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

Abstract N 6 ‐methyladenosine (m 6 A) 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 m 6 A sites of individual transcripts in plants. Here, programmable m 6 A editing tools are developed by combining CRISPR/dCas13(Rx) with the methyltransferase GhMTA ( T argeted RNA M ethylation E ditor, TME ) or the demethyltransferase GhALKBH10 ( T argeted RNA D emethylation E ditor, TDE ). These editors enable efficient deposition or removal of m 6 A 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 m 6 A levels by 24%–76%, while the TME editor increases m 6 A enrichment, ranging from 1.37‐ to 2.51‐fold. Furthermore, installation and removal of m 6 A modifications play opposing roles in regulating GhECA1 and GhDi19 mRNA transcripts, which may be attributed to the fact that their m 6 A 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 m 6 A editors can be applied to study the function of specific m 6 A modifications and have the potential for future applications in crop improvement.