Direct Mutagenesis of Thousands of Genomic Targets Using Microarray-Derived Oligonucleotides

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.