Photocleavable Guide RNA for Photocontrolled CRISPR/Cas9 System

Abstract

Objective: The development of CRISPR/Cas-based gene-editing systems having a higher efficacy and specificity, and capable of changing activity in response to light irradiation is an urgent problem. A promising approach to this problem is to modify CRISPR/Cas components, in particular guide RNA, by introducing photocleavable linkers. We developed an approach to the synthesis of photocleavable single guide RNA (sgRNA) for the CRISPR/Cas9 system containing linkers on the basis of 1-(2-nitrophenyl)-1,2-ethanediol. Such photomodified guide RNAs are cleaved under UV irradiation, thereby inactivating the CRISPR/Cas9 system. Methods: Automatic solid-phase phosphoramidate method was used for photomodified sgRNA synthesis. Model plasmid was used for designed system testing. Results and Discussion: We obtained three variants of photomodified sgRNA with different photolinker positions. Evidence was obtained showing that the sgRNA with the photolinker introduced in the protein Cas9 site of binding and hairpin formation is able to effectively guide Cas9 nuclease for target DNA cleavage before UV irradiation and lose its activity after irradiation. The conditions of controllable 40% cleavage of a model target DNA were chosen. Conclusions: The work presents the results of photocleavable sgRNA design and usage as a component of photoregulated CRISPR/Cas9 system. The developed approach makes possible specific inactivation of the CRISPR/Cas9 gene editing system in a specific time moment in a definite place. The photoregulation of the gene-editing system not only allows one to reduce undesirable off-target effects, but also forms the basis for genetic disease therapy.