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

N⁶ -methyladenonsine (m⁶ A) is the most prevalent internal modification of messenger RNA (mRNA) and plays critical roles in mRNA processing and metabolism. However, perturbation of individual m⁶ A modification to reveal its function and the phenotypic effects is still lacking in Plants. Here, we describe the construction and characterization of programmable m⁶ A editing tools by fusing the m⁶ 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 m⁶ A sites on mRNAs. We demonstrated that our m⁶ A editors could efficiently and specifically deposit and remove m⁶ 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 m⁶ 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 m⁶ A editing tools can be applied to study the functions of individual m⁶ A modifications in Plants, and may also have potential applications for future crop improvement.

Arabidopsis; CRISPR/dCas13a; N6-methyladenonsine; m6A editing; plant growth.