Development of Marker-free CRISPR/Cas9 System in Filamentous Fungi

Abstract

The filamentous fungus of Penicillium species emerges as a promising producer of cellulolytic enzymes that are essential for lignocellulosic biomass degradation. However, efficient genome editing and manipulations are limited due to the restricted availability of selection markers. This study aims to overcome this limitation by developing a marker-free CRISPR/Cas9-based genome editing tool to knock out the existing selection markers, hence, enhancing cellulase production through targeted genetic manipulations. Earlier in the laboratory, the carbon catabolite repressor gene, mig1 (or creA), was deleted in P. funiculosum and P. marneffei to enhance cellulase production. Additionally, the genes for different cellulases, such as cellobiohydrolase I and lytic polysaccharide mono-oxygenase, were over-expressed to enhance biomass degradation. In the current study, the strategy utilized focuses on the deletion of the pyrG gene to enable auxotrophic selection and subsequent deletion of existing antibiotic resistance markers by simultaneously complementing the pyrG. Using the CRISPR/Cas9 system, we deleted the pyrG gene in P. funiculosum and P. marneffei efficiently without the need for antibiotic selection. Subsequent complementation of pyrG using an episomal plasmid system restored the uridine auxotrophy, allowing for further genetic manipulations. This genome editing was confirmed via PCR screening and sequencing, thus representing the efficiency of the developed system. After genome editing, the episomal plasmid was subsequently cured to make the system free from any marker. This study provides a marker-free CRISPR/Cas9 system for efficient genetic manipulation of the cellulolytic fungi, thus allowing for strain improvement to enhance enzyme production.