title: CRISPR/Cas9 Based Genome Editing of Penicillium chrysogenum.
abstract: CRISPR/Cas9 based systems have emerged as versatile platforms for precision genome editing in a wide range of organisms. Here we have developed powerful CRISPR/Cas9 tools for marker-based and marker-free genome modifications in Penicillium chrysogenum, a model filamentous fungus and industrially relevant cell factory. The developed CRISPR/Cas9 toolbox is highly flexible and allows editing of new targets with minimal cloning efforts. The Cas9 protein and the sgRNA can be either delivered during transformation, as preassembled CRISPR-Cas9 ribonucleoproteins (RNPs) or expressed from an AMA1 based plasmid within the cell. The direct delivery of the Cas9 protein with in vitro synthesized sgRNA to the cells allows for a transient method for genome engineering that may rapidly be applicable for other filamentous fungi. The expression of Cas9 from an AMA1 based vector was shown to be highly efficient for marker-free gene deletions.
authors.affiliation: Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen , Nijenborgh 7, 9747 AG, Groningen, The Netherlands.
authors.forename: C
authors.initials: C
authors.lastname: Pohl
authors.affiliation: Synthetic Biology and Cell Engineering, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen , Nijenborgh 7, 9747 AG, Groningen, The Netherlands.
authors.forename: J A K W
authors.initials: JA
authors.lastname: Kiel
authors.affiliation: Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen , Nijenborgh 7, 9747 AG, Groningen, The Netherlands.
authors.forename: A J M
authors.initials: AJ
authors.lastname: Driessen
authors.affiliation: Synthetic Biology and Cell Engineering, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen , Nijenborgh 7, 9747 AG, Groningen, The Netherlands.|DSM Biotechnology Centre , Alexander Fleminglaan 1, 2613 AX, Delft, The Netherlands.
authors.forename: R A L
authors.initials: RA
authors.lastname: Bovenberg
authors.affiliation: Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen , Nijenborgh 7, 9747 AG, Groningen, The Netherlands.|DSM Biotechnology Centre , Alexander Fleminglaan 1, 2613 AX, Delft, The Netherlands.
authors.forename: Y
authors.initials: Y
authors.lastname: Nygård
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D000076987:CRISPR-Associated Protein 9; D064113:CRISPR-Cas Systems; D004260:DNA Repair; D004720:Endonucleases / Q000235:genetics; D017353:Gene Deletion; D000072669:Gene Editing / Q000379:methods*; D018390:Gene Targeting / Q000379:methods; D005819:Genetic Markers; D005822:Genetic Vectors; D016681:Genome, Fungal; D009841:Oligonucleotides / Q000235:genetics; D010408:Penicillium chrysogenum / Q000235:genetics*; D000094704:RNA, Guide, CRISPR-Cas Systems
chemical_list: D001426:Bacterial Proteins; D005819:Genetic Markers; D009841:Oligonucleotides; D000094704:RNA, Guide, CRISPR-Cas Systems; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases
keywords: CRISPR/Cas9; Penicillium chrysogenum; RNP; genome editing; marker-free gene deletion