Targeted Gene Disruption Via CRISPR/Cas9 Ribonucleoprotein Complexes in Fusarium oxysporum

Ambika Pokhrel; Seungyeon Seo; Qiang Wang; Jeffrey J Coleman

doi:10.1007/978-1-0716-1795-3_7

Targeted Gene Disruption Via CRISPR/Cas9 Ribonucleoprotein Complexes in Fusarium oxysporum

Methods Mol Biol. 2022:2391:75-87. doi: 10.1007/978-1-0716-1795-3_7.

Authors

Ambika Pokhrel¹, Seungyeon Seo¹, Qiang Wang², Jeffrey J Coleman^#³

Affiliations

¹ Department of Entomology and Plant Pathology, Auburn University, Auburn, AL, USA.
² State Key Laboratory of Crop Stress Biology for Arid Areas and College of Plant Protection, Northwest A&F University, Yangling, Shaanxi, China.
³ Department of Entomology and Plant Pathology, Auburn University, Auburn, AL, USA. jjcoleman@auburn.edu.

^# Contributed equally.

PMID: 34686978
DOI: 10.1007/978-1-0716-1795-3_7

Abstract

Characterization of a gene of interest frequently relies on generation of a mutant as a critical component. Transformation to disrupt a gene has been previously accomplished by several methods in Fusarium oxysporum. Here we provide a detailed method to generate a gene mutation mediated by a CRISPR/Cas9 ribonucleoprotein (RNP) complex. The Cas9 RNP cleaves the DNA at the target site, and during DNA repair integration of a dominant selectable marker is incorporated via homologous recombination generating the desired gene disruption.

Keywords: CRISPR; Cas9; Homologous recombination; Mutation.

Publication types

Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

CRISPR-Cas Systems* / genetics
Fungal Proteins
Fusarium* / genetics
Fusarium* / metabolism
Homologous Recombination
Ribonucleoproteins / genetics
Ribonucleoproteins / metabolism

Substances

Fungal Proteins
Ribonucleoproteins