Tailor‐made CRISPR/Cas system for highly efficient targeted gene replacement in the rice blast fungus

TLDR

CRISPR/Cas RNA‑guided nucleases create sequence‑specific DNA double‑strand breaks for genome editing, and the system—comprising Cas9 and a single‑guide RNA—is simple and efficient, yet its use in filamentous fungi has not been validated. The study aims to establish a CRISPR/Cas system in *Pyricularia oryzae* and to enable high‑efficiency targeted gene replacement, thereby opening avenues for CRISPR/Cas applications in filamentous fungi. They engineered a codon‑optimized Cas9 and used the endogenous RNAP III U6 promoter and a RNAP II fungal promoter to express sgRNA in *Pyricularia oryzae*. The system successfully recognized target sequences and achieved high‑efficiency homologous recombination‑mediated gene replacement. Biotechnol.

Abstract

ABSTRACT 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. © 2015 Wiley Periodicals, Inc.