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Multiplexed tracking of combinatorial genomic mutations in engineered cell populations.
Zeitoun, Ramsey I; Garst, Andrew D; Degen, George D; Pines, Gur; Mansell, Thomas J; Glebes, Tirzah Y; Boyle, Nanette R; Gill, Ryan T.
Afiliación
  • Zeitoun RI; Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, Colorado, USA.
  • Garst AD; Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, Colorado, USA.
  • Degen GD; Department of Chemical Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania, USA.
  • Pines G; Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, Colorado, USA.
  • Mansell TJ; Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, Colorado, USA.
  • Glebes TY; Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, Colorado, USA.
  • Boyle NR; Department of Chemical and Biological Engineering, Colorado School of Mines, Golden, Colorado, USA.
  • Gill RT; Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, Colorado, USA.
Nat Biotechnol ; 33(6): 631-7, 2015 Jun.
Article en En | MEDLINE | ID: mdl-25798935
Multiplexed genome engineering approaches can be used to generate targeted genetic diversity in cell populations on laboratory timescales, but methods to track mutations and link them to phenotypes have been lacking. We present an approach for tracking combinatorial engineered libraries (TRACE) through the simultaneous mapping of millions of combinatorially engineered genomes at single-cell resolution. Distal genomic sites are assembled into individual DNA constructs that are compatible with next-generation sequencing strategies. We used TRACE to map growth selection dynamics for Escherichia coli combinatorial libraries created by recursive multiplex recombineering at a depth 10(4)-fold greater than before. TRACE was used to identify genotype-to-phenotype correlations and to map the evolutionary trajectory of two individual combinatorial mutants in E. coli. Combinatorial mutations in the human ES2 ovarian carcinoma cell line were also assessed with TRACE. TRACE completes the combinatorial engineering cycle and enables more sophisticated approaches to genome engineering in both bacteria and eukaryotic cells than are currently possible.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Variación Genética / Ingeniería Genética / Escherichia coli / Mutación Límite: Humans Idioma: En Revista: Nat Biotechnol Asunto de la revista: BIOTECNOLOGIA Año: 2015 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Estados Unidos
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Variación Genética / Ingeniería Genética / Escherichia coli / Mutación Límite: Humans Idioma: En Revista: Nat Biotechnol Asunto de la revista: BIOTECNOLOGIA Año: 2015 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Estados Unidos