Bonde, Mads T.Kosuri, SriramGenee, Hans J.Sarup-Lytzen, KiraChurch, GeorgeSommer, Morten O.A.Wang, Harris H.2015-02-022014Bonde, Mads T., Sriram Kosuri, Hans J. Genee, Kira Sarup-Lytzen, George M. Church, Morten O.A. Sommer, and Harris H. Wang. 2014. “Direct Mutagenesis of Thousands of Genomic Targets Using Microarray-Derived Oligonucleotides.” ACS Synthetic Biology 4 (1): 17-22. doi:10.1021/sb5001565. http://dx.doi.org/10.1021/sb5001565.2161-5063http://nrs.harvard.edu/urn-3:HUL.InstRepos:13890740Multiplex 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.en-USgenome engineeringMAGEmetabolic engineeringmicroarraylibrary synthesisJournal Article2015-02-0210.1021/sb5001565