Efficient gene editing in Neurospora crassa with CRISPR technology

Abstract

BackgroundEfficient 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.FindingsIn 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.ConclusionCRISPR/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.Electronic supplementary materialThe online version of this article (doi:10.1186/s40694-015-0015-1) contains supplementary material, which is available to authorized users.

DNA Replication Is Required for Circadian Clock Function by Regulating Rhythmic Nucleosome Composition

“…The histone H2B is a major component of the nucleosome, and its level should reflect the amount of the DNA in cells. We created a reporter construct (H2B::LUC) in which the codon-optimized firefly luciferase (Gooch et al, 2008) was fused in-frame to the 3′ end of the endogenous Neurospora h2B locus by CRISPR-Cas9 genome editing (Matsu-ura et al, 2015). As shown in Figure 1A–B, the level of bioluminescence of the H2B::LUC strain exhibited a circadian rhythm in constant darkness (DD), suggesting that the amount of chromatin accumulated rhythmically in cells.…”

The circadian system as an organizer of metabolism

“…A filamentous fungus, Neurospora is originally best known for the one gene, one enzyme hypothesis (Beadle and Tatum, 1941). With the manifold similarities between the Neurospora and animal circadian systems, a fully sequenced and well annotated genome (Galagan et al, 2003), facile recombineering with 98% efficiency, functional genomics, a genome scale metabolic model, a high throughput knock out project (Colot et al, 2006; Dreyfuss et al, 2013; Dunlap et al, 2007), and the development of a CRISPR system (Matsu-ura et al, 2015), Neurospora has become one of the most tractable model organisms for the study of chronobiology at the level of the cell.…”

Genes Linked to Production of Secondary Metabolites in Talaromyces atroroseus Revealed Using CRISPR-Cas9

“…CRISPR-Cas9-based genetic engineering has recently been implemented in Aspergillus nidulans by us [4], and by others in several other species of filamentous fungi such as Trichoderma reesei [5], Neurospora crassa [6], Magnaporta oryzae [7], and Penicillium chrysogenum [8]. Our system for A .…”