The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system has become a powerful tool for functional genomics in plants. The RNA-guided nuclease can be used to not only generate precise genomic mutations, but also to manipulate gene expression when present as a deactivated protein (dCas9). In this study, we describe a vector toolkit for analyzing dCas9-mediated activation (CRISPRa) or inactivation (CRISPRi) of gene expression in maize protoplasts. An improved maize protoplast isolation and transfection method is presented, as well as a description of dCas9 vectors to enhance or repress maize gene expression. We anticipate that this maize protoplast toolkit will streamline the analysis of gRNA candidates and facilitate genetic studies of important trait genes in this transformation-recalcitrant plant.

中文翻译：

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用于玉米基因功能分析的 CRISPR/dCas9 工具包

成簇规则间隔短回文重复序列 (CRISPR)/Cas9 系统已成为植物功能基因组学的强大工具。RNA 引导的核酸酶不仅可用于产生精确的基因组突变，而且当以失活蛋白 (dCas9) 形式存在时还可操纵基因表达。在本研究中，我们描述了一种用于分析玉米原生质体中 dCas9 介导的基因表达激活 (CRISPRa) 或失活 (CRISPRi) 的载体工具包。提出了一种改进的玉米原生质体分离和转染方法，以及增强或抑制玉米基因表达的 dCas9 载体的描述。我们预计该玉米原生质体工具包将简化 gRNA 候选物的分析，并促进这种转化顽抗植物中重要性状基因的遗传研究。