CRISPR-Cas9 is an emerging genome editing tool for reverse genetics in plants. However, its application for functional study of non-coding RNAs in plants is still at its infancy. Despite being a major class of non-coding RNAs, the biological roles of circle RNAs (circRNAs) remain largely unknown in plants. Previous plant circRNA studies have focused on identification and annotation of putative circRNAs, with their functions largely uninvestigated by genetic approaches. Here, we applied a multiplexed CRISPR-Cas9 strategy to efficiently acquire individual null mutants for four circRNAs in rice. We showed each of these rice circRNA loci (Os02circ25329, Os06circ02797, Os03circ00204 and Os05circ02465) can be deleted at 10% or higher efficiency in both protoplasts and stable transgenic T0 lines. Such high efficiency deletion enabled the generation of circRNA null allele plants without the CRISPR-Cas9 transgene in the T1 generation. Characterization of the mutants reveals these circRNAs’ participation in salt stress response during seed germination and in particular the Os05circ02465 null mutant showed high salt tolerance. Notably, the seedlings of the Os06circ02797 mutant showed rapid growth phenotype after seed germination with the seedlings containing higher chlorophyll A/B content. Further molecular and computational analyses suggested a circRNA–miRNA–mRNA regulatory network where Os06circ02797 functions to bind and sequester OsMIR408, an important and conserved microRNA in plants. This study not only presents genetic evidence for the first time in plants that certain circRNAs may serve as sponges to negatively regulate miRNAs, a phenomenon previously demonstrated in mammalian cells, but also provides important insights for improving agronomic traits through gene editing of circRNA loci in crops.

中文翻译：

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通过 CRISPR-Cas9 有效删除多个环状 RNA 基因座，揭示了 Os06circ02797 是水稻中 OsMIR408 的假定海绵


CRISPR-Cas9 是一种新兴的植物反向遗传学基因组编辑工具。然而，其在植物非编码RNA功能研究中的应用仍处于起步阶段。尽管环状 RNA (circRNA) 是一类主要的非编码 RNA，但其在植物中的生物学作用仍然很大程度上未知。之前的植物 circRNA 研究主要集中在假定的 circRNA 的识别和注释上，其功能基本上没有通过遗传方法进行研究。在这里，我们应用多重 CRISPR-Cas9 策略来有效获取水稻中四种 circRNA 的单个无效突变体。我们证明，这些水稻 circRNA 位点（ Os02circ25329 、 Os06circ02797 、 Os03circ00204和Os05circ02465 ）在原生质体和稳定转基因 T0 系中都可以以 10% 或更高的效率删除。这种高效的缺失使得能够在 T1 代中产生没有 CRISPR-Cas9 转基因的 circRNA 无效等位基因植物。突变体的表征揭示了这些 circRNA 在种子萌发过程中参与盐胁迫反应，特别是Os05circ02465无效突变体表现出高耐盐性。值得注意的是， Os06circ02797突变体的幼苗在种子萌发后表现出快速生长表型，并且幼苗含有较高的叶绿素A/B含量。进一步的分子和计算分析表明，存在一个 circRNA-miRNA-mRNA 调控网络，其中Os06circ02797 的功能是结合和隔离OsMIR408 ，OsMIR408 是植物中重要且保守的 microRNA。 这项研究不仅首次在植物中提供了遗传证据，表明某些 circRNA 可能充当海绵负向调节 miRNA（这种现象之前在哺乳动物细胞中得到证实），而且还为通过对作物中的 circRNA 位点进行基因编辑来改善农艺性状提供了重要的见解。 。