Precision RNA Base Editing in Plant Organelles Using Engineered Synthetic P-type PPR Editing Factors

RNA editing, particularly cytidine-to-uridine conversions in plant organelles, plays a crucial role in regulating gene expression. 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 from a moss mitochondrial editing factor, PPR56. These designer PPR editors (dPPRe) were tested in Escherichia coli and Nicotiana benthamiana chloroplasts and mitochondria, demonstrating efficient and precise de novo RNA editing. Transcriptome-wide analysis of the most efficient chloroplastic dPPRe revealed minimal off-target effects, with only three non-target 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. Overall design: To investigate the effect of dPPRerpl2 on chloroplast gene expression and RNA editing, we transiently expressed this construct with the P19 silencing suppressor in Nicotiana benthamiana leave. P19 construct was expressed alone as a control. dPPRerpl2 was expressed from two vectors (pGWB2 and pMDC32). We extracted total RNA at 3 dpi from the experimental (3 biological replicates for each vector) and control (3 biologial replicates) samples for differential gene expression analysis by RNA-seq.