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

Farren J. Isaacs; Peter A. Carr; Harris H. Wang; Marc J. Lajoie; Bram Sterling; Laurens Kraal; Andrew C. Tolonen; Tara A. Gianoulis; Daniel B. Goodman; Nikos B. Reppas; Christopher J. Emig; Duhee Bang; Samuel J. Hwang; Michael C. Jewett; Joseph M. Jacobson; George M. Church

doi:10.1126/science.1205822

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

Farren J. Isaacs¹,*,†‡,
Peter A. Carr²,³,*,‡§,
Harris H. Wang¹,⁴,⁵,⁶,*,
Marc J. Lajoie¹,⁷,
Bram Sterling²,³,
Laurens Kraal¹,
Andrew C. Tolonen¹,
Tara A. Gianoulis¹,⁶,
Daniel B. Goodman¹,⁵,
Nikos B. Reppas⁸,
Christopher J. Emig⁹,
Duhee Bang¹⁰,
Samuel J. Hwang¹¹,
Michael C. Jewett¹,¹²,
Joseph M. Jacobson²,³,
George M. Church¹,⁶

¹Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
²Center for Bits and Atoms, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
³MIT Media Lab, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
⁴Program in Biophysics, Harvard University, Cambridge, MA 02138, USA.
⁵Program in Medical Engineering and Medical Physics, Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA 02139, USA.
⁶Wyss Institute, Harvard University, Cambridge, MA 02115, USA.
⁷Program in Chemical Biology, Harvard University, Cambridge, MA 02138, USA.
⁸Joule Unlimited, Cambridge, MA 02139, USA.
⁹Department of Bioengineering, Stanford University, Palo Alto, CA 94305, USA.
¹⁰Department of Chemistry, Yonsei University, Shinchon 134, Seoul 120-749, Korea.
¹¹Department of Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
¹²Department of Chemical and Biological Engineering and Chemistry of Life Processes Institute, Northwestern University, Evanston, IL 60208, USA.

↵‡To whom correspondence should be addressed. E-mail: farren.isaacs{at}yale.edu (F.J.I.); carr{at}mit.edu (P.A.C.)

↵* 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.

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Science 15 Jul 2011:
Vol. 333, Issue 6040, pp. 348-353
DOI: 10.1126/science.1205822

Farren J. Isaacs

Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.

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For correspondence: farren.isaacs@yale.edu carr@mit.edu

Peter A. Carr

Center for Bits and Atoms, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.MIT Media Lab, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

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For correspondence: farren.isaacs@yale.edu carr@mit.edu

Harris H. Wang

Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.Program in Biophysics, Harvard University, Cambridge, MA 02138, USA.Program in Medical Engineering and Medical Physics, Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA 02139, USA.Wyss Institute, Harvard University, Cambridge, MA 02115, USA.

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Marc J. Lajoie

Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.Program in Chemical Biology, Harvard University, Cambridge, MA 02138, USA.

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Bram Sterling

Center for Bits and Atoms, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.MIT Media Lab, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

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Laurens Kraal

Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.

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Andrew C. Tolonen

Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.

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Tara A. Gianoulis

Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.Wyss Institute, Harvard University, Cambridge, MA 02115, USA.

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Daniel B. Goodman

Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.Program in Medical Engineering and Medical Physics, Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA 02139, USA.

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Nikos B. Reppas

Joule Unlimited, Cambridge, MA 02139, USA.

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Christopher J. Emig

Department of Bioengineering, Stanford University, Palo Alto, CA 94305, USA.

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Duhee Bang

Department of Chemistry, Yonsei University, Shinchon 134, Seoul 120-749, Korea.

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Samuel J. Hwang

Department of Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

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Michael C. Jewett

Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.Department of Chemical and Biological Engineering and Chemistry of Life Processes Institute, Northwestern University, Evanston, IL 60208, USA.

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Joseph M. Jacobson

Center for Bits and Atoms, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.MIT Media Lab, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

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George M. Church

Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.Wyss Institute, Harvard University, Cambridge, MA 02115, USA.

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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.

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

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