title: I-SceI enzyme mediated integration (SEMI) for fast and efficient gene targeting in Trichoderma reesei.
abstract: We previously showed that creation of a double strand DNA break (DSB) by expressing I-SceI in an engineered Trichoderma reesei (Hypocrea jecorina) strain containing a I-SceI recognition site improved transformation and homologous integration efficiencies. In this study, we further improved homologous integration frequencies by combining I-SceI mediated double strand break with disruption of the tku70 gene. The inability of the tku70 mutant to repair a I-SceI mediated DSB via NHEJ was used to force integration of an expression cassette with homologous flanks surrounding the DSB site. Besides expressing I-SceI from a plasmid, we also show that adding I-SceI enzyme during transformation was successful to generate DSBs. The I-SceI enzyme mediated integration, or SEMI, in combination with a Δtku70 mutant has a synergistic effect on homologous recombination efficiencies as 90-100% of the transformants exhibited integration of the expression cassette at the homologous site.
authors: Molecular Microbiology and Biotechnology, Institute of Biology Leiden, Leiden University, Kluyver Centre for Genomics of Industrial Fermentation, Sylviusweg 72, 2333 BE Leiden, The Netherlands.
authors: Jean Paul
authors: JP
authors: Ouedraogo
authors: Molecular Microbiology and Biotechnology, Institute of Biology Leiden, Leiden University, Kluyver Centre for Genomics of Industrial Fermentation, Sylviusweg 72, 2333 BE Leiden, The Netherlands.
authors: Mark
authors: M
authors: Arentshorst
authors: Dupont Industrial Biosciences, Archimedesweg 30, 2333 CN Leiden, The Netherlands.
authors: Igor
authors: I
authors: Nikolaev
authors: Dupont Industrial Biosciences, Archimedesweg 30, 2333 CN Leiden, The Netherlands.
authors: Sharief
authors: S
authors: Barends
authors: Molecular Microbiology and Biotechnology, Institute of Biology Leiden, Leiden University, Kluyver Centre for Genomics of Industrial Fermentation, Sylviusweg 72, 2333 BE Leiden, The Netherlands. Electronic address: A.F.J.Ram@biology.leidenuniv.nl.
authors: Arthur F J
authors: AF
authors: Ram
mesh_terms: D003001:Cloning, Molecular; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics*; D005656:Fungal Proteins / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics*
chemical_list: D005656:Fungal Proteins; D015252:Deoxyribonucleases, Type II Site-Specific
keywords: Ade1; Double strand break; Homologous integration; Hypocrea jecorina; Non-homologous-end-joining