Nature 460, 894-898 (13 August 2009) | doi:10.1038/nature08187; Received 6 March 2009; Accepted 29 May 2009; Published online 26 July 2009

Programming cells by multiplex genome engineering and accelerated evolution

Harris H. Wang1,2,3,8, Farren J. Isaacs1,8, Peter A. Carr4,5, Zachary Z. Sun6, George Xu6, Craig R. Forest7 & George M. Church1

  1. Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA
  2. Program in Biophysics, Harvard University, Cambridge, Massachusetts 02138, USA
  3. Program in Medical Engineering Medical Physics, Harvard-MIT Division of Health Sciences and Technology,
  4. The Center for Bits and Atoms,
  5. Media Lab, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA
  6. Harvard College, Cambridge, Massachusetts 02138, USA
  7. George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332, USA
  8. These authors contributed equally to this work.

Correspondence to: Harris H. Wang1,2,3,8Farren J. Isaacs1,8 Correspondence and requests for materials should be addressed to H.H.W. (Email: hhwang@genetics.med.harvard.edu) or F.J.I. (Email: farren@alumni.upenn.edu).

The breadth of genomic diversity found among organisms in nature allows populations to adapt to diverse environments1, 2. However, genomic diversity is difficult to generate in the laboratory and new phenotypes do not easily arise on practical timescales3. Although in vitro and directed evolution methods4, 5, 6, 7, 8, 9 have created genetic variants with usefully altered phenotypes, these methods are limited to laborious and serial manipulation of single genes and are not used for parallel and continuous directed evolution of gene networks or genomes. Here, we describe multiplex automated genome engineering (MAGE) for large-scale programming and evolution of cells. MAGE simultaneously targets many locations on the chromosome for modification in a single cell or across a population of cells, thus producing combinatorial genomic diversity. Because the process is cyclical and scalable, we constructed prototype devices that automate the MAGE technology to facilitate rapid and continuous generation of a diverse set of genetic changes (mismatches, insertions, deletions). We applied MAGE to optimize the 1-deoxy-d-xylulose-5-phosphate (DXP) biosynthesis pathway in Escherichia coli to overproduce the industrially important isoprenoid lycopene. Twenty-four genetic components in the DXP pathway were modified simultaneously using a complex pool of synthetic DNA, creating over 4.3 billion combinatorial genomic variants per day. We isolated variants with more than fivefold increase in lycopene production within 3 days, a significant improvement over existing metabolic engineering techniques. Our multiplex approach embraces engineering in the context of evolution by expediting the design and evolution of organisms with new and improved properties.