Enhanced or Reversible RNA N6-Methyladenosine Editing by Red/Far-red Light induction

The RNA N6-methyladenosine (m6A) modification is a critical regulator of various biological processes, but precise and dynamic control of m6A remains a challenge. In this work, we present a red/far-red light-inducible m6A editing system that enables efficient and reversible modulation of m6A levels with minimal off-target effects. By engineering the CRISPR dCas13 protein and sgRNA with two pairs of light-inducible heterodimerizing proteins, ΔphyA/FHY1 and Bphp1/PspR2, we achieved targeted recruitment of m6A effectors. This system significantly enhances m6A writing efficiency, and allows dynamic regulation of m6A deposition and removal on specific transcripts, such as SOX2 and ACTB. Notably, reversible m6A editing was achieved through cyclic modulation at a single target site, demonstrating the ability to influence mRNA expression and modulate the differentiation state of human embryonic stem cells. This optogenetic platform offers a precise, versatile tool for cyclic and reversible m6A regulation, with broad implications for understanding RNA biology and its potential applications in research and medicine. This study comprehensively assessed the alterations and consistency of RNA m6A modification in HEK293T cells. Utilizing the m6A sequencing results, we investigated differential genes across various RNA types.

RNA N6-甲基腺嘌呤（RNA N6-methyladenosine，m6A）修饰是多种生物学过程的关键调控因子，但精准且动态地调控m6A水平仍是一项挑战。本研究构建了一套红光/远红光诱导型m6A编辑系统，可实现脱靶效应极低的高效、可逆m6A水平调控。通过对CRISPR dCas13蛋白与单向导RNA（sgRNA）进行工程化改造，并引入两对光诱导异源二聚化蛋白ΔphyA/FHY1与Bphp1/PspR2，本研究成功实现了m6A效应因子的靶向招募。该系统显著提升了m6A的写入效率，可对SOX2、ACTB等特定转录本的m6A沉积与移除过程进行动态调控。值得注意的是，仅需对单个靶位点进行循环调控即可实现可逆的m6A编辑，证明该系统能够影响mRNA表达并调控人胚胎干细胞的分化状态。这套光遗传平台为循环式、可逆的m6A调控提供了精准且通用的工具，对解析RNA生物学及其在科研与医学领域的潜在应用具有广泛意义。本研究全面评估了HEK293T细胞中RNA m6A修饰的变化与一致性。利用m6A测序结果，我们分析了不同RNA类型间的差异基因。