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Science
Vol. 333 no. 6040 pp. 348-353
DOI: 10.1126/science.1205822
  • Report

Precise Manipulation of Chromosomes in Vivo Enables Genome-Wide Codon Replacement

  1. George M. Church1,6
  1. 1Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
  2. 2Center for Bits and Atoms, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
  3. 3MIT Media Lab, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
  4. 4Program in Biophysics, Harvard University, Cambridge, MA 02138, USA.
  5. 5Program in Medical Engineering and Medical Physics, Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA 02139, USA.
  6. 6Wyss Institute, Harvard University, Cambridge, MA 02115, USA.
  7. 7Program in Chemical Biology, Harvard University, Cambridge, MA 02138, USA.
  8. 8Joule Unlimited, Cambridge, MA 02139, USA.
  9. 9Department of Bioengineering, Stanford University, Palo Alto, CA 94305, USA.
  10. 10Department of Chemistry, Yonsei University, Shinchon 134, Seoul 120-749, Korea.
  11. 11Department of Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
  12. 12Department of Chemical and Biological Engineering and Chemistry of Life Processes Institute, Northwestern University, Evanston, IL 60208, USA.
  1. ↵‡To whom correspondence should be addressed. E-mail: farren.isaacs{at}yale.edu (F.J.I.); carr{at}mit.edu (P.A.C.)

  1. ↵* These authors contributed equally to this work.

  • ↵† Present address: Department of Molecular, Cellular and Developmental Biology, Systems Biology Institute, Yale University, New Haven, CT 06520, USA.

  • ↵§ Present address: Lincoln Laboratory, Massachusetts Institute of Technology, Lexington, MA 02420, USA.

Abstract

We present genome engineering technologies that are capable of fundamentally reengineering genomes from the nucleotide to the megabase scale. We used multiplex automated genome engineering (MAGE) to site-specifically replace all 314 TAG stop codons with synonymous TAA codons in parallel across 32 Escherichia coli strains. This approach allowed us to measure individual recombination frequencies, confirm viability for each modification, and identify associated phenotypes. We developed hierarchical conjugative assembly genome engineering (CAGE) to merge these sets of codon modifications into genomes with 80 precise changes, which demonstrate that these synonymous codon substitutions can be combined into higher-order strains without synthetic lethal effects. Our methods treat the chromosome as both an editable and an evolvable template, permitting the exploration of vast genetic landscapes.

  • Received for publication 18 March 2011.
  • Accepted for publication 20 May 2011.
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