The improvement of crop yield and quality is an eternal theme to face climate change and population growth. The key to improving crop varieties lies in precisely manipulating gene expression. Recent advancements in CRISPR/Cas9 technology have made gene knockout increasingly straightforward, yet for genes related to important agronomic traits, it is crucial to regulate their expression levels appropriately. Complete knockout often results in defects in other aspects. In addition, many agronomic traits require upregulation of target gene expression for their improvement. Therefore, the development of novel methods for precise upregulation or downregulation of gene expression, without altering gene protein sequences or introducing new genome fragments, will significantly bolster the technical foundation for crop genetic improvement.

N ⁶-methyladenosine (m⁶A) is the most abundant and reversible internal chemical modification in eukaryotic mRNA, which is installed, removed, and recognized by methyltransferases (writers), demethylases (erasers), and m⁶A-binding proteins (readers), respectively (Tang et al., 2023). Currently, two types of m⁶A methyltransferases have been identified in plants: multiprotein complexes and a single protein. The multiprotein complex includes MTA, MTB, FIP37, VIRILIZER (VIR), HAKAI, and HIZ2 (HAKAI interacting zinc finger protein 2), which catalyze the majority of m⁶A modifications in mRNA (Parker et al., 2021; Ruzicka et al., 2017; Shen et al., 2016; Zhang et al., 2022; Zhong et al., 2008). The single protein FIONA1 also exhibits methyltransferase activity in Arabidopsis (Wang et al., 2022; Xu et al., 2022), catalyzing approximately 10% of m⁶A modifications in mRNA. Several m⁶A demethylases, which belong to the Fe (II)/α - kg dependent dioxygenase superfamily, have been identified in plants, including Arabidopsis AtALKBH10B and AtALKBH9B (Martinez-Perez et al., 2017), rice OsALKBH9 (Tang et al., 2024), and tomato SlALKBH2 (Zhou et al., 2019). m⁶A is recognized by m⁶A-binding proteins, such as ECTs in Arabidopsis, which contains the YTH domain. In plants, the ratio of m⁶A/A in poly A⁺ RNA varied among different tissues, with a range of 0.36–0.75% in Arabidopsis and 0.52–0.67% in rice, suggesting its high abundance (Wang et al., 2024). At the transcriptome level, m⁶A sites are primarily enriched within the 3′-untranslated region (3′ UTR), followed by the coding DNA sequence (CDS) and 5′-untranslated region (5′ UTR). Recent studies have demonstrated the crucial roles of m⁶A in regulating gene expression in plants, primarily by influencing mRNA stability, translation, and 3′ UTR processing (Tang et al., 2023). Among them, mRNA stability regulation is one of the primary functions of m⁶A, which involves two aspects: acceleration of RNA decay or preservation of RNA stability, depending on the specific m⁶A-binding proteins. Recent studies combining proteomics and m⁶A analysis have shown that m⁶A in the untranslated regions is negatively correlated with protein abundance, suggesting that m⁶A in UTR is likely to inhibit protein abundance in plants (Li et al., 2024). Therefore, manipulating m⁶A could lead to an increase in protein abundance. m⁶A modifications have also been found to play crucial roles in plant biology, such as embryo development, floral transition, stem cell fate determination, pollen development, fruit ripening, photomorphogenesis, circadian clock, nitrate signaling, and responses to biotic and abiotic stress (Tang et al., 2023). Given the high abundance and crucial roles of m⁶A modifications in gene expression regulating, altering m⁶A modifications in genes holds promise as a strategy for enhancing crop agronomic traits.

In mammals, the m⁶A demethylase FTO, known as an obesity gene, plays a crucial role in regulating body weight. Researchers have genetically engineered rice and potato to express FTO. In field experiments, the yield and biomass of genetically engineered rice and potato increased by approximately 50% (Yu et al., 2021). Further research indicates that the expression of FTO in rice promotes root growth, tiller bud formation, photosynthetic efficiency, and drought resistance, and these phenotypes are dependent on the m⁶A demethylase activity of FTO (Yu et al., 2021). In strawberries, inhibiting the expression of m⁶A methyltransferase genes FveMTA or FveMTB can delay fruit ripening, while upregulating the expression of either gene can accelerate fruit ripening, suggesting that manipulating m⁶A modification can regulate the ripening time of strawberries (Zhou et al., 2021). The above research suggests that manipulating the m⁶A modification level overall can enhance the agronomic traits of crops.

In addition to altering the overall m⁶A modification level, researchers have also endeavored to edit m⁶A modification levels on individual genes. In plants, a precise editing system for plant mRNA m⁶A has been successfully developed by fusing dCas13a with the plant m⁶A methyltransferase core complex MTA-MTB or the mammalian demethylase ALKBH5 (Shi et al., 2024). By specifically editing the m⁶A modification on the SHR transcript, which is a key gene for root development, it was found that an increase in the m⁶A modification level of the SHR transcript can promote the enlargement of the aboveground and root parts of plants, increase leaf area, plant height, biomass, and grain yield, thereby promoting plant growth (Shi et al., 2024). In cotton, similar m⁶A editing tools have also been developed, combining CRISPR/dCas13(Rx) with the methyltransferase GhMTA (Targeted RNA Methylation Editor, TME) or the demethyltransferase GhALKBH10 (Targeted RNA Demethylation Editor, TDE) (Yu et al., 2024). Using TME editor, the m⁶A level of GhDi19 transcript increased, and the plants with increased m⁶A levels results in a significant increase in root length and enhanced drought resistance. Both works indicate that manipulating the m⁶A modification level of key genes can regulate plant phenotype and improve agronomic traits.

According to current research, three strategies have been proposed for enhancing crop agronomic traits through m⁶A modification. (1) Altering RNA m⁶A modification regulatory proteins (RMRPs) (methyltransferases, demethylases, and recognition proteins) and their interacting proteins. Given that m⁶A regulatory proteins typically regulate a multitude of substrate genes, this strategy exhibits a high degree of randomness and uncertainty in improving agronomic traits, and may frequently result in other phenotypic abnormalities; (2) Conducting m⁶A editing by fusing Cas13 (dCas13) with RNA-modified methyltransferase or demethylase in specific mRNA. However, the RNA-based m⁶A editing strategy poses limitations in crop breeding applications: the m⁶A editing vector must be maintained in the offspring, otherwise the editing effect of m⁶A cannot be preserved. Currently, they are not suitable for practical breeding applications; (3) Editing m⁶A motif of specific gene to remove m⁶A modifications. For example, using a base editor to accurately replace m⁶A-modified adenine on DNA. Regarding the m⁶A modification site on the UTR, the CRISPR/Cas9 system can be employed to disrupt the m⁶A modification motif on the UTR, thereby eliminating the m⁶A modification. This strategy involves directly altering m⁶A motifs on DNA, enabling the production of m⁶A editing materials without transgenic vectors in offspring, which can be utilized in breeding applications.

Fine-tuning gene expression is of great significance for crop improvement. m⁶A, the most abundant modification in mRNAs, plays crucial roles in regulating gene expression, positioning it as a potential tool for manipulating gene expression. With the advent of new m⁶A sequencing methods, mapping m⁶A at single base resolution is no longer an obstacle. The primary limitation of m⁶A application is the insufficient mining of functional m⁶A sites. The most effective approach to explore functional m⁶A sites in crops involves studying the biological functions of m⁶A methyltransferases, demethylases, or m⁶A binding proteins in crop development, biotic and abiotic stress response, and other processes. However, fewer RMRPs have been identified in crops so far, and future research should strengthen the identification and functional research of crop RMRPs. In summary, because of the vital roles of m⁶A in regulating gene expression post-transcriptionally, the m⁶A editing strategy holds significant potential for crop improvement.