Efficient gene editing in Neurospora crassa with CRISPR technology

Abstract

Efficient gene editing is a critical tool for investigating molecular mechanisms of cellular processes and engineering organisms for numerous purposes ranging from biotechnology to medicine. Recently developed RNA-guided CRISPR/Cas9 technology has been used for efficient gene editing in various organisms, but has not been tested in a model filamentous fungus, Neurospora crassa . In this report, we demonstrate efficient gene replacement in a model filamentous fungus, Neurospora crassa , with the CRISPR/Cas9 system. We utilize Cas9 endonuclease and single crRNA:tracrRNA chimeric guide RNA (gRNA) to: (1) replace the endogenous promoter of clr - 2 with the β - tubulin promoter, and (2) introduce a codon optimized fire fly luciferase under the control of the gsy - 1 promoter at the csr - 1 locus. CLR-2 is one of the core transcription factors that regulate the expression of cellulases, and GSY-1 regulates the conversion of glucose into glycogen. We show that the β - tubulin promoter driven clr - 2 strain shows increased expression of cellulases, and gsy - 1 - luciferase reporter strain can be easily screened with a bioluminescence assay. CRISPR/Cas9 system works efficiently in Neurospora crassa , which may be adapted to Neurospora natural isolates and other filamentous fungi. It will be beneficial for the filamentous fungal research community to take advantage of CRISPR/Cas9 tool kits that enable genetic perturbations including gene replacement and insertions.