Abstract
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome editing has become a promising approach for efficient and versatile genetic engineering in various organisms; however, simple and precise nucleotide modification methods in filamentous fungi have been restricted to double crossover type homologous recombination (HR). In this study, we developed a novel genome editing strategy via single crossover-mediated HR in the model filamentous fungus Pyricularia (Magnaporthe) oryzae. This method includes the CRISPR/Cas9 system and a donor vector harboring a single homology arm with point mutations at the CRISPR/Cas9 cleavage site. Using this strategy, we demonstrated highly efficient and freely programmable base substitutions within the desired genomic locus, and target gene disrupted mutants were also obtained via a shortened (100–1000 bp) single homology arm. We further demonstrated that this method allowed a one-step GFP gene knock-in at the C-terminus of the targeted gene. Since the genomic recombination does not require an intact protospacer-adjacent motif within the donor construct and any additional modifications of host components, this method can be used in various filamentous fungi for CRISPR/Cas9-based basic and applied biological analyses.
Highlights
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems derived from prokaryotic adaptive immune system have emerged as RNA-guided revolutionary genome editing technologies[1]
To validate whether the crossover type homologous recombination (HR) can be induced by targeted double-strand break (DSB) in the genome of P. oryzae, we further developed this system by modifying pRS-yellow fluorescent protein (YFP)::BSD, for detecting the crossover type HR
The bialaphos resistant gene cassette was inserted into the pRS-YFP::BSD vector, which resulted in pRS-YFP::BSD-bar (Fig. 1a)
Summary
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems derived from prokaryotic adaptive immune system have emerged as RNA-guided revolutionary genome editing technologies[1]. HR-mediated targeted gene modifications can induce more precise editing by adding homologous DNA templates; the construction of a target vector is often laborious, and its efficiency depends on the host and its DNA repair property. In addition to these two repair pathways, microhomology-mediated end joining (MMEJ) repair has been used for targeted gene disruptions and www.nature.com/scientificreports/. Pyricularia oryzae (Magnaporthe oryzae) is a model filamentous fungus, which causes destructive fungal diseases in rice This fungus comprises mononuclear haploid cells and has been established as a basic tool for the analysis of DSB repair and genome editing[13,21,22,23]. We established simplified and free genome editing strategies for targeted gene disruption, base substitution, and knock-in in the filamentous fungus
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
