Highly efficient single base editing in Aspergillus niger with CRISPR/Cas9 cytidine deaminase fusion

Abstract

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.

Keywords: Aspergillus niger; Base editing; CRISPR/Cas9; Cytidine deaminase.