用于大豆疫霉的CRISPR/Cas9介导的基因敲除和原位互补系统

CRISPR/Cas9-Mediated Gene-Knockout and In Situ Complementation System for Phytophthora sojae.

作者信息

Qiu Min, Chen Nan, Wang Yuanchao

机构信息

Sanya Institute of Nanjing Agricultural University, Department of Plant Pathology, Nanjing Agricultural University, Nanjing, Jiangsu, China.

The Key Laboratory of Plant Immunity, Nanjing Agricultural University, Nanjing, Jiangsu, China.

出版信息

Methods Mol Biol. 2025;2892:69-82. doi: 10.1007/978-1-0716-4330-3_5.

DOI:10.1007/978-1-0716-4330-3_5

PMID:39729269

Abstract

The establishment of reliable and efficient systems for genome editing in Phytophthora is very important for studying gene functions. Here, step-by-step methods for CRISPR/Cas9-based gene knockout and in situ complementation for Phytophthora sojae are presented. These steps include the sgRNA design, Cas9-sgRNA plasmid construction, homologous replacement, complementation vector construction, P. sojae transformation, and detection of mutations for both gene knockout and in situ complementation. These methods may also potentially be adapted for other Phytophthora species.

摘要

建立可靠且高效的疫霉菌基因组编辑系统对于研究基因功能非常重要。本文介绍了基于CRISPR/Cas9的大豆疫霉菌基因敲除和原位互补的逐步方法。这些步骤包括sgRNA设计、Cas9-sgRNA质粒构建、同源替换、互补载体构建、大豆疫霉菌转化以及基因敲除和原位互补的突变检测。这些方法也可能适用于其他疫霉菌物种。