Gentzel Irene N, Park Chan Ho, Bellizzi Maria, Xiao Guiqing, Gadhave Kiran R, Murphree Colin, Yang Qin, LaMantia Jonathan, Redinbaugh Margaret G, Balint-Kurti Peter, Sit Tim L, Wang Guo-Liang
Department of Plant Pathology, The Ohio State University, 483B Kottman Hall, 2021 Coffey Road, Columbus, OH 43210 USA.
Department of Entomology and Plant Pathology, North Carolina State University, Raleigh, NC 27695 USA.
Plant Methods. 2020 Oct 2;16:133. doi: 10.1186/s13007-020-00675-5. eCollection 2020.
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.
成簇规律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。这种RNA引导的核酸酶不仅可用于产生精确的基因组突变，还可在作为失活蛋白（dCas9）存在时用于操纵基因表达。
在本研究中，我们描述了一个载体工具包，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）。本文介绍了一种改进的玉米原生质体分离和转染方法，以及用于增强或抑制玉米基因表达的dCas9载体的描述。
我们预计，这个玉米原生质体工具包将简化gRNA候选物的分析，并促进这种难以转化的植物中重要性状基因的遗传研究。
