CRISPR/dCas13(Rx) Derived RNA N<sup>6</sup>‐methyladenosine (m<sup>6</sup>A) Dynamic Modification in Plant
Lu Yu, Muna Alariqi, Baoqi Li, Amjad Hussain, Huifang Zhou, Qiongqiong Wang, Fuqiu Wang, Guanying Wang, Xiangqian Zhu, Fengjiao Hui, Xiyan Yang, Xinhui Nie, Xianlong Zhang, Shuangxia Jin
Abstract
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.