Programming cells by multiplex genome engineering and accelerated evolution

By Harris H. Wang, Farren J. Isaacs, P. Carr, Zachary Z. Sun, G. Xu, C. Forest, George M. Church. Nature, 2009

1,521 citations recorded by the source corpus.

The breadth of genomic diversity found among organisms in nature allows populations to adapt to diverse environments. However, genomic diversity is difficult to generate in the laboratory and new phenotypes do not easily arise on practical timescales. Although in vitro and directed evolution methods 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.

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/5bfa218cf08f39c4f956f8fe330f9d41fcc0b76d

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Citations in the corpus

Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. (2013, methodology): “coli MG1655 strain was transformed with a temperature-sensitive plasmid pKD46 that contained l-Red recombination proteins (Exo, Beta, and Gam) (Wang et al., 2009).”
Adaptive laboratory evolution – principles and applications for biotechnology (2013, neutral): “coli and accelerate evolution [180].”
Sustainable life support on Mars – the potential roles of cyanobacteria (2015, methodology): “2002) and multiplex genome engineering (Wang et al. 2009).”
Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System (2015, methodology): “The recombination efficiency might be improved by using ssDNA as the donor DNA, as -Red-like proteins also facilitate the recombination of smaller ssDNA fragments, such as those used in MAGE.”
Synthetic biology: applications come of age (2010, methodology): “In an alternative method of relieving regulatory control over the large number of DXP pathway components, Wang et al.121 diversified and, as a result, optimized the native DXP biosynthetic pathway in E. coli (see the figure, part a).”