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Programming cells by multiplex genome engineering and accelerated evolution
Open accessAbstract
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.
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“(c) epistasis is a major factor for adaptive optimization of gene regulation [89,157].”
From Adaptive laboratory evolution—Principles and applications for biotechnology (2013) · cites this paper as [157]
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“coli mutator strains,(4) the co-opting of B cells for somatic hypermutation of exogenous genes,(5) recombination-mediated library mutagenesis by transformation,(6) a phage-based system for continuous mutagenesis,(7) and a Multiplex Automated Genome Engineering technology (MAGE) have all been reported.(8) None of these…”
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“We thank the National Institutes of Health (GM077596), Roy J. Carver Charitable Trust (13-4257), and Carl R. Woese Institute for Genomic Biology at the University of Illinois at Urbana-Champaign for financial support in our development and application of genome engineering technologies.”
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TALENs-Assisted Multiplex Editing for Accelerated Genome Evolution To Improve Yeast Phenotypes
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Self-citationNatural reassignment of CUU and CUA sense codons to alanine in Ashbya mitochondria
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“Enabled by genome engineering technologies, such as multiplex automated genome engineering (6), conjugative assembly genome engineering (7) and de novo genome synthesis (8), editing and rewriting the genetic code have emerged as an exciting topic in synthetic biology.”
BackgroundSelf-citationSynthetic Biology for Space Exploration: Promises and Societal Implications
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“evolution of organisms with new and improved properties (27).”
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“…a considerable combinatorial challenge for complex multigenic traits, and, thus, application of artificial selection has yet been limited to single proteins or pathways with photometric readouts (Arnold, 1993; Wang et al , 2009; Lee et al , 2013; Chen et al , 2018; van Tatenhove-Pel et al , 2020).”
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“The short length and context-independence of TISs make them particularly useful for engineering regulatory branch points of cellular metabolism, similar to earlier reports from MAGE-derived replacement of SD sequences in bacteria (60).”
BackgroundHighly Combinatorial Genetic Interaction Analysis Reveals a Multi-Drug Transporter Influence Network
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“coli and yeast, methods have been designed to combinatorially modify multiple loci in a population of cells (DiCarlo et al., 2013; Wang et al., 2009), and these may be extended to allow largescale strain isolation and phenotyping (Zeitoun et al.”
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Self-citationHost Organisms:Bacillus subtilis
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Design Automation for Synthetic Biological Systems
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“‘‘Systems’’ can then be created by composing Devices.”
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“The pORTMAGE system, which allows an efficient directed genome editing in E. coli (27), was used to introduce into the naive ancestor strains the mutations fixed in the genes maeA and lpd during the evolution experiments.”
MethodInfluentialEvolutionary repair reveals an unexpected role of the tRNA modification m1G37 in aminoacylation
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A Single-Component Blue Light-Induced System Based on EL222 in Yarrowia lipolytica
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Metadata
- Authors
- Wang, H. H., Isaacs, F. J., Carr, P. A., Sun, Z. Z., Xu, G., Forest, C. R., & Church, G. M.
- Affiliations
- Harvard University (US)
- MIT Media Lab (US)
- Georgia Institute of Technology (US)
- Corresponding author
- Wang, Harris H.
- Isaacs, Farren J.
- ORCID
- Venue
- Nature
- Published
- 2009-07-26
- Volume
- 460
- Issue
- 7257
- Pages
- 894-898
- ISSN
- 0028-0836 (ISSN-L)
- 1476-4687
- PMC
- PMC4590770
- OpenAlex
- W2144779133
- Cited by
- 1,529
- References
- 32
- Citations by year
- 2026: 56 · 2025: 60 · 2024: 70 · 2023: 58 · 2022: 97 · 2021: 94 · 2020: 88 · 2019: 96 · 2018: 119 · 2017: 113 · 2016: 121 · 2015: 141 · 2014: 114 · 2013: 94 · 2012: 81
- FWCI
- 27.54
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Nature (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- RePEc: Research Papers in Economics (submittedVersion)
- Funders
- National Institutes of Health (DP5OD009172)
- National Defense Science and Engineering Graduate
- Hansjörg Wyss Institute for Biologically Inspired Engineering, Harvard University
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gene Regulatory Network Analysis
- Keywords
- accelerated evolution, directed evolution, genome editing, lycopene production, isoprenoid biosynthesis, Escherichia coli, synthetic DNA, metabolic engineering
- MeSH terms
- Lycopene, Alleles, Biotechnology, Carotenoids, Chromosomes, Bacterial, DNA, Escherichia coli, Pentosephosphates, Genetic Variation, Genome, Bacterial, Directed Molecular Evolution
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