RECODE: a programmable guide-free C-to-U RNA editing tool

Programmable RNA cytidine deaminase tools have been developed to convert cytidine-to-uridine (C-to-U) using CRISPR systems with guide RNAs. These tools, however, have limitations such as low editing efficiency, limited targetable sequence flexibility, and off-target RNA editing. Here, we present a novel guide-free C-to-U editing tool, named RECODE (RNA Editor for C-to-U with an Optimized DYW Enzyme), based on the RNA-binding pentatricopeptide repeat proteins, naturally fused to a C-terminal DYW cytidine deaminase domain. The RECODE specificity domain was engineered to enable retargeting, while its length and sequence were optimized to reduce off-target effects. Further optimization of the C-terminal catalytic region increased both the editing activity and the translation of the edited RNA. We showed that RECODE efficiently edits a wide range of targets in human cells, without affecting adjacent cytidines. It achieved over 50% editing efficiency for most sites, except those with an upstr..., Alignment of C-terminal domains and construction of a phylogenetic tree
To design the C-terminal domain, the non-DYW:KP proteins identified in hornwort, lycophyte, and fern transcriptomes (Ichinose et al., 2022) were aligned using MAFFT in L-INS-i mode (v7.407) (Katoh and Standley, 2013). Alignments were then trimmed using TrimAL (v1.4.rev15) (Capella-Gutiérrez et al., 2009) with a minimum conservation threshold and a gap threshold of 20%. A tree was built for the trimmed alignment using IQ-TREE (v.2.0.3) (Minh et al., 2020) with the JTT+F+I+G4 substitution model, selected using the in-built automated test (Kalyaanamoorthy et al., 2017). The branch support was computed with 1000 bootstrap replicates. The tree was visualized using iTOL (Letunic and Bork, 2016).
Transfection
For the design of the PG and WW C-terminal domains and the analysis of several PPR code combinations (at the exogenous rpoA editing site), HEK293T cells were plated in 24-well plates (Thermo Fisher Scientifi..., , # RECODE: A programmable guide-free C-to-U RNA editing tool

[https://doi.org/10.5061/dryad.cjsxksngv](https://doi.org/10.5061/dryad.cjsxksngv)

## Description of the data and file structure

### Phylogenetic tree

#### File: tree-P2toDYW-all_al_trim.fa

**Description:** Fasta format of a multiple amino acid sequence alignment of non-DYW:KP protein sequences identified in hornwort, lycophyte, and fern transcriptomes. This alignment was generated using MAFFT in L-INS-i mode and trimmed using TrimAL.

#### File: tree-P2toDYW-all_al_trim.fa.treefile

**Description:** Newick format tree of the non-KP C-terminal domain sequences found in hornwort, lycophyte, and fern transcriptomes (Supplementary Figure 1) generated using tree-P2toDYW-all_al_trim.fa file. The branch support was computed with 1000 bootstrap replicates. 

The synthetic DYW:PG and DYW:WW C-terminal domains used in this study were designed on DYW subgroups identified in this tree.

### Chromatograms

#### File: chromatogramsv3.x...,