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

N6-methyladenonsine (m6A) is the most prevalent modification on mRNA and plays critical roles in mRNA processing and metabolism. However, how the m6A modification on individual gene of interest regulates gene function and the links to phenotypic outcome in plants are mostly unknown. Here, we described the construction and characterization of programmable m6A editing tools by fusing the m6A writer, core catalytical domain of MTA and MTB complex, and eraser, ALKBH5, to catalytically dead Cas13a (dCas13a), respectively, for targeting methylation and demethylation of specific mRNA. We demonstrated that our m6A editors could efficiently and specifically add and remove the m6A modification on specific RNA transcript in both Nicotiana benthamiana and Arabidopsis. Moreover, targeting SHORT-ROOT (SHR) transcript with methylation editor could significantly increase its m6A levels with limited off-target effects and enhance its expression, giving rise to induced plant growth with enlarged leaf size and root, increased plant height, plant biomass and total grain weight in Arabidopsis. Collectively, these findings suggest that our programmable m6A editing tools can be applied to study m6A modification of specific genes in plants, and might also have great potential applications for crop improvement in future Overall design: The MeRIP-seq is based on the protocols previously described. Total RNA from Arabidopsis inflorescences was isolated by TRIzol reagent, and 100 µg total RNA was polyA-selected to get ~2 µg intact mRNA using Dynabeads mRNA Purification Kit (Invitrogen). All the isolated mRNA was chemically fragmented using NEBNext Magnesium RNA Fragmentation Module (New England Biolabs). We aliquoted ~300 ng fragmented mRNA used as input for MeRIP-seq. The remaining fragmented mRNA was added into 500 µl IP buffer, incubated with 5 µg anti-m6A antibody (Synaptic Systems) and 5 µl of RNasin Plus RNase Inhibitor (Promega) and tumbled at 4 °C for 2 h. Then, the reaction mixture was incubated with protein A/G magnetic beads (MedChemExpress) and tumbled at 4°C for 1 h. Then, the reaction mixture was washed twice with IP buffer, twice with low-salt IP buffer, and twice with high-salt IP buffer at 4 °C for 10 min for each. After extensive washing, the m6A-enriched RNA was purified by TRIzol reagent with glycol blue coprecipitant (Invitrogen). All the input and IP RNA were used for RNA library construction using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs) according to the manufacturer's instructions. All the RNA libraries were sequenced (Paired-end 150nt) on an Illumina NovaSeq 6000 platform at Novogene, China.