Department of Plant Science and Landscape Architecture, University of Maryland, College Park, Maryland.
Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, Maryland.
Curr Protoc. 2022 Feb;2(2):e365. doi: 10.1002/cpz1.365.
CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein)-mediated genome editing has revolutionized fundamental research and plant breeding. Beyond gene editing, CRISPR/Cas systems have been repurposed as a platform for programmable transcriptional regulation. Catalytically inactive Cas variants (dCas), when fused with transcriptional activation domains, allow for specific activation of any target gene in the genome without inducing DNA double-strand breaks. CRISPR activation enables simultaneous activation of multiple genes, holding great promise in the identification of gene regulatory networks and rewiring of metabolic pathways. Here, we describe a simple protocol for constructing a dCas9-mediated multiplexed gene activation system based on the CRISPR-Act3.0 system. The resulting vectors are tested in rice protoplasts. © 2022 Wiley Periodicals LLC. Basic Protocol 1: sgRNA design and construction of CRISPR-Act3.0 vectors for multiplexed gene activation Basic Protocol 2: Determining the activation efficiency of CRISPR-Act3.0 vectors using rice protoplasts.
CRISPR/Cas（成簇规律间隔短回文重复/CRISPR 相关蛋白）介导的基因组编辑彻底改变了基础研究和植物育种。除了基因编辑外，CRISPR/Cas 系统还被重新用作可编程转录调控的平台。当无催化活性的 Cas 变体（dCas）与转录激活结构域融合时，可以在不诱导 DNA 双链断裂的情况下特异性激活基因组中的任何靶基因。CRISPR 激活可以同时激活多个基因，在鉴定基因调控网络和重排代谢途径方面具有巨大的应用前景。在这里，我们描述了一种基于 CRISPR-Act3.0 系统构建 dCas9 介导的多重基因激活系统的简单方案。所得载体在水稻原生质体中进行测试。© 2022 威利父子公司。基本方案 1：sgRNA 设计和基于 CRISPR-Act3.0 载体的多重基因激活构建基本方案 2：使用水稻原生质体确定 CRISPR-Act3.0 载体的激活效率。
