Liu Junqi, Gunapati Samatha, Mihelich Nicole T, Stec Adrian O, Michno Jean-Michel, Stupar Robert M
Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, MN, USA.
Methods Mol Biol. 2019;1917:217-234. doi: 10.1007/978-1-4939-8991-1_16.
CRISPR/Cas9 mediated genome editing technology has experienced rapid advances in recent years and has been applied to a wide variety of plant species, including soybean. Several platforms have been developed for designing and cloning of single CRISPR targets or multiple targets in a single destination vector. This chapter provides an updated working protocol for applying CRISPR/Cas9 technology to target a single gene or multiple genes simultaneously in soybean. We describe two platforms for cloning single CRISPR targets and multiplexing targets, respectively, and reagent delivery methodologies. The protocols address crucial limiting steps that can limit CRISPR editing in soybean hairy roots, composite plants, and tissue culture-based regenerated whole plants. To date, transgenic soybean plants with mutagenesis in up to three target genes have been obtained with this procedure.
近年来，CRISPR/Cas9介导的基因组编辑技术取得了快速进展，并已应用于包括大豆在内的多种植物物种。已经开发了几个平台，用于在单个目的载体中设计和克隆单个CRISPR靶点或多个靶点。本章提供了一种最新的工作方案，用于应用CRISPR/Cas9技术在大豆中同时靶向单个基因或多个基因。我们分别描述了用于克隆单个CRISPR靶点和多重靶点的两个平台以及试剂递送方法。该方案解决了可能限制大豆毛状根、复合植物和基于组织培养的再生整株植物中CRISPR编辑的关键限制步骤。迄今为止，通过该程序已获得了在多达三个靶基因中发生诱变的转基因大豆植株。
