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

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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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Journal

Journal ID (nlm-journal-id): 0404511

Journal ID (pubmed-jr-id): 7473

Journal ID (nlm-ta): Science

Journal ID (iso-abbrev): Science

Title: Science (New York, N.Y.)

ISSN (Print): 0036-8075

ISSN (Electronic): 1095-9203

Publication date Nihms-submitted: 29 June 2016

Publication date (Print): 15 July 2011

Publication date PMC-release: 15 June 2017

Volume: 333

Issue: 6040

Pages: 348-353

Affiliations

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

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

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

[4 ]Program in Biophysics, Harvard University, Cambridge, MA 02138, USA

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

[6 ]Wyss Institute, Harvard University, Cambridge, MA 02115, USA

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

[8 ]Joule Unlimited, Cambridge, MA 02139, USA

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

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

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

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

Author notes

[‡ ]To whom correspondence should be addressed. E-mail: farren.isaacs@ 123456yale.edu (F.J.I.); carr@ 123456mit.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.

Article

Accession ID: PMC5472332 Pmcid ID: PMC5472332 Pmc-uid ID: 5472332 Manuscript ID: nihpa606570

DOI: 10.1126/science.1205822

PMC ID: 5472332

PubMed ID: 21764749

SO-VID: 9570a308-3bb6-4b25-a047-dfd12cab4f51

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