Work

A Simplified Guide RNA Synthesis Protocol for SNAP- and Halo-Tag-Based RNA Editing Tools

Year: 2025

Type: article

Abstract: SNAP-tag and Halo-tag have been employed to achieve targeted RNA editing by directing the deaminase domain of human ADAR to specific sites in the transcriptome. This targeting is facilitated by short guide RNAs (gRNAs) complementary to the target transcript, which are chemically modified with benzylguanine or chloroalkane moieties to enable covalent binding to the respective self-labeling enzymes. However, broad application of this approach has been limited by challenges such as low scalability, the requirement for specialized chemical expertise and equipment, and labor-intensive protocols. In this study, we introduce streamlined, efficient protocols for the synthesis and purification of these linkers, suitable for SNAP-tag and Halo-tag applications, without the need for advanced chemical equipment. Our methods enable linker coupling in a kit-like manner and support the high-yield production of modified gRNAs. We demonstrate that the newly synthesized linkers and gRNA designs perform similarly to previously published constructs with regard to RNA editing efficiency. Moreover, large-scale production of modified gRNAs facilitates their use in studies involving cellular uptake and in vivo experiments.

Source: Molecules

DOI: 10.3390/molecules30051049

Authors: Daniel Tobias Hofacker, Sebastian Kalkuhl, Jana Franziska Schmid, Shubhangi Singh, Thorsten Stafforst

Institution: University of Tübingen

Language: English

Topic: RNA regulation and disease

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: gold

Estimated APC paid: $3,258

Funders: Deutsche Forschungsgemeinschaft, Volkswagen Foundation, Eberhard Karls Universität Tübingen