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

Report

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.

See allHide authors and affiliations

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

Farren J. Isaacs

1Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.

Find this author on Google Scholar
Find this author on PubMed
Search for this author on this site
For correspondence: farren.isaacs@yale.edu carr@mit.edu

Peter A. Carr

2Center for Bits and Atoms, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

3MIT Media Lab, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

Find this author on Google Scholar
Find this author on PubMed
Search for this author on this site
For correspondence: farren.isaacs@yale.edu carr@mit.edu

Harris H. Wang

1Department of Genetics, Harvard Medical School, Boston, MA 02115, 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.

Find this author on Google Scholar
Find this author on PubMed
Search for this author on this site

Marc J. Lajoie

1Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.

7Program in Chemical Biology, Harvard University, Cambridge, MA 02138, USA.

Find this author on Google Scholar
Find this author on PubMed
Search for this author on this site

Bram Sterling

2Center for Bits and Atoms, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

3MIT Media Lab, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

Find this author on Google Scholar
Find this author on PubMed
Search for this author on this site

Laurens Kraal

1Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.

Find this author on Google Scholar
Find this author on PubMed
Search for this author on this site

Andrew C. Tolonen

1Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.

Find this author on Google Scholar
Find this author on PubMed
Search for this author on this site

Tara A. Gianoulis

1Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.

6Wyss Institute, Harvard University, Cambridge, MA 02115, USA.

Find this author on Google Scholar
Find this author on PubMed
Search for this author on this site

Daniel B. Goodman

1Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.

5Program in Medical Engineering and Medical Physics, Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA 02139, USA.

Find this author on Google Scholar
Find this author on PubMed
Search for this author on this site

Nikos B. Reppas

8Joule Unlimited, Cambridge, MA 02139, USA.

Find this author on Google Scholar
Find this author on PubMed
Search for this author on this site

Christopher J. Emig

9Department of Bioengineering, Stanford University, Palo Alto, CA 94305, USA.

Find this author on Google Scholar
Find this author on PubMed
Search for this author on this site

Duhee Bang

10Department of Chemistry, Yonsei University, Shinchon 134, Seoul 120-749, Korea.

Find this author on Google Scholar
Find this author on PubMed
Search for this author on this site

Samuel J. Hwang

11Department of Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

Find this author on Google Scholar
Find this author on PubMed
Search for this author on this site

Michael C. Jewett

1Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.

12Department of Chemical and Biological Engineering and Chemistry of Life Processes Institute, Northwestern University, Evanston, IL 60208, USA.

Find this author on Google Scholar
Find this author on PubMed
Search for this author on this site

Joseph M. Jacobson

2Center for Bits and Atoms, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

3MIT Media Lab, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

Find this author on Google Scholar
Find this author on PubMed
Search for this author on this site

George M. Church

1Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.

6Wyss Institute, Harvard University, Cambridge, MA 02115, USA.

Find this author on Google Scholar
Find this author on PubMed
Search for this author on this site

Article
Figures & Data
Info & Metrics
eLetters
PDF

You are currently viewing the abstract.

View Full Text

Log in to view the full text

via AAAS login

AAAS login provides access to Science for AAAS members, and access to other journals in the Science family to users who have purchased individual subscriptions.

Become an AAAS Member
Activate your Account
Purchase Access to Other Journals in the Science Family
Account Help

As a service to the community, this article is available for free. Existing users log in.

Become a AAAS member
Account help

Log in through your institution

More options

Purchase digital access to this article

Download and print this article for your personal scholarly, research, and educational use.

Purchase this issue in print

Buy a single issue of Science for just $15 USD.

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.

View Full Text

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

Abstract

Science

Article Tools