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.
- Genome engineering
- Metabolic engineering
- Library synthesis
Bonde, M., Kosuri, S., Genee, H. J., Sarup-Lytzen, K., Church, G. M., Sommer, M., & Wang, H. H. (2015). Direct Mutagenesis of Thousands of Genomic Targets using Microarray-derived Oligonucleotides. A C S Synthetic Biology, 4(1), 17-22. https://doi.org/10.1021/sb5001565