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

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.

Fig 1. Subcellular localization of Cas9-eGFP protein in F. graminearum.

GFP fluorescence was colocalized with histone H1-RFP in protoplasts. The results indicated that SV40 NLS is functional for the nuclear localization of Cas9 protein in this fungus. Scale bars, 20 μm.

Fig 2. Schematic representation of the in vitro cleavage assay of preassembled Cas9 RNP.

(a) Schematic representation of sgRNA design for in vitro cleavage assay and preassembled Cas9 RNP-mediated transformation. The two sgRNAs were designed to cleave two sites, S1 (49–71 bp) and S2 (6433–6455 bp), respectively. The protospacer adjacent motif sequence is required for Cas9 to recognize the target site. (b) In vitro cleavage assay. The results showed the activity of preassembled Cas9 RNP.

Fig 3. Construction of deletion mutants by Cas9 RNP-mediated transformation with different homology arm sizes.

(a) Schematic representation of PKS12 deletion. Three constructs with different homology arm sizes (1 kb, 270 bp, and 50 bp) were generated for PKS12 deletion. (b) Colony morphologies of F. graminearum strains generated during Cas9 RNA-mediated transformation. (c) Each randomly chosen plate of disrupted phenotypes was counted as transformants in regeneration media. Expected phenotypes were later calculated as frequencies.

Fig 4. An identical deletion strategy was applied to FGSG_04274 to demonstrate the replication of deletion efficiencies via RNP complexes.

(a) Schematic representation of sgRNA design for preassembled Cas9 RNP-mediated transformation. The two sgRNAs were designed to cleave two sites, S3 (18–40 bp) and S4 (1093–1115 bp). (b) Deletion mutants were identified by the dark red color and abnormal shapes of the colonies.