title: A new variant of self-excising β-recombinase/six cassette for repetitive gene deletion and homokaryon purification in Neurospora crassa.
abstract: In a previous study, we developed a cassette employing a bacterial β-recombinase acting on six recognition sequences (β-rec/six), which allowed repetitive site-specific gene deletion and marker recycling in Neurospora crassa. However, only one positive selection marker was used in the cassette. A tedious subsequent procedure was needed to purify homokaryons due to the lack of a negative selection after cassette eviction. Additionally, the endoxylanase xylP promoter from Penicillium chrysogenum used in the construct was not strongly regulated in N. crassa, which led to low efficiency in cassette eviction. Herein we report an improved variant of the self-excising β-recombinase/six cassette for repetitive gene deletions in N. crassa using a native endoxylanase gh10-2 promoter from N. crassa, plus the introduction of a bidirectional selection marker to facilitate homokaryon selection using a thymidine kinase (tk) gene (negative selection) in addition to the phosphinothricin resistance gene (bar(r)) (positive selection).
authors.affiliation: Biological and Agricultural Engineering Department, University of California, Davis, One Shields Avenue, Davis, CA 95616, United States.
authors.forename: Edyta
authors.initials: E
authors.lastname: Szewczyk
authors.affiliation: Department of Plant Pathology, University of California, Davis, One Shields Avenue, Davis, CA 95616, United States; United States Department of Agriculture, Agricultural Research Service, Davis, CA 95616, United States.
authors.forename: Takao
authors.initials: T
authors.lastname: Kasuga
authors.affiliation: Biological and Agricultural Engineering Department, University of California, Davis, One Shields Avenue, Davis, CA 95616, United States. Electronic address: jzfan@ucdavis.edu.
authors.forename: Zhiliang
authors.initials: Z
authors.lastname: Fan
mesh_terms: D000613:Aminobutyrates / Q000494:pharmacology; D025141:Drug Resistance, Fungal; D055786:Gene Knockout Techniques / Q000379:methods*; D005827:Genetics, Microbial / Q000379:methods*; D016254:Mutagenesis, Insertional / Q000379:methods*; D009492:Neurospora crassa / Q000235:genetics*; D011401:Promoter Regions, Genetic; D045522:Recombinases / Q000378:metabolism*; D012641:Selection, Genetic; D013937:Thymidine Kinase / Q000235:genetics / Q000378:metabolism
chemical_list: D000613:Aminobutyrates; D045522:Recombinases; C003121:phosphinothricin; D013937:Thymidine Kinase
keywords: Endoxylanase gh10-2 inducible promoter; Marker recycling; Negative selection marker; Neurospora crassa; Repetitive gene deletion; Thymidine kinase