Department of Plant Science and Landscape Architecture, University of Maryland, College Park, MD, USA.
Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD, USA.
Methods Mol Biol. 2023;2698:27-40. doi: 10.1007/978-1-0716-3354-0_3.
The CRISPR/Cas system has emerged as a versatile platform for sequence-specific genome engineering in plants. Beyond genome editing, CRISPR/Cas systems, based on nuclease-deficient Cas9 (dCas9), have been repurposed as an RNA-guided platform for transcriptional regulation. CRISPR activation (CRISPRa) represents a novel gain-of-function (GOF) strategy, conferring robust over-expression of the target gene within its native chromosomal context. The CRISPRa systems enable precise, scalable, and robust RNA-guided transcription activation, holding great potential for a variety of fundamental and translational research. In this chapter, we provide a step-by-step guide for efficient gene activation in Arabidopsis based on a highly robust CRISPRa system, CRISPR-Act3.0. We present detailed procedures on the sgRNA design, CRISPR-Act3.0 system construction, Agrobacterium-mediated transformation of Arabidopsis using the floral dip method, and identification of desired transgenic plants.
CRISPR/Cas 系统已成为植物中用于特定基因组工程的多功能平台。除了基因组编辑，基于核酸酶缺陷型 Cas9（dCas9）的 CRISPR/Cas 系统已被重新用作 RNA 引导的转录调控平台。CRISPR 激活（CRISPRa）代表一种新颖的功能获得（GOF）策略，在其天然染色体背景下赋予靶基因的强大过表达。CRISPRa 系统能够实现精确、可扩展和强大的 RNA 引导转录激活，为各种基础研究和转化研究带来了巨大的潜力。在本章中，我们基于高度稳健的 CRISPRa 系统 CRISPR-Act3.0，提供了在拟南芥中进行高效基因激活的分步指南。我们介绍了 sgRNA 设计、CRISPR-Act3.0 系统构建、使用花浸法进行拟南芥的农杆菌介导转化，以及鉴定所需的转基因植物的详细步骤。
