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.