Marker-Free Crispr/Cas9 Based Genome Editing of Penicillium Chrysogenum

CRISPR/Cas9 systems have emerged as versatile platforms for precise genome editing in a wide range of organisms. CRISPR/Cas9 tools offer high flexibility in target selection and the possibility to introduce genetic alterations without the introduction of epitopic sequences. These tools may in the future replace marker-based gene editing in filamentous fungi.

Here, we describe CRISPR/Cas9 tools for rapid genome engineering of Penicillium chrysogenum. We have developed powerful CRISPR/Cas9 tools for marker-based and marker-free gene modifications in P. chrysogenum, a model filamentous fungi but also an industrially relevant cell factory. The developed CRISPR/Cas9 toolbox is highly flexible and allows editing of new targets with minimal cloning efforts.

We have used the CRISPR/Cas9 tools for metabolic engineering of P. chrysogenum, for improved secondary metabolite (SM) production. We use a modular cloning approach for building novel fungal expression cassettes and complete pathways in Escherichia coli and we have created a library of genetic parts (including promoters, activators, transcription factors, terminators, fluorescent reporters and integration sites) for rapid and standardized assembly of genetic constructs and deletion cassettes.

CRISPR/Cas9 based technologies are expected to significantly advance the research and exploitation of novel natural products and the development of P. chrysogenum as an industrial chassis organism. A major obstacle in the exploration of the SM clusters in filamentous fungi has been the lack of efficient genome editing tools, therefore the CRISPR/Cas9 technology is expected to become a major driver in exploitation of fungal diversity.