Lowder Levi G, Malzahn Aimee, Qi Yiping
Department of Biology, East Carolina University, Howell Science Complex, Greenville, NC, 27858, USA.
Department of Plant Science and Landscape Architecture, University of Maryland, College Park, MD, 20742, USA.
Methods Mol Biol. 2018;1676:197-214. doi: 10.1007/978-1-4939-7315-6_12.
Besides genome editing, the CRISPR-Cas9-based platform provides a new way of engineering artificial transcription factors (ATFs). Multiplex of guide RNA (gRNA) expression cassettes holds a great promise for many useful applications of CRISPR-Cas9. In this chapter, we provide a detailed protocol for building advanced multiplexed CRISPR-dCas9-Activator/repressor T-DNA vectors for carrying out transcriptional activation or repression experiments in plants. We specifically describe the assembly of multiplex T-DNA vectors that can express multiple gRNAs to activate a silenced gene, or to repress two independent miRNA genes simultaneously in Arabidopsis. We then describe a "higher-order" vector assembly method for increased multiplexing capacity. This higher-order assembly method in principle allows swift stacking of gRNAs cassettes that are only limited by the loading capacity of a cloning or expression vector.
除了基因组编辑外，基于CRISPR-Cas9的平台还为工程化人工转录因子（ATF）提供了一种新方法。多重引导RNA（gRNA）表达盒在CRISPR-Cas9的许多有用应用中具有巨大潜力。在本章中，我们提供了一个详细的方案，用于构建先进的多重CRISPR-dCas9-激活子/抑制子T-DNA载体，以在植物中进行转录激活或抑制实验。我们具体描述了多重T-DNA载体的组装，该载体可以表达多个gRNA来激活一个沉默基因，或在拟南芥中同时抑制两个独立的miRNA基因。然后，我们描述了一种用于提高多重能力的“高阶”载体组装方法。这种高阶组装方法原则上允许快速堆叠gRNA盒，而这仅受克隆或表达载体的装载能力限制。
