The DOI matches its Crossref record; 32 of the 32 references it cites were found in a registry or an index.
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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Cites
In vitro selection of RNA molecules that bind specific ligands
Ellington, A. D., & Szostak, J. W. (1990) · Nature · doi:10.1038/346818a0 · Cited by 9,845 · cited 1×
“7); (2) induce cells for allelic replacement via 42 uC heat shock for 15 min; (3) chill cells at 4 uC to halt cellular metabolism; (4) wash cells through 15–20 iterations of filtration and resuspension with dH2O; (5) mix cell suspension and synthetic DNA; (6) deliver DNA into cells by electroporation; and (7) resuspen…”
BackgroundEnvironmental genome shotgun sequencing of the sargasso sea
Venter, J. C., Remington, K., Heidelberg, J. F., Halpern, A. L., Rusch, D., Eisen, J. A., Wu, D., Paulsen, I., Nelson, K. E., Nelson, W., et al. (2004) · Science · doi:10.1126/science.1093857 · Cited by 4,376 · cited 3×
“The device then executed repeatedly and continuously through the following steps: (1) grow cells at 30 uC to a pre-set density (that is, OD600 nm of 0.”
BackgroundMethodAn efficient recombination system for chromosome engineering in Escherichia coli
Yu, D., Ellis, H. M., Lee, E.-C., Jenkins, N. A., Copeland, N. G., & Court, D. L. (2000) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.100127597 · Cited by 1,688
Comparative Metagenomics of Microbial Communities
Tringe, S. G., von Mering, C., Kobayashi, A., Salamov, A. A., Chen, K., Chang, H. W., Podar, M., Short, J. M., Mathur, E. J., Detter, J. C., et al. (2005) · Science · doi:10.1126/science.1107851 · Cited by 1,677 · cited 3×
“Oligonucleotides used in the MAGE process contained the following modifications: (1) 30–110 bp in length for optimization experiments; (2) different numbers of phosphorothioated bases; and (3) degenerate nucleotides as described elsewhere in this paper.”
BackgroundMethodAccurate Multiplex Polony Sequencing of an Evolved Bacterial Genome
Shendure, J., Porreca, G. J., Reppas, N. B., Lin, X., McCutcheon, J. P., Rosenbaum, A. M., Wang, M. D., Zhang, K., Mitra, R. D., & Church, G. M. (2005) · Science · doi:10.1126/science.1117389 · Cited by 1,402
Self-citationEvolution Experiments with Microorganisms: The Dynamics and Genetic Bases of Adaptation
Elena, S. F., & Lenski, R. E. (2003) · Nature Reviews Genetics · doi:10.1038/nrg1088 · Cited by 1,386 · cited 3×
“Oligonucleotides used in the MAGE process contained the following modifications: (1) 30–110 bp in length for optimization experiments; (2) different numbers of phosphorothioated bases; and (3) degenerate nucleotides as described elsewhere in this paper.”
BackgroundMethodComplete Chemical Synthesis, Assembly, and Cloning of a Mycoplasma genitalium Genome
Gibson, D. G., Benders, G. A., Andrews-Pfannkoch, C., Denisova, E. A., Baden-Tillson, H., Zaveri, J., Stockwell, T. B., Brownley, A., Thomas, D. W., Algire, M. A., et al. (2008) · Science · doi:10.1126/science.1151721 · Cited by 1,243
A new logic for DNA engineering using recombination in Escherichia coli
Zhang, Y., Buchholz, F., Muyrers, J. P. P., & Stewart, A. F. (1998) · Nature Genetics · doi:10.1038/2417 · Cited by 1,225
Randomization of genes by PCR mutagenesis.Preprint
Cadwell, R. C., & Joyce, G. F. (1992) · Genome Research · doi:10.1101/gr.2.1.28 · Cited by 1,082
DINAMelt web server for nucleic acid melting prediction
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Cited by
Sorted by citations
Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression
Qi, L. S., Larson, M. H., Gilbert, L. A., Doudna, J. A., Weissman, J. S., Arkin, A. P., & Lim, W. A. (2013) · Cell · doi:10.1016/j.cell.2013.02.022 · Cited by 5,016
“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).”
MethodCreation of a Bacterial Cell Controlled by a Chemically Synthesized Genome
Gibson, D. G., Glass, J. I., Lartigue, C., Noskov, V. N., Chuang, R.-Y., Algire, M. A., Benders, G. A., Montague, M. G., Ma, L., Moodie, M. M., et al. (2010) · Science · doi:10.1126/science.1190719 · Cited by 2,638
Isoprenoid Pathway Optimization for Taxol Precursor Overproduction in Escherichia coli
Ajikumar, P. K., Xiao, W., Tyo, K. E. J., Wang, Y., Simeon, F., Leonard, E., Mucha, O., Too, H., Pfeifer, B. A., & Stephanopoulos, G. N. (2010) · Science · doi:10.1126/science.1191652 · Cited by 1,681
Synthetic biology: applications come of age
Khalil, A. S., & Collins, J. J. (2010) · Nature Reviews Genetics · doi:10.1038/nrg2775 · Cited by 1,413
“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).”
MethodMultigene editing in the Escherichia coli genome via the CRISPR-Cas9 system
Jiang, Y., Chen, B., Duan, C., Sun, B., Yang, J., & Yang, S. (2015) · Applied and Environmental Microbiology · doi:10.1128/aem.04023-14 · Cited by 1,362
“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.”
MethodInfluential
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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