CRISPR/dCas13(Rx) Derived RNA N 6 -methyladenosine (m 6 A) Dynamic Modification in Plant.

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 (Targeted RNA Methylation Editor, TME) or the demethyltransferase GhALKBH10 (Targeted RNA Demethylation Editor, 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.