CRISPR/Cas9 Based Genome Editing of Penicillium chrysogenum
Journal article, 2016
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.
Oligonucleotides/genetics
*genome editing
RNA
Genetic Markers
Fungal
Genome
*CRISPR-Cas Systems
*marker-free gene deletion
Penicillium chrysogenum/*genetics
Genetic Vectors
*Penicillium chrysogenum
Gene Editing/*methods
Gene Deletion
*Rnp
Bacterial Proteins/genetics
*CRISPR/Cas9
Gene Targeting/methods
Guide
Endonucleases/genetics
DNA Repair
Author
C. Pohl
University of Groningen
J. A. Kiel
University of Groningen
A. J. Driessen
University of Groningen
R. A. Bovenberg
DSM Biotechnology Centre
University of Groningen
Yvonne Nygård
University of Groningen
ACS Synthetic Biology
2161-5063 (eISSN)
Vol. 5 7 754-64Subject Categories
Other Industrial Biotechnology
DOI
10.1021/acssynbio.6b00082