Enhanced multiplex genome engineering through co-operative oligonucleotide co-selection

Nucleic Acids Res. 2012 Sep 1;40(17):e132. doi: 10.1093/nar/gks455. Epub 2012 May 25.


Genome-scale engineering of living organisms requires precise and economical methods to efficiently modify many loci within chromosomes. One such example is the directed integration of chemically synthesized single-stranded deoxyribonucleic acid (oligonucleotides) into the chromosome of Escherichia coli during replication. Herein, we present a general co-selection strategy in multiplex genome engineering that yields highly modified cells. We demonstrate that disparate sites throughout the genome can be easily modified simultaneously by leveraging selectable markers within 500 kb of the target sites. We apply this technique to the modification of 80 sites in the E. coli genome.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Chromosomes, Bacterial
  • Escherichia coli / genetics
  • Genetic Engineering / methods*
  • Genome, Bacterial
  • Oligonucleotides / chemistry*


  • Oligonucleotides