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

Min Qiu; Nan Chen; Yuanchao Wang

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

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

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

Authors

Min Qiu^{1

2

3}, Nan Chen¹, Yuanchao Wang^{4

5

6}

Affiliations

¹ Sanya Institute of Nanjing Agricultural University, Department of Plant Pathology, Nanjing Agricultural University, Nanjing, Jiangsu, China.
² The Key Laboratory of Plant Immunity, Nanjing Agricultural University, Nanjing, Jiangsu, China.
³ Key Laboratory of Soybean Disease and Pest Control (Ministry of Agriculture and Rural Affairs), Nanjing Agricultural University, Nanjing, Jiangsu, China.
⁴ Sanya Institute of Nanjing Agricultural University, Department of Plant Pathology, Nanjing Agricultural University, Nanjing, Jiangsu, China. [email protected].
⁵ The Key Laboratory of Plant Immunity, Nanjing Agricultural University, Nanjing, Jiangsu, China. [email protected].
⁶ Key Laboratory of Soybean Disease and Pest Control (Ministry of Agriculture and Rural Affairs), Nanjing Agricultural University, Nanjing, Jiangsu, China. [email protected].

PMID: 39729269
DOI: 10.1007/978-1-0716-4330-3_5

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.

Keywords: CRISPR/Cas9; Gene complementation; Gene editing; Gene knockout; Phytophthora sojae.

MeSH terms

CRISPR-Cas Systems*
Gene Editing* / methods
Gene Knockout Techniques* / methods
Genetic Complementation Test
Genetic Vectors / genetics
Phytophthora* / genetics
Plasmids / genetics
RNA, Guide, CRISPR-Cas Systems / genetics

Substances

RNA, Guide, CRISPR-Cas Systems