title: I-SceI-mediated scarless gene modification via allelic exchange in Clostridium.
abstract: Although gene disruption in Clostridium spp. with the TargeTron technology is much more effective than single-crossover integration, it cannot achieve gene modification via allelic exchange. Here, we developed a targeted, nonpolar, scarless gene modification system based on the I-SceI endonuclease. First, a replicative plasmid containing homology arms on either side of the target sequence and I-SceI recognition sites was integrated into the Clostridium chromosome, resulting in single-crossover integrants containing a mutant allele. Second, the cells were transformed with plasmids containing the synthetic gene (sceC) encoding the I-SceI enzyme, resulting in double-stranded breaks at the I-SceI recognition sites, which stimulated homologous recombination and yielded double-crossover mutants. Application of the method was demonstrated by deleting two genes (adc and glcG) from C. acetobutylicum ATCC 824 and one gene (adc) from C. beijerinckii NCIMB 8052, and by introducing point mutations into xylR of C. beijerinckii NCIMB 8052. The double-crossover mutants displayed similar fermentation phenotypes to those constructed with the TargeTron technology.
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China; Graduate University of the Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Ning
authors.initials: N
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
authors.forename: Lijun
authors.initials: L
authors.lastname: Shao
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China; Shanghai Research and Development Center of Industrial Biotechnology, Shanghai 201201, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Jiang
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Gu
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China; Graduate University of the Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Qi
authors.initials: Q
authors.lastname: Li
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China; Graduate University of the Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Jinle
authors.initials: J
authors.lastname: Liu
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China; Shanghai Collaborative Innovation Center for Biomanufacturing Technology, Shanghai 200237, China.
authors.forename: Weihong
authors.initials: W
authors.lastname: Jiang
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China; Shanghai Research and Development Center of Industrial Biotechnology, Shanghai 201201, China; Shanghai Collaborative Innovation Center for Biomanufacturing Technology, Shanghai 200237, China. Electronic address: syang@sibs.ac.cn.
authors.forename: Sheng
authors.initials: S
authors.lastname: Yang
mesh_terms: D000483:Alleles; D001426:Bacterial Proteins / Q000235:genetics*; D003013:Clostridium / Q000235:genetics*; D015253:Deoxyribonucleases, Type I Site-Specific / Q000378:metabolism*; D005818:Genetic Engineering / Q000379:methods*; D059765:Homologous Recombination; D009154:Mutation; D010957:Plasmids / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D015253:Deoxyribonucleases, Type I Site-Specific
keywords: Clostridium; Gene modification; Homologous recombination; I-SceI