An Approach to the Synthesis of Cyclic Photocleavable RNA for Photoactivatable CRISPR/Cas9 System

Abstract

Objective: The development of controllable gene editing systems based on the CRISPR/Cas technology is a problem of immediate interest in modern molecular biology and genetic engineering. An interesting solution of this problem is modification of guide RNA by introduction of photocleavable linkers. Methods: We developed an approach to the synthesis of cyclic photocleavable guide crRNA for the CRISPR/Cas9 system with a photolinker based on 1-(2-nitrophenyl)-1,2-ethanediol (PL). In cyclic form, such guide RNA is not functional. Upon irradiation by UV-light, such guide RNA is linearized and CRISPR/Cas9 system is activated. Two chemical methods for the cyclization of RNA were tested: Michael reaction (thiol-maleimide condensation) and Cu-catalyzed azide-alkyne cycloaddition (CuAAC, click-chemistry reaction). For this purpose, 5′,3′-modified RNA containing reactive groups were prepared. Results and Discussion: The advantages of the CuAAC reaction for cyclic RNA preparation was demonstrated. Efficiency of cyclic RNA depends on their secondary structure and on the ability of reactive groups to move closer. A series of photocleavable and control non-cleavable cyclic crRNA was obtained. It was shown that cyclic crRNAs guide Cas9 nuclease for plasmid cleavage less efficiently, but linearization of photocleavable cyclic crRNA increases the extent of plasmid cleavage. Conclusions: The developed approach allows to synthesize cyclic photocleavable RNA that can be used for spatiotemporal activation of guide RNA for gene editing. Photoregulation of gene editing will allow minimizing the off-target effects.