Site-directed RNA base editing enables the transient and dosable change of genetic information and represents a recent strategy to manipulate cellular processes, paving ways to novel therapeutic modalities. While tools to introduce adenosine-to-inosine changes have been explored quite intensively, the engineering of precise and programmable tools for cytidine-to-uridine editing is somewhat lacking behind. Here we demonstrate that the cytidine deaminase domain evolved from the ADAR2 adenosine deaminase, taken from the RESCUE-S tool, provides very efficient and highly programmable editing when changing the RNA targeting mechanism from Cas13-based to SNAP-tag-based. Optimization of the guide RNA chemistry further allowed to dramatically improve editing yields in the difficult-to-edit 5′-CCN sequence context thus improving the substrate scope of the tool. Regarding editing efficiency, SNAP-CDAR-S outcompeted the RESCUE-S tool clearly on all tested targets, and was highly superior in perturbing the β-catenin pathway. NGS analysis showed similar, moderate global off-target A-to-I and C-to-U editing for both tools.

Site-directed RNA base editing enables the transient and dosable change of genetic information and represents a recent strategy to manipulate cellular processes, paving ways to novel therapeutic modalities. While tools to introduce adenosine-to-inosine changes have been explored quite intensively, the engineering of precise and programmable tools for cytidine-to-uridine editing is somewhat lacking behind. Here we demonstrate that the cytidine deaminase domain evolved from the ADAR2 adenosine deaminase, taken from the RESCUE-S tool, provides very efficient and highly programmable editing when changing the RNA targeting mechanism from Cas13-based to SNAP-tag-based. Optimization of the guide RNA chemistry further allowed to dramatically improve editing yields in the difficult-to-edit 5prime-CCN sequence context thus improving the substrate scope of the tool. Regarding editing efficiency, SNAP-CDAR-S outcompeted the RESCUE-S tool clearly on all tested targets, and was highly superior in perturbing the beta-catenin pathway. NGS analysis showed similar, moderate global off-target A-to-I and C-to-U editing for both tools.