Programmable RNA N⁶-methyladenosine editing with CRISPR/dCas13a in plants

N-6-methyladenonsine (m(6)A) is the most prevalent internal modification of messenger RNA (mRNA) and plays critical roles in mRNA processing and metabolism. However, perturbation of individual m(6)A modification to reveal its function and the phenotypic effects is still lacking in plants. Here, we describe the construction and characterization of programmable m(6)A editing tools by fusing the m(6)A writers, the core catalytic domain of the MTA and MTB complex, and the AlkB homologue 5 (ALKBH5) eraser, to catalytically dead Cas13a (dCas13a) to edit individual m(6)A sites on mRNAs. We demonstrated that our m(6)A editors could efficiently and specifically deposit and remove m(6)A modifications on specific RNA transcripts in both Nicotiana benthamiana and Arabidopsis thaliana. Moreover, we found that targeting SHORT-ROOT (SHR) transcripts with a methylation editor could significantly increase its m(6)A levels with limited off-target effects and promote its degradation. This leads to a boost in plant growth with enlarged leaves and roots, increased plant height, plant biomass, and total grain weight in Arabidopsis. Collectively, these findings suggest that our programmable m(6)A editing tools can be applied to study the functions of individual m(6)A modifications in plants, and may also have potential applications for future crop improvement.