MicroRNAs (miRNAs) are 20‐24 nucleotides (nt) small RNAs functioning in eukaryotes. The length and sequence of miRNAs are not only related to the biogenesis of miRNAs but are also important for downstream physiological processes like ta‐siRNA production. To investigate these roles, it is informative to create small mutations within mature miRNA sequences. We used both TALENs (transcription activator‐like effector nucleases) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 (Cas9) to introduce heritable base pair mutations in mature miRNA sequences. For rice, TALEN constructs were built targeting five different mature miRNA sequences and yielding heritable mutations. Among the resulting mutants, mir390 mutant showed a severe defect in the shoot apical meristem (SAM), a shootless phenotype, which could be rescued by the wild‐type MIR390 . Small RNA sequencing showed the two base pair deletion in mir390 substantially interfered with miR390 biogenesis. In Arabidopsis, CRISPR/Cas9‐mediated editing of the miR160* strand confirmed that the asymmetric structure of miRNA is not a necessary determinant for secondary siRNA production. CRISPR/Cas9 with double‐guide RNAs successfully generated mir160a null mutants with fragment deletions, at a higher efficiency than a single‐guide RNA. The difference between the phenotypic severity of miR160a mutants in Col‐0 versus Ler backgrounds highlights a diverged role for miR160a in different ecotypes. Overall, we demonstrated that TALENs and CRISPR/Cas9 are both effective in modifying miRNA precursor structure, disrupting miRNA processing and generating miRNA null mutant plants.

中文翻译：

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TALENs和CRISPR / Cas9对miRNA序列的破坏会诱导产生不同长度的miRNA。

MicroRNA（miRNA）是在真核生物中起作用的20-24个核苷酸（nt）小RNA。miRNA的长度和序列不仅与miRNA的生物发生有关，而且对下游生理过程（如ta-siRNA生产）也很重要。为了研究这些作用，在成熟的miRNA序列中产生小的突变是有益的。我们同时使用了TALENs（转录激活因子样效应子核酸酶）和成簇的规则间隔的短回文重复序列（CRISPR）/ CRISPR相关蛋白9（Cas9）在成熟的miRNA序列中引入了可遗传的碱基对突变。对于水稻，构建了针对五种不同成熟miRNA序列并产生可遗传突变的TALEN构建体。在产生的突变体中，mir390该突变体显示出芽顶分生组织（SAM）的严重缺陷，即无芽表型，可以通过野生型MIR390挽救。小RNA测序表明，mir390中的两个碱基对缺失严重干扰了miR390的生物发生。在拟南芥中，CRISPR / Cas9介导的miR160 *链编辑证实了miRNA的不对称结构不是产生次级siRNA的必要决定因素。具有双向导RNA的CRISPR / Cas9成功产生了具有片段缺失的mir160a null突变体，其效率高于单向导RNA。miR160a表型严重程度之间的差异Col-0与Ler背景下的突变体突显了miR160a在不同生态类型中的作用不同。总体而言，我们证明TALENs和CRISPR / Cas9均可有效修饰miRNA前体结构，破坏miRNA加工并生成miRNA无效突变植物。