CRISPR/dCas13(Rx) Derived RNA N6‐methyladenosine (m6A) Dynamic Modification in Plant

Abstract N6‐methyladenosine (m6A) is the most prevalent internal modification of mRNA and plays an important role in regulating plant growth. However, there is still a lack of effective tools to precisely modify m6A sites of individual transcripts in plants. Here, programmable m6A editing tools are developed by combining CRISPR/dCas13(Rx) with the methyltransferase GhMTA (Targeted RNA Methylation Editor, TME) or the demethyltransferase GhALKBH10 (Targeted RNA Demethylation Editor, TDE). These editors enable efficient deposition or removal of m6A modifications at targeted sites of endo‐transcripts GhECA1 and GhDi19 within a broad editing window ranging from 0 to 46 nt. TDE editor significantly decreases m6A levels by 24%–76%, while the TME editor increases m6A enrichment, ranging from 1.37‐ to 2.51‐fold. Furthermore, installation and removal of m6A modifications play opposing roles in regulating GhECA1 and GhDi19 mRNA transcripts, which may be attributed to the fact that their m6A sites are located in different regions of the genes. Most importantly, targeting the GhDi19 transcript with TME editor plants results in a significant increase in root length and enhanced drought resistance. Collectively, these m6A editors can be applied to study the function of specific m6A modifications and have the potential for future applications in crop improvement.