Rapid and Cost-Effective Digital Quantification of RNA Editing and Maturation in Organelle Transcripts

RNA editing and maturation are critical regulatory mechanisms in plant organelles, yet their quantification remains technically challenging. Traditional Sanger sequencing lacks sensitivity and reproducibility, whereas advanced next-generation sequencing (NGS) approaches, such as rRNA-depleted RNA-seq or targeted amplicon-seq, involve high costs, complex workflows, and limited accessibility. To address these limitations, we developed a rapid and cost-effective long-read sequencing approach, termed premium PCR sequencing, for digital quantification of RNA-editing and intron retention events in targeted chloroplast transcripts. This method combines multiplexed high-fidelity PCR amplification with Oxford Nanopore sequencing and custom in-house Perl and Python scripts for streamlined data processing, including barcode-based demultiplexing, strand reorientation, alignment to a pseudo-genome, manual editing-site inspection, and splicing variant identification and comparison. Using this platform, we analyzed the ndhB and ndhD transcripts, two chloroplast NAD(P)H dehydrogenase genes with the highest number of known editing sites, in an inducible CRISPR interference (iCRISPRi) system targeting MORF2 , a key RNA-editing factor. Our results revealed MORF2 dosage-dependent reductions in C-to-U editing efficiency, with significant defects observed in the strongly repressed P1-12 line. Moreover, we identified an accumulation of intron-retaining ndhB transcripts, specifically in Dex-treated iCRISPRi lines, indicating impaired chloroplast splicing functions upon MORF2 suppression. The platform achieves single-molecule resolution, robust reproducibility, and high read coverage across biological replicates at a fraction of the cost of lncRNA-seq, making it broadly accessible. This study establishes premium PCR sequencing as a versatile, scalable, and affordable tool for targeted post-transcriptional analysis in plant organelles and expands our understanding of MORF2’s role in chloroplast RNA maturation.