Journal
BIOTECHNOLOGY AND BIOENGINEERING
Volume 112, Issue 12, Pages 2543-2549Publisher
WILEY
DOI: 10.1002/bit.25662
Keywords
engineered nuclease; CRISPR; Cas system; genome editing; gene targeting; filamentous fungi; rice blast fungus
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Funding
- Japan Society for the Promotion of Science (JSPS) [24.11224]
- [15K18647]
- Grants-in-Aid for Scientific Research [15K18647] Funding Source: KAKEN
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CRISPR/Cas-derived RNA-guided nucleases (RGNs) that can generate DNA double-strand breaks (DSBs) at a specific sequence are widely used for targeted genome editing by induction of DSB repair in many organisms. The CRISPR/Cas system consists of two components: a single Cas9 nuclease and a single-guide RNA (sgRNA). Therefore, the system for constructing RGNs is simple and efficient, but the utilization of RGNs in filamentous fungi has not been validated. In this study, we established the CRISPR/Cas system in the model filamentous fungus, Pyricularia oryzae, using Cas9 that was codon-optimized for filamentous fungi, and the endogenous RNA polymerase (RNAP) III U6 promoter and a RNAP II fungal promoter for the expression of the sgRNA. We further demonstrated that RGNs could recognize the desired sequences and edit endogenous genes through homologous recombination-mediated targeted gene replacement with high efficiency. Our system will open the way for the development of various CRISPR/Cas-based applications in filamentous fungi. Biotechnol. Bioeng. 2015;112: 2543-2549. (c) 2015 Wiley Periodicals, Inc.
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