通过有效的 CRISPR-Act3.0 系统靶向激活拟南芥基因。

Targeted Activation of Arabidopsis Genes by a Potent CRISPR-Act3.0 System.

机构信息

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.

出版信息

Methods Mol Biol. 2023;2698:27-40. doi: 10.1007/978-1-0716-3354-0_3.

DOI:10.1007/978-1-0716-3354-0_3

PMID:37682467

Abstract

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.

摘要

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。