RNA editing plays a key posttranscriptional role in gene expression. Existing studies on cytidine-to-uridine RNA editing in plants have focused on maize (), rice (), and Arabidopsis (). However, the importance and regulation of RNA editing in several critical agronomic processes are not well understood, a notable example of which is fruit ripening. Here, we analyzed the expression profile of 33 RNA editing factors and identified 11 putative tomato () fruit ripening-related factors. A rapid virus-induced gene silencing assay indicated that the organelle RNA recognition motif-containing protein SlORRM4 affected tomato fruit ripening. Knocking out SlORRM4 expression using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 genome editing strategy delayed tomato fruit ripening by lowering respiratory rate and ethylene production. Additionally, the expression of numerous genes associated with fruit ripening and mitochondrial functions changed significantly when was knocked out. Moreover, the loss of function significantly reduced RNA editing of many mitochondrial transcripts, leading to low-level expression of some core subunits that are critical for mitochondrial complex assembly (i.e. Nad3, Cytc1, and COX II). Taken together, these results indicate that SlORRM4 is involved in RNA editing of transcripts in ripening fruit that influence mitochondrial function and key aspects of fruit ripening.

Plant organellar RNA editing is a distinct type of post-transcriptional RNA modification that is critical for plant development. We showed previously that the RNA editing factor SlORRM4 is required for mitochondrial function and fruit ripening in tomato (Solanum lycopersicum). However, a comprehensive atlas of the RNA editing mediated by SlORRM4 is lacking. We observed that SlORRM4 is targeted to both chloroplasts and mitochondria, and its knockout results in pale-green leaves and delayed fruit ripening. Using high-throughput sequencing, we identified 12 chloroplast editing sites and 336 mitochondrial editing sites controlled by SlORRM4, accounting for 23% of chloroplast sites in leaves and 61% of mitochondrial sites in fruits, respectively. Analysis of native RNA immunoprecipitation sequencing revealed that SlORRM4 binds to 31 RNA targets; 19 of these targets contain SlORRM4-dependent editing sites. Large-scale analysis of putative SlORRM4-interacting proteins identified SlRIP1b, a RIP/MORF protein. Moreover, functional characterization demonstrated that SlRIP1b is involved in tomato fruit ripening. Our results indicate that SlORRM4 binds to RNA targets and interacts with SlRIP1b to broadly affect RNA editing in tomato organelles. These results provide insights into the molecular and functional diversity of RNA editing factors in higher plants.

Gene-editing systems have emerged as bioengineering tools in recent years. Classical gene-editing systems include zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9), and these tools allow specific sequences to be targeted and edited. Various modified gene-editing systems have been established based on classical gene-editing systems. Base editors (BEs) can accurately carry out base substitution on target sequences, while prime editors (PEs) can replace or insert sequences. CRISPR systems targeting mitochondrial genomes and RNA have also been explored and established. Multiple gene-editing techniques based on CRISPR/Cas9 have been established and applied to genome engineering. Modified gene-editing systems also make transgene-free plants more readily available. In this review, we discuss the modifications made to gene-editing systems in recent years and summarize the capabilities, deficiencies, and applications of these modified gene-editing systems. Finally, we discuss the future developmental direction and challenges of modified gene-editing systems.