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[优秀文章推荐]植物MicroRNA编码基因高效定向编辑及遗传表达分析
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Targeting rice endogenous microRNA genes with CRISPR-Cas9. 研究论文，对CRISPR-Cas9核酸酶在植物microRNA编码基因定向编辑高效实现、突变体准确鉴定、不同基因型编辑事件、遗传表型等关键问题进行了有效解读，为植物microRNA编码基因定向编辑提供了基础数据。 microRNA是一类普遍存在于真核生物中，长度约为19-24个核苷酸的非编码内源小分子RNA。植物microRNA主要分布在基因间隔区和内含子区，由RNA聚合酶II转录生成初级转录本microRNA（pri-miRNA），并经多步加工获得成熟microRNA分子，降解或翻译抑制对应靶基因mRNA，在植物生长发育、表观遗传、应对生物及非生物胁迫等方面起着重要作用。然而，microRNA相关研究工作中，基于RNAi的knock-down策略并不可行，而T-DNA插入突变体也十分有限，很大程度上限制了microRNA具体生物功能的研究。 近年来基于基因组编辑的植物目标基因定向敲除突变体创制工作已取得有效进展，但相对于广泛报道的蛋白编码基因，植物microRNA编码基因却因其功能单元长度有限（成熟microRNA长度主要为19-24bp）、编码区域内存在反向重复相似序列、相关microRNA存在高相似度冗余成员等序列、结构、数目上的复杂性，使得针对microRNA编码基因进行活性定向编辑核酸酶设计、活性评价、编辑事件检出及获得都存在一定困难。其特殊的的基因组结构特性，定向编辑工作开展相对有限。 近年来，电子科技大学植物基因组工程实验室聚焦植物基因组定向编辑开展了有效工作，基于前期开发的高效CRISPR-Cas9系统（Lowder et al., 2015, Plant Physiology; Tang et al., 2016, Molecular Plant）及SSCP定向编辑检测技术（Zheng et al., 2016, Plant Cell Reports），针对水稻microRNA编码基因（包括含高相似度冗余成员的microRNA家族）有效实现了定向编辑突变体材料创制、筛选、鉴定工作： 1、针对对水稻中OsMIR408、OsMIR528编码基因核心功能区实现了有效定向编辑，获得了修饰位点稳定遗传的传代材料（T0代定向敲除效率为48%-89%），明确了其表型及基因型关联关系。同时，高效CRISPR-Cas9配合高灵敏SSCP筛选的策略同样适于具有冗余家族成员的microRNA编码基因OsMIR815a/b/c、OsMIR820a/b/c基因家族不同成员的定向编辑，可有效创制microRNA家族冗余成员多基因聚合定向编辑材料。 SSCP is superior to RFLP on genotyping mutations at microRNA loci. a, RFLP (upper panel) and SSCP (lower panel) analysis of 10 independent OsMIR528-sgRNA02 T0 lines. Note PCR products were digested by SphI in the RFLP analysis. b, Sanger sequencing of the target site in the OsMIR528-sgRNA02 T0 lines. c, SSCP analysis of 6 independent OsMIR528-sgRNA01 T0 lines. Note 3 independent leaves of each T0 plant were genotyped. d, Sanger Sequencing of the target site in the OsMIR528-sgRNA01 T0 lines. 2、通过目标microRNA定向敲除材料基因型及表型关联分析，发现成熟microRNA区间的1-2bp缺失/插入突变并不影响其生物功能表现，只有发生3bp及以上DNA缺失（或插入）突变时，目标microRNA生物功能才受到明显影响。针对OsMIR528位点缺失突变体，进行microRNA-seq高通量分析，发现1bp插入突变基因组位点通过加工获得了一个有别于野生型的新OsMIR528成熟小RNA分子，可以行使与野生型microRNA分子类似的功能，为相关microRNA定向编辑及功能研究工作提供了有效借鉴。 Generation of functionally redundant new microRNAs with genome editing. a, 1-bp indels in mature OsMIR528 don’t abolish miRNA function. b, 3-bp deletion in mature OsMIR528 abolishes function. c, Illustration of pri-MIR528 produced in OsMIR528-sgRNA02-08 mutant lines. Mutagenized target regions are highlighted in green shade. d, Detection of a new microRNA, OsMIR528’, in the 1-bp insertion mutant line by RNA-seq. TPM, trans per million. 3、进一步针对不同OsMIR408、OsMIR528定向敲除基因型突变体进行microRNA-seq高通量测序，发现突变体microRNA转录组发生了显著变化（对应的mRNA-seq转录组分析也表现类似的变化），在一定程度上揭示了植物microRNA转录组表达的网络调控现象。 Targeted knocking out specific microRNAs results in global expression perturbation of microRNAs and their putative targets. a, targeted knock out of OsMIR408 and OsMIR528 results in global expression and perturbation of microRNAs. b,OsMIR408 and OsMIR528 induce each other as revealed by RNA-seq analysis. c, Knocking out OsMIR408 or OsMIR528 boosts expression of OsMIR397 and OsMIR398 as validated by qRT-PCR analysis. 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. |
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