Fig. 1

Schematic view of plant m6A editors (PMEs). (A-B) Schematic diagram of the construct for PMEs in Arabidopsis. ALKBH-CD represents human m6A demethylase ALKBH5 catalytic domain (66–292 aa); dLwaCas13a-msfGFP represents inactive dLwaCas13a (R474A and R1046A)-msfGFP; the XTEN linker separates dLwaCas13a-msfGFP and ALKBH-CD. dLwaCas13a-msfGFP-ALKBH-CD construct with two conserved nuclear localization signals (NLS) was driven by CMV 35 S promoter. RNA polymerase III promoter (AtU6) was employed to express crRNAs. For PME-WS-H and PME-FSS-H, double ribozyme system was used for crRNAs processing. DR indicates direct repeat and T indicates target for PMEs. (C) A self-cleaving double ribozyme system for precise processing of mature crRNAs. The upper graph is the predicted secondary structures o of the pre-crRNA, containing a Hammerhead ribozyme at the 5′-end (light yellow background), the sequence-specific crRNA portion in the middle (light blue background), and a HDV ribozyme at the 3′-end (light yellow background). The mature crRNA is released from the pre-crRNA through self-catalyzed processing. The mature crRNA contains direct repeat (universal for all crRNAs) and spacer (complementary to the target sequences)