CRISPR-Cas12a-Based Diagnostics of Wheat Fungal Diseases

Abstract

Fusarium head blight (FHB) of wheat, mainly caused by <i>Fusarium graminearum</i> (<i>F. graminearum</i>) infection, reduces crop yield and contaminates grain with mycotoxins. We report a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a-based nucleic acid assay for an early and rapid diagnosis of wheat FHB. Guide RNA (gRNA) was screened for highly specific recognition of polymerase chain reaction (PCR) amplicon of the internal transcribed spacer (ITS) region and the transcription elongation factor 1α (<i>EF1α</i>) of <i>F. graminearum</i>. The trans-activation of Cas12a protein cleaves the single-stranded DNA probes with the terminal fluorophore and quencher groups, thus allowing us to report the presence of ITS and <i>EF1α</i> of <i>F. graminearum</i>. Owing to the dual recognition process through PCR primers and gRNA hybridization, the approach realized specific discrimination of <i>F. graminearum</i> from other pathogenic fungi. It also allowed us to detect as low as 1 fg/μL total DNA from <i>F. graminearum</i>, which is sufficient to diagnose a 4 day <i>F. graminearum</i> infection. CRISPR-Cas12a-based nucleic acid assay promises the molecular diagnosis of crop diseases and broadens the application of CRISPR tools.