control of genome editing and regulatory events are still desirable. Here, we report a spatiotemporal and efficient control of CRISPR/Cas9- and Cas12a-mediated editing with conformational restricted gRNAs. This approach relied on only two or three pre-installed photo-labile substituents followed by an intramolecular cyclization, representing a robust synthetic method in comparison to the heavily modified linear gRNAs which often required extensive screening and time-consuming optimization. The tactic could direct precise cleavage of GFP and VEGFA within a predefined cutting region without notable editing leakage in live cells. We also achieved light-mediated MSTN gene editing in embryos, wherein a new bow-knot-type gRNA was constructed with excellent OFF/ON switch efficiency. Overall, our work provides a significantly new strategy in CRISPR/Cas editing with modified circular guide RNAs to precisely manipulate where and when genes are edited.