Engineering Programmable Tools to Target the Transcriptome

Programmable RNA-guided tools for gene editing attracted a lot of interest during the last years. However, there are safety concerns related to off-target edits, and immunogenicity, and gene editing fails when the introduced mutation is lethal or genetically compensated. For the last years, our lab has been pioneering several alternative strategies that are based on site-directed A-to-I editing of specific transcripts.

Specifically, we developed the SNAP-ADARs approach that applies a unique and novel assembly strategy to generate artificial RNA-guided riboproteins. Recently, we demonstrated the simultaneous A-to-I editing of several endogenous transcripts, with high efficiency (up to 90%), high potency, sufficient duration, and high precision [1]. Yet unpublished, we will report on improvements of the approach and their application on the manipulation of signaling proteins. We aim to point out that the SNAP-tag approach is a simple and convenient general RNA-targeting approach, orthogonal to current Cas approaches and may allow for the targeted writing and erasing of further epitranscriptomic marks.

Furthermore, we develop genetically encodable guideRNAs [2] and chemically stabilized oligonucleotides [3] that enable the recruitment of endogenous ADARs for site-directed RNA editing with high potential for clinical translation.

[1] Vogel et al., Efficient and Precise Editing of Endogenous Transcripts with SNAP-tagged ADARs. Nat. Meth. 15, 535 (2018).

[2] Wettengel, J., Reautschnig, J., Geisler, S., Kahle, P. J., Stafforst, T. Harnessing human ADAR2 for RNA repair – Recoding a PINK1 mutation rescues mitophagy. Nucl. Acids Res. 45, 2797-2808 (2017).

[3] T. Merkle, S. Merz, P. Reautschnig, A. Blaha, Q. Li, P. Vogel, J. Wettengel, J. B. Li, T. Stafforst. Precise RNA editing by recruiting endogenous ADARs with antisense oligonucleotides. Nature Biotech. 37, 133-38 (2019).