CRISPR-Cas9 Based Genome Editing Reveals New Insights into MicroRNA Function and Regulation in Rice.
CRISPR-Cas9 Based Genome Editing Reveals New Insights into MicroRNA Function and Regulation in Rice.
复制标题
基于 CRISPR-Cas9 的基因组编辑揭示了水稻 MicroRNA 功能和调控的新见解
DOI：
10.3389/fpls.2017.01598
复制
发表时间：
2017
影响因子：
5.6
通讯作者：
Zhang Y
中科院分区：
文献类型：
--
作者：
Zhou J;Deng K;Cheng Y;Zhong Z;Tian L;Tang X;Tang A;Zheng X;Zhang T;Qi Y;Zhang Y
microRNAs（miRNAs）是一类在植物发育和逆境应答中起重要作用的非编码小分子RNA。由于缺乏合适的敲除工具，miRNA基因的功能丧失分析传统上具有挑战性。在本研究中，CRISPR-Cas9靶向水稻中的单个miRNA基因（OsMIR 408和OsMIR 528）和miRNA基因家族（miR 815 a/B/c和miR 820 a/B/c）。我们发现单链构象多态性（SSCP）是一种比限制性片段长度多态性（RFLP）更可靠的方法，用于鉴定CRISPR-Cas9产生的突变体。在所有测试的miRNA靶位点，再生的T0系中靶向诱变的频率范围为48%至89%。在miRNA 528的情况下，三个独立的向导RNA（gRNA）在确认的突变株系中均产生双等位基因突变。当两种gRNA同时作用时，T0水稻株系中的miRNA基因很容易被缺失，缺失频率高达60%。因此，我们证明了CRISPR-Cas9是敲除植物miRNA的有效工具。成熟miRNA区域中的单碱基对（bp）插入/缺失突变（indels）可导致功能冗余的miRNA的产生。发现成熟miRNA或互补miRNA* 处的大缺失容易废除miRNA功能。利用OsMIR 408和OsMIR 528的突变体，我们发现敲除单个miRNA可以导致许多其他看似无关的miRNA表达谱的改变。在对OsMIR 528的案例研究中，我们发现它是盐胁迫下的正调节因子。我们的工作不仅为CRISPR-Cas9靶向miRNA提供了经验指导，还为水稻中miRNA的功能和复杂的交叉调控带来了新的见解。
MicroRNAs (miRNAs) are small non-coding RNAs that play important roles in plant development and stress responses. Loss-of-function analysis of miRNA genes has been traditionally challenging due to lack of appropriate knockout tools. In this study, single miRNA genes (OsMIR408 and OsMIR528) and miRNA gene families (miR815a/b/c and miR820a/b/c) in rice were targeted by CRISPR-Cas9. We showed single strand conformation polymorphism (SSCP) is a more reliable method than restriction fragment length polymorphism (RFLP) for identifying CRISPR-Cas9 generated mutants. Frequencies of targeted mutagenesis among regenerated T0 lines ranged from 48 to 89% at all tested miRNA target sites. In the case of miRNA528, three independent guide RNAs (gRNAs) all generated biallelic mutations among confirmed mutant lines. When targeted by two gRNAs, miRNA genes were readily to be deleted at a frequency up to 60% in T0 rice lines. Thus, we demonstrate CRISPR-Cas9 is an effective tool for knocking out plant miRNAs. Single-base pair (bp) insertion/deletion mutations (indels) in mature miRNA regions can lead to the generation of functionally redundant miRNAs. Large deletions at either the mature miRNA or the complementary miRNA* were found to readily abolish miRNA function. Utilizing mutants of OsMIR408 and OsMIR528, we find that knocking out a single miRNA can result in expression profile changes of many other seemingly unrelated miRNAs. In a case study on OsMIR528, we reveal it is a positive regulator in salt stress. Our work not only provides empirical guidelines on targeting miRNAs with CRISPR-Cas9, but also brings new insights into miRNA function and complex cross-regulation in rice.
登录
查看更多内容
影响因子：
3.7
作者：
Fahlgren N;Howell MD;Kasschau KD;Chapman EJ;Sullivan CM;Cumbie JS;Givan SA;Law TF;Grant SR;Dangl JL;Carrington JC
通讯作者：
Carrington JC
影响因子：
4.6
作者：
Romualdi, C;Bortoluzzi, S;Danieli, GA
通讯作者：
Danieli, GA
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle
影响因子：
11.6
作者：
Guo, Changkui;Xu, Yunmin;Wu, Gang
通讯作者：
Wu, Gang
用于多重植物基因组编辑和转录调控的 CRISPR/Cas9 工具箱
影响因子：
7.4
作者：
Lowder, Levi G.;Zhang, Dengwei;Qi, Yiping
通讯作者：
Qi, Yiping