Manipulating gene translation in plants by CRISPR–Cas9-mediated genome editing of upstream open reading frames
Manipulating gene translation in plants by CRISPR–Cas9-mediated genome editing of upstream open reading frames
复制标题
DOI：
10.1038/s41596-019-0238-3
复制
发表时间：
2020-01
期刊：
影响因子：
14.8
通讯作者：
Xiaomin Si;Huawei Zhang;Yanpeng Wang;Kunling Chen;Caixia Gao
中科院分区：
文献类型：
--
作者：
Xiaomin Si;Huawei Zhang;Yanpeng Wang;Kunling Chen;Caixia Gao
基因表达受多个过程调控，mRNA翻译成蛋白质是一个特别关键的步骤。上游开放阅读框（uORFs）是真核生物基因中的一个上游顺式元件，通常抑制下游一级ORFs（pORFs）的翻译。在这里，我们描述了一种通过用CRISPR-Cas9系统编辑内源性uORF来微调植物中基因翻译的方案。我们提出的方法很容易产生转基因无突变后代。我们提供了使用双荧光素酶报告系统预测uORF并测试其对下游pORF的影响的详细方案，设计和构建单向导RNA（sgRNA）-Cas9载体，鉴定转基因无突变体，并最终比较突变体和对照中靶基因的mRNA、蛋白质和表型水平。预测uORF并确认其在原生质体中的作用仅需2-3周，4个月内即可获得具有控制不同pORF翻译水平的编辑靶uORF的无转基因突变体。与以往的方法不同，我们的策略实现了对无转基因衍生物中基因翻译的微调，从而加速了基因功能的分析和作物性状的改良。
Gene expression is regulated by multiple processes, and the translation of mRNAs into proteins is an especially critical step. Upstream open reading frames (uORFs) are widespreadcis-elements in eukaryotic genes that usually suppress the translation of downstream primary ORFs (pORFs). Here, we describe a protocol for fine-tuning gene translation in plants by editing endogenous uORFs with the CRISPR–Cas9 system. The method we present readily yields transgene-freeuorfmutant offspring. We provide detailed protocols for predicting uORFs and testing their effects on downstream pORFs using a dual-luciferase reporter system, designing and constructing single guide RNA (sgRNA)–Cas9 vectors, identifying transgene-freeuorfmutants, and finally comparing the mRNA, protein and phenotypic levels of target genes inuorfmutants and controls. Predicting uORFs and confirming their effects in protoplasts takes only 2–3 weeks, and transgene-free mutants with edited target uORFs controlling different levels of pORF translation can be obtained within 4 months. Unlike previous methods, our strategy achieves fine-tuning of gene translation in transgene-free derivatives, which accelerates the analysis of gene function and the improvement of crop traits.