Abstract
Aspergillus niger and other filamentous fungi are widely used in industry, but efficient genetic engineering of these hosts remains nascent. For example, while molecular genetic tools have been developed, including CRISPR/Cas9, facile genome engineering of A. niger remains challenging. To address these challenges, we have developed a simple Cas9-based gene targeting method that provides selectable, iterative, and ultimately marker-free generation of genomic deletions and insertions. This method leverages locus-specific “pop-out” recombination to suppress off-target integrations. We demonstrated the effectiveness of this method by targeting the phenotypic marker albA and validated it by targeting the glaA and mstC loci. After two selection steps, we observed 100% gene editing efficiency across all three loci. This method greatly reduces the effort required to engineer the A. niger genome and overcomes low Cas9 transformations efficiency by eliminating the need for extensive screening. This method represents a significant addition to the A. niger genome engineering toolbox and could be adapted for use in other organisms. It is expected that this method will impact several areas of industrial biotechnology, such as the development of new strains for the secretion of heterologous enzymes and the discovery and optimization of metabolic pathways.
Highlights
The recombinant production of enzymes at high titers using various hosts, such as filamentous fungi, is an important aspect affecting costs for many commercial applications today, including pharmaceuticals [1], food processing [2], biofuels [3], and detergents
Our approach relies on the induction of a genomic double stranded breaks (DSBs) with a targetable Cas9/small guide RNA (sgRNA) complex, incorporation of a selectable marker via homologous recombination (HR), and selection of pyrG-containing mutants by culturing in the absence of uracil/uridine
We have designed and demonstrated a technique that efficiently edits the genome of A. niger based on CRISPR/Cas9
Summary
The recombinant production of enzymes at high titers using various hosts, such as filamentous fungi, is an important aspect affecting costs for many commercial applications today, including pharmaceuticals [1], food processing [2], biofuels [3], and detergents. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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