SummaryIn recent years, the CRISPR‐Cas9 nuclease has been used to knock out MicroRNA (miRNA) genes in plants, greatly promoting the study of miRNA function. However, due to its propensity for generating small insertions and deletions, Cas9 is not well‐suited for achieving a complete knockout of miRNA genes. By contrast, CRISPR‐Cas12a nuclease generates larger deletions, which could significantly disrupt the secondary structure of pre‐miRNA and prevent the production of mature miRNAs. Through the case study of OsMIR390 in rice, we confirmed that Cas12a is a more efficient tool than Cas9 in generating knockout mutants of a miRNA gene. To further demonstrate CRISPR‐Cas12a‐mediated knockout of miRNA genes in rice, we targeted nine OsMIRNA genes that have different spaciotemporal expression and have not been previously investigated via genetic knockout approaches. With CRISPR‐Cas12a, up to 100% genome editing efficiency was observed at these miRNA loci. The resulting larger deletions suggest Cas12a robustly generated null alleles of miRNA genes. Transcriptome profiling of the miRNA mutants, as well as phenotypic analysis of the rice grains revealed the function of these miRNAs in controlling gene expression and regulating grain quality and seed development. This study established CRISPR‐Cas12a as an efficient tool for genetic knockout of miRNA genes in plants.

中文翻译：

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一种高效的 CRISPR-Cas12a 介导的植物 MicroRNA 敲除策略


摘要近年来，CRISPR-Cas9核酸酶被用于敲除植物体内的MicroRNA（miRNA）基因，极大地促进了miRNA功能的研究。然而，由于其产生小插入和缺失的倾向，Cas9 不太适合实现 miRNA 基因的完全敲除。相比之下，CRISPR-Cas12a 核酸酶产生更大的缺失，这可能会显著破坏 pre-miRNA 的二级结构并阻止成熟 miRNA 的产生。通过水稻中 OsMIR390 的案例研究，我们证实 Cas12a 在产生 miRNA 基因敲除突变体方面是比 Cas9 更有效的工具。为了进一步证明 CRISPR-Cas12a 介导的水稻中 miRNA 基因敲除，我们靶向了 9 个 OsMIRNA 基因，这些基因具有不同的空间时间表达，以前没有通过基因敲除方法进行过研究。使用 CRISPR-Cas12a，在这些 miRNA 位点观察到高达 100% 的基因组编辑效率。由此产生的较大缺失表明 Cas12a 稳健地生成了 miRNA 基因的无效等位基因。miRNA 突变体的转录组分析以及稻粒的表型分析揭示了这些 miRNA 在控制基因表达和调节谷物品质和种子发育方面的功能。本研究确定了 CRISPR-Cas12a 是植物中 miRNA 基因基因敲除的有效工具。