Genome editing using preassembled CRISPR-Cas9 ribonucleoprotein complexes in Fusarium graminearum

PLoS One. 2022 Jun 3;17(6):e0268855. doi: 10.1371/journal.pone.0268855. eCollection 2022.

Abstract

Genome editing using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system has greatly facilitated the genetic analysis of fungal pathogens. The head blight fungus, Fusarium graminearum, causes destructive losses of economically important cereal crops. The recent development of the CRISPR-Cas9 system for use with F. graminearum has enabled more efficient genome editing. In this study, we described a CRISPR-Cas9-based genome-editing tool for the direct delivery of preassembled Cas9 ribonucleoproteins (RNPs) into the protoplasts of F. graminearum. The use of RNPs significantly increased both the number of transformants and percentage of transformants in which the target gene was successfully replaced with a selectable marker. We showed that a single double-strand DNA break mediated by the Cas9 ribonucleoprotein was sufficient for gene deletion. In addition, short-homology recombination required only 50 base pair regions flanking the target gene. The high efficiency of Cas9 RNPs enables large-scale functional analysis, the identification of essential genes, and gene deletion that is difficult with conventional methods. We expect that our approach will accelerate genetic studies of F. graminearum.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems / genetics
  • Fusarium* / genetics
  • Gene Editing* / methods
  • Ribonucleoproteins / genetics

Substances

  • Ribonucleoproteins

Supplementary concepts

  • Fusarium graminearum

Grants and funding

This work was supported by the National Research Foundation of Korea (Nos. 2021R1C1C1004200, 2021R1A2C1004673, and 2021R1I1A1A01040292) and Cooperative Research Program for Agriculture Science and Technology Development (No. PJ016243022021), Rural Development Administration, Republic of Korea. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.