In-vitro CRISPR/Cas9 system for genome editing of Aspergillus niger based on removable bidirectional selection marker AmdS.

In-vitro CRISPR/Cas9 system for genome editing of Aspergillus niger based on removable bidirectional selection marker AmdS.

Biotechnol Appl Biochem. 2020 Jul 30;:

Authors: Nan Y, Ouyang L, Chu J

Abstract
We established an in-vitro CRISPR (Clustered regularly interspaced short palindromic repeats)-associated RNA-guided DNA endonucleases (Cas9) system to efficiently produce specific genome editing in Aspergillus niger, using a novel recyclable, bidirectional selection marker gene amdS without the need of prior production of an amdS mutant. The donor DNA plasmid consisted of amdS open reading frame (ORF), promoter, terminator, and directional repeats (DRs) flanking sequences. It was co-transformed with recombinant nuclease Cas9 and the sgRNA, which targets to the pigment gene olvA of A. niger strain CBS513.88. The positive olive transformants, other than the wild type strain, were able to grow on the media containing acetamide as the sole nitrogen source and Cesium chloride (CsCl). Furthermore, culturing the transformants on media with fluoroacetamide and urea allowed a loop-out of the amdS expression cassette by recombining the flanking directional repeats. This study confirmed the facts that the endogenous amdS can be used as a dominant marker and that it can be removed by counter-selection in gene editing of A. niger. The proposed in-vitro CRISPR/Cas9 method offers a powerful tool for marker-free genetic manipulation of filamentous fungi industrial-specific strains. This article is protected by copyright. All rights reserved.

PMID: 32729961 [PubMed – as supplied by publisher]

Source: Industry