title: Direct mutagenesis of thousands of genomic targets using microarray-derived oligonucleotides.
abstract: Multiplex Automated Genome Engineering (MAGE) allows simultaneous mutagenesis of multiple target sites in bacterial genomes using short oligonucleotides. However, large-scale mutagenesis requires hundreds to thousands of unique oligos, which are costly to synthesize and impossible to scale-up by traditional phosphoramidite column-based approaches. Here, we describe a novel method to amplify oligos from microarray chips for direct use in MAGE to perturb thousands of genomic sites simultaneously. We demonstrated the feasibility of large-scale mutagenesis by inserting T7 promoters upstream of 2585 operons in E. coli using this method, which we call Microarray-Oligonucleotide (MO)-MAGE. The resulting mutant library was characterized by high-throughput sequencing to show that all attempted insertions were estimated to have occurred at an average frequency of 0.02% per locus with 0.4 average insertions per cell. MO-MAGE enables cost-effective large-scale targeted genome engineering that should be useful for a variety of applications in synthetic biology and metabolic engineering.
authors.affiliation: Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , DK-2970 Hørsholm, Denmark.
authors.forename: Mads T
authors.initials: MT
authors.lastname: Bonde
authors.forename: Sriram
authors.initials: S
authors.lastname: Kosuri
authors.forename: Hans J
authors.initials: HJ
authors.lastname: Genee
authors.forename: Kira
authors.initials: K
authors.lastname: Sarup-Lytzen
authors.forename: George M
authors.initials: GM
authors.lastname: Church
authors.forename: Morten O A
authors.initials: MO
authors.lastname: Sommer
authors.forename: Harris H
authors.initials: HH
authors.lastname: Wang
mesh_terms: D017123:Bacteriophage T7 / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D005818:Genetic Engineering / Q000379:methods*; D016680:Genome, Bacterial; D060847:Metabolic Engineering; D016296:Mutagenesis; D020411:Oligonucleotide Array Sequence Analysis; D009876:Operon; D011401:Promoter Regions, Genetic; D058615:Synthetic Biology
keywords: MAGE; genome engineering; library synthesis; metabolic engineering; microarray