De novo RNA base editing in plant organelles with engineered synthetic P-type PPR editing factors

In plant mitochondria and chloroplasts, cytidine-to-uridine RNA editing is necessary for the production of functional proteins. While natural PLS-type PPR proteins are specialized in this process, synthetic PPR proteins offer significant potential for targeted RNA editing. In this study, we engineered chimeric editing factors by fusing synthetic P-type PPR guides with the DYW cytidine deaminase domain of a moss mitochondrial editing factor, PPR56. These designer PPR editors (dPPRe) elicited efficient and precise de novo RNA editing in Escherichia coli as well as in the chloroplasts and mitochondria of Nicotiana benthamiana. Chloroplast transcriptome-wide analysis of the most efficient dPPRe revealed minimal off-target effects, with only three nontarget C sites edited due to sequence similarity with the intended target. This study introduces a novel and precise method for RNA base editing in plant organelles, paving the way for new approaches in gene regulation applicable to plants and potentially other organisms.