A rapid and efficient in planta assay for assessment of RNA editing factor performance.

Land plant mitochondria and plastids are endosymbiotic organelles that possess their own, reduced genomes. Gene expression is largely regulated at the transcript level, predominantly by nuclear-encoded proteins. Notably, up to thousands of cytidines in organellar RNAs are specifically edited to uridines, mostly restoring conserved genetic information. Key players in this process are nuclear-encoded pentatricopeptide repeat (PPR) proteins, which bind target RNAs in a one-PPR:one-nucleotide manner, following a two-amino-acid-based code. Cytidine deamination is catalyzed by intrinsic or trans-acting DYW domains. The modularity and programmability of PPR-RNA interactions make PPR editing factors attractive candidates for the development of biotechnological tools. However, RNA target selection is not determined by the 'PPR code' alone, hampering the generation of highly effective and specific editors. Refining such tools requires efficient assays to dissect the determinants of editor-RNA interaction. Here, we introduce a rapid assay to assess PPR editor specificity through transient expression in the cytosol of Nicotiana benthamiana, which offers a large and diverse transcriptome that enables extensive off-target editing. Profiling of these off-targets provides comprehensive information on an editor's target selectivity. Compared with a previous setup in the model moss Physcomitrium patens, which relies on stable transformation, the required experimental time is reduced from months to days. We demonstrate the power of the system by characterizing the off-targets of the moss editor PpPPR56. The new assay allows rapid functional assessment of editing factor variants and removes a bottleneck in understanding editor-RNA interactions, which is a prerequisite for the rational improvement of PPR-based RNA editing tools.