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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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The Use of Cell-free Protein Synthesis to Push the Boundaries of Synthetic Biology
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Self-citationGenome-driven cell engineering review: in vivo and in silico metabolic and genome engineering
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“Genome recoding is possible due to the development of MAGE (multiplex automated genome engineering) [120] and CAGE (conjugative assembly genome engineering) [8,121], and subsequently REXER [129].”
BackgroundEvolution-aided engineering of plant specialized metabolism
Irfan, M., Chavez, B. G., Rizzo, P., D’Auria, J. C., & Moghe, G. D. (2021) · aBIOTECH · doi:10.1007/s42994-021-00052-3 · Cited by 15
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Multi-Input Distributed Classifiers for Synthetic Genetic Circuits
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Deciphering Combinatorial Genetics
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Using Synthetic Biology to Understand the Evolution of Gene Expression
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“To assess lycopene production, we co-transformed E. coli with a pBbB1k-dxs-idi-crtE-crtI-crtB plasmid, a single crRNA plasmid and a dCas13-expressing plasmid (pBbA2c-dCas13a) (Supplementary Table S1) ( 48 ).”
MethodConstruction of Escherichia Coli Cell Factories for Production of Organic Acids and Alcohols
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High-throughput metabolic engineering of Yarrowia lipolytica through gene expression tuning
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Application of CRISPR/Cas System in the Metabolic Engineering of Small Molecules
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Efficient and iterative retron-mediated in vivo recombineering in Escherichia coli
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Design of artificial small regulatory trans-RNA for gene knockdown in Bacillus subtilis
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“Traditionally, genome engineering strategies such as random mutagenesis [20], gene-knockout [21], and multiplex automated genome engineering (MAGE) [22] are wildly used in manipulating bacteria to improve the yields of valuable chemicals.”
Background
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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