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 widespread cis-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-free uorf mutant 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-free uorf mutants, and finally comparing the mRNA, protein and phenotypic levels of target genes in uorf mutants 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.

基因表达受多个过程的调节，将 mRNA 翻译成蛋白质是一个特别关键的步骤。上游开放阅读框 (uORF) 是真核基因中普遍存在的顺式元件，通常抑制下游初级 ORF (pORF) 的翻译。在这里，我们描述了一种通过使用 CRISPR–Cas9 系统编辑内源 uORF 来微调植物基因翻译的协议。我们提出的方法很容易产生无转基因的uorf突变后代。我们提供了用于预测 uORF 并使用双荧光素酶报告系统测试其对下游 pORF 的影响、设计和构建单向导 RNA (sgRNA)–Cas9 载体、识别无转基因uorf的详细方案突变体，最后比较uorf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，并且可以在 4 个月内获得具有编辑目标 uORF 控制不同水平 pORF 翻译的无转基因突变体。与以前的方法不同，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善。