Efficient gene editing in with CRISPR technology.

BACKGROUND

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, .

FINDINGS

In this report, we demonstrate efficient gene replacement in a model filamentous fungus, , with the CRISPR/Cas9 system. We utilize Cas9 endonuclease and single crRNA:tracrRNA chimeric guide RNA (gRNA) to: (1) replace the endogenous promoter of - with the - promoter, and (2) introduce a codon optimized fire fly under the control of the - promoter at the - 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 - promoter driven - strain shows increased expression of cellulases, and -- reporter strain can be easily screened with a bioluminescence assay.

CONCLUSION

CRISPR/Cas9 system works efficiently in , 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.