biostudies-literatureZhang DDefense Advanced Research Projects AgencyNational Institute of General Medical SciencesNIGMS NIH HHS4487-4495https://www.ebi.ac.uk/biostudies/studies/S-EPMC9469474biostudies-literatureUnknown144(10)Chemical cross-linking enables rapid identification of RNA-protein and RNA-nucleic acid inter- and intramolecular interactions. However, no method exists to site-specifically and covalently cross-link two user-defined sites within an RNA. Here, we develop RNA-CLAMP, which enables site-specific and enzymatic cross-linking (clamping) of two selected guanine residues within an RNA. Intramolecular clamping can disrupt normal RNA function, whereas subsequent photocleavage of the cross-linker restores activity. We used RNA-CLAMP to clamp two stem loops within the single-guide RNA (sgRNA) of the CRISPR-Cas9 gene editing system via a photocleavable cross-linker, completely inhibiting gene editing. Visible light irradiation cleaved the cross-linker and restored gene editing with high spatiotemporalJournal of the American Chemical SocietySite-Specific and Enzymatic Cross-Linking of sgRNA Enables Wavelength-Selectable Photoactivated Control of CRISPR Gene Editing.PMC9469474R35 GM141939R01 GM123285HR0011-18-2-0039Zhang DLi ZLiu LTota EJin SPiao XZhang XDevaraj NKFu XDfalseSite-Specific and Enzymatic Cross-Linking of sgRNA Enables Wavelength-Selectable Photoactivated Control of CRISPR Gene Editing.Chemical cross-linking enables rapid identification of RNA-protein and RNA-nucleic acid inter- and intramolecular interactions. However, no method exists to site-specifically and covalently cross-link two user-defined sites within an RNA. Here, we develop RNA-CLAMP, which enables site-specific and enzymatic cross-linking (clamping) of two selected guanine residues within an RNA. Intramolecular clamping can disrupt normal RNA function, whereas subsequent photocleavage of the cross-linker restores activity. We used RNA-CLAMP to clamp two stem loops within the single-guide RNA (sgRNA) of the CRISPR-Cas9 gene editing system via a photocleavable cross-linker, completely inhibiting gene editing. Visible light irradiation cleaved the cross-linker and restored gene editing with high spatiotemporal2022-01-01T00:00:00Z2022 Mar2025-04-18T20:48:28.764Z2025-04-07T08:47:13.828ZS-EPMC94694743525757510.1021/jacs.1c12166