MicroRNAs (miRNAs) play key roles in biological processes in plants, such as stress resistance, yet can hardly be quantified by an enzyme-involved terminal polymerization process due to their 2′-O-methyl modifications at the 3′-terminal. Herein, we proposed a CRISPR/Cas14a-based rolling circle amplification (termed Cas14R) assay, allowing reverse transcription-free and demethylation-free detection of plant miRNAs with single-nucleotide resolution. The employment of target-templated rolling circle amplification circumvents the extension of the unaccessible 2′-O-methyl group at the 3′-terminal. Particularly, the activated Cas14a confers the trans-cleavage activity for identifying target single-stranded DNA sequences without the necessity of the protospacer adjacent motif, generalizing the detection of miRNA sequences and the integration of different isothermal amplification techniques. Ultimately, the Cas14R assay has been applied to profile miR156a to evaluate the ripeness process of banana, indicating its feasibility in analyzing the roles of miRNAs in biological processes of plants.

中文翻译：

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基于 CRISPR/Cas14a 的等温扩增，用于分析植物微 RNA

MicroRNA (miRNA) 在植物的生物过程中发挥关键作用，例如抗逆性，但由于其3'-末端的 2'- O-甲基修饰，很难通过酶参与的末端聚合过程进行量化。在此，我们提出了一种CRISPR / Cas14基于- ř olling环扩增（称为Cas14R）测定法，允许植物miRNA的反转录和无脱甲基化检测用单核苷酸分辨率。目标模板滚环扩增的使用规避了不可访问的 2'- O的延伸3'-末端的-甲基。特别是，激活的 Cas14a 赋予了识别目标单链 DNA 序列的反式切割活性，无需原始间隔区相邻基序，推广了 miRNA 序列的检测和不同等温扩增技术的整合。最终，Cas14R 检测已被应用于配置 miR156a 以评估香蕉的成熟过程，表明其在分析 miRNA 在植物生物过程中的作用方面的可行性。