Direct Mutagenesis of Thousands of Genomic Targets using Microarray-derived Oligonucleotides

Mads Bonde, Sriram Kosuri, Hans Jasper Genee, Kira Sarup-Lytzen, George M. Church, Morten Sommer, Harris H. Wang

Research output: Contribution to journalJournal articleResearchpeer-review

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 loci 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.
Original languageEnglish
JournalA C S Synthetic Biology
Volume4
Issue number1
Pages (from-to)17-22
Number of pages6
ISSN2161-5063
DOIs
Publication statusPublished - 2015

Keywords

  • Genome engineering
  • MAGE
  • Metabolic engineering
  • Microarray
  • Library synthesis

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