Classic genome editing tools including ZFN， TALEN， and CRISPR/Cas9 rely on DNA double-strand breaks for genome editing. To prevent the potential hazard caused by double-strand breaks (DSBs)， a series of single base editing tools that convert cytidine (C) to thymine (T) without DSBs have been developed extensively in multiple species. Herein， we report for the first time that C was converted to T with a high frequency in the filamentous fungi Aspergillus niger by fusing cytidine deaminase and Cas9 nickase. Using the CRISPR/Cas9-dependent base editor and inducing nonsense mutations via single base editing， we inactivated the uridine auxotroph gene pyrG and the pigment gene fwnA with an efficiency of 47.... More
Classic genome editing tools including ZFN， TALEN， and CRISPR/Cas9 rely on DNA double-strand breaks for genome editing. To prevent the potential hazard caused by double-strand breaks (DSBs)， a series of single base editing tools that convert cytidine (C) to thymine (T) without DSBs have been developed extensively in multiple species. Herein， we report for the first time that C was converted to T with a high frequency in the filamentous fungi Aspergillus niger by fusing cytidine deaminase and Cas9 nickase. Using the CRISPR/Cas9-dependent base editor and inducing nonsense mutations via single base editing， we inactivated the uridine auxotroph gene pyrG and the pigment gene fwnA with an efficiency of 47.36%-100% in A.niger. At the same time， the single-base editing results of the non-phenotypic gene prtT showed an efficiency of 60%. The editable window reached 8 bases (from C2 to C9 in the protospacer) in A. niger. Overall， we successfully constructed a single base editing system in A. niger. This system provides a more convenient tool for investigating gene function in A. niger， and provides a new tool for genetic modification in filamentous fungi.
