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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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Arranz‐Gibert, P., Vanderschuren, K., & Isaacs, F. J. (2018) · Current Opinion in Chemical Biology · doi:10.1016/j.cbpa.2018.07.020 · Cited by 64
“Another study by Amiram et al. combined the use of multiplex automated genome engineering (MAGE) in a genomically recoded organism (GRO; see next section) to produce 25-fold more active MjRS enzymes capable of introducing 30 instances of an nsAA in a single protein [13 ].”
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“Conceptually, approaches like multiplex automated genome engineering (MAGE) allow for parallelization of directed genetic modifications and screening, decreasing the time to finding an optimized strain (Wang et al., 2009).”
MethodSynthetic biology: Tools to design microbes for the production of chemicals and fuels
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Kang, Z., Zhang, C., Zhang, J., Jin, P. K., Zhang, J., Du, G., & Chen, J. (2014) · Applied Microbiology and Biotechnology · doi:10.1007/s00253-014-5569-y · Cited by 63
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“coli, harboring a temperature-inducible λRed recombination system, and optimized for efficient genome recombineering, was used as the host.(Wang et al., 2009) To enable the removal of the genes encoding the EcTyrRS/tRNA pair, we either used gene cassettes selectable using antibiotics (gentamycin or zeocin), or the dua…”
MethodDabirian, Y., Teixeira, P. G., Nielsen, J., Siewers, V., & David, F. (2019) · ACS Synthetic Biology · doi:10.1021/acssynbio.9b00118 · Cited by 62
Engineering biological systems toward a sustainable bioeconomy
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MethodSelf-citationSynthetic biology strategies toward heterologous phytochemical production
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“3a) , the two major approaches, multiplex automated genome engineering (MAGE) using λ-red recombination 8 and conjugative assembly genome engineering (CAGE) 13 , have been widely used in prokaryotes and have demonstrated a high degree of multiplexability.”
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Usmani, Z., Sharma, M., Sudheer, S. M., Gupta, V. K., & Bhat, R. (2020) · Current Genomics · doi:10.2174/1389202921999200330152007 · Cited by 63
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MethodSelf-citationMethods and applications for assembling large DNA constructs
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Harris, D. C., & Jewett, M. C. (2012) · Current Opinion in Biotechnology · doi:10.1016/j.copbio.2012.02.002 · Cited by 58
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Roquet, N., & Lu, T. K. (2014) · Biotechnology Journal · doi:10.1002/biot.201300258 · Cited by 58
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BackgroundSilver as a Bactericidal Coating for Biomedical Implants
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“Most notably, the majority of previously reported systems achieved high efficiencies of replacement of single nucleotides through the use of strains defective in mismatch repair (MMR) (by deletion of mutS, encoding one subunit of the MutHLS methyldirected mismatch repair system) (4,5).”
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Self-citationRandom and cyclical deletion of large DNA segments in the genome of Pseudomonas putida
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“elimination of, in most cases, one or a small number of genes at a time in a given strain (Wang et al., 2009).”
BackgroundDesign and development of synthetic microbial platform cells for bioenergy
Lee, S. J., Lee, S. J., & Lee, D.-W. (2013) · Frontiers in Microbiology · doi:10.3389/fmicb.2013.00092 · Cited by 57
“…addition, lipids (i.e., triacylglycerols) in microalgae serve as an attractive biofuel feedstock, which can be converted to biodiesel through not only transesterification in biorefining (He et al., 2012) but also genetically interrupting starch biosynthesis (Wang et al., 2009b; Work et al., 2010).”
Codon Compression Algorithms for Saturation Mutagenesis
Pines, G., Pines, A., Garst, A. D., Zeitoun, R. I., Lynch, S. A., & Gill, R. T. (2014) · ACS Synthetic Biology · doi:10.1021/sb500282v · Cited by 54
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Photosynthetic terpene hydrocarbon production for fuels and chemicals
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BackgroundHarnessing mutagenic homologous recombination for targeted mutagenesis in vivo by TaGTEAM
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Genomic Recoding Broadly Obstructs the Propagation of Horizontally Transferred Genetic Elements
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“For pRK2, we performed ten cycles of MAGE in a +UAG+RF1 background and then conjugated to +UAG+RF1 or DUAGDRF1 strains to identify mutations that recover plasmid maintenance and conjugative efficiency.”
InfluentialSelf-citationToward a genetic tool development pipeline for host-associated bacteria
Waller, M. C., Bober, J. R., Nair, N. U., & Beisel, C. L. (2017) · Current Opinion in Microbiology · doi:10.1016/j.mib.2017.05.006 · Cited by 55
“With the development of recursive editing strategies [10,38,39], recombineering could achieve efficient multiplexed genome editing without direct selection of the”
MethodStructure and mechanism of the Red recombination system of bacteriophage λ
Caldwell, B. J., & Bell, C. E. (2019) · Progress in Biophysics and Molecular Biology · doi:10.1016/j.pbiomolbio.2019.03.005 · Cited by 56
“A particularly powerful extension of oligonucleotide recombineering has been developed, called MAGE for multiplex automated genome engineering, in which degenerate libraries of oligos directed towards multiple target sites are used in an iterative, selective manner to manipulate and evolve entire biosynthetic pathways…”
BackgroundAccelerating enzyme discovery and engineering with high-throughput screening
Bozkurt, E. U., Ørsted, E. C., Volke, D. C., & Nikel, P. I. (2024) · Natural Product Reports · doi:10.1039/d4np00031e · Cited by 54
Evolution of a chimeric aspartate kinase for L-lysine production using a synthetic RNA device
Wang, J., Gao, D., Yu, X., Li, W., & Qi, Q. (2015) · Applied Microbiology and Biotechnology · doi:10.1007/s00253-015-6615-0 · Cited by 54
Microbial production strategies and applications of lycopene and other terpenoids
Ma, T., Deng, Z., & Liu, T. (2015) · World Journal of Microbiology and Biotechnology · doi:10.1007/s11274-015-1975-2 · Cited by 54
“2011), multiplex automated genome engineering (MAGE) for largescale programming (Wang et al. 2009), and metabolomics have been coupled with genetic standardization and modern cloning techniques to make this goal attainable (George et al.”
MethodRelating supply network structure to productive efficiency: A multi-stage empirical investigation
Kao, T., Simpson, N., Shao, B. B. M., & Lin, W. T. (2016) · European Journal of Operational Research · doi:10.1016/j.ejor.2016.11.008 · Cited by 54
Hayes, C. A., Dalia, T. N., & Dalia, A. B. (2017) · Molecular Microbiology · doi:10.1111/mmi.13646 · Cited by 49
“…media to select for integration of the selected product (i.e., to select for the recombinogenic subpopulation) and then screened for integration of the unselected product(s) via multiplex allele-specific colony PCR (MASC-PCR) exactly as previously described (Wang et al., 2009; Dalia et al., 2014b).”
BackgroundMethodInfluentialEngineering an Optogenetic CRISPRi Platform for Improved Chemical Production
Wu, P., Chen, Y., Liu, M., Xiao, G., & Yuan, J. (2020) · ACS Synthetic Biology · doi:10.1021/acssynbio.0c00488 · Cited by 54
Strategies to Enhance Periplasmic Recombinant Protein Production Yields in Escherichia coli
Karyolaimos, A., & de Gier, J.-W. L. (2021) · Frontiers in Bioengineering and Biotechnology · doi:10.3389/fbioe.2021.797334 · Cited by 45
Wang, J., Li, C., Jiang, T., & Yan, Y. (2022) · Metabolic Engineering · doi:10.1016/j.ymben.2022.11.004 · Cited by 53
Towards Reassigning the Rare AGG Codon in Escherichia coli
Zeng, Y., Wang, W., & Liu, W. R. (2014) · ChemBioChem · doi:10.1002/cbic.201400075 · Cited by 61
Continuous directed evolution for strain and protein engineering
d’Oelsnitz, S., & Ellington, A. D. (2018) · Current Opinion in Biotechnology · doi:10.1016/j.copbio.2017.12.020 · Cited by 53
Long, M., Xu, M., Ma, Z., Pan, X., You, J., Hu, M., Shao, Y., Yang, T., Zhang, X., & Rao, Z. (2020) · Science Advances · doi:10.1126/sciadv.aba2383 · Cited by 53
In the business of base editors: Evolution from bench to bedside
Porto, E. M., & Komor, A. C. (2023) · PLoS Biology · doi:10.1371/journal.pbio.3002071 · Cited by 53
“Directed evolution facilitates the enhancement or alteration of the activity of a given protein [9–11].”
BackgroundOvercoming Challenges in Engineering the Genetic Code
Lajoie, M. J., Söll, D. G., & Church, G. M. (2015) · Journal of Molecular Biology · doi:10.1016/j.jmb.2015.09.003 · Cited by 54
Self-citationBatth, T. S., Singh, P., Ramakrishnan, V. R., de Sousa, M. M. L., Chan, L. J. G., Tran, H. M., Luning, E. G., Pan, E. H. Y., Vuu, K. M., Keasling, J. D., et al. (2014) · Metabolic Engineering · doi:10.1016/j.ymben.2014.08.004 · Cited by 51
Conditional DNA repair mutants enable highly precise genome engineering
Nyerges, Á., Csörgő, B., Nagy, I., Latinovics, D., Szamecz, B., Pósfai, G., & Pál, C. (2014) · Nucleic Acids Research · doi:10.1093/nar/gku105 · Cited by 52
“From these techniques, oligonucleotide (oligo)mediated allelic replacement has been optimized toward multiplexing and automation (2).”
MethodReisch, C. R., & Prather, K. L. J. (2017) · Current Protocols in Molecular Biology · doi:10.1002/cpmb.29 · Cited by 52
“Any method can be used to make electrocompetent cells, although we prefer the glycerolmannitol density step method because it is quick and yields cells with consistent time constants upon electroporation (Warren, 2011).”
MethodIterative genome editing of Escherichia coli for 3-hydroxypropionic acid production
Liu, R., Liang, L., Choudhury, A., Bassalo, M. C., Garst, A. D., Tarasava, K., & Gill, R. T. (2018) · Metabolic Engineering · doi:10.1016/j.ymben.2018.04.007 · Cited by 52
Targeted Nucleotide Editing Technologies for Microbial Metabolic Engineering
Arazoe, T., Kondo, A., & Nishida, K. (2018) · Biotechnology Journal · doi:10.1002/biot.201700596 · Cited by 54
Multiplex Genome Engineering Methods for Yeast Cell Factory Development
Malcı, K., Walls, L. E., & Rios‐Solis, L. (2020) · Frontiers in Bioengineering and Biotechnology · doi:10.3389/fbioe.2020.589468 · Cited by 52
Systems Metabolic Engineering of Escherichia coli
Choi, K. R., Shin, J. H., Cho, J. S., Yang, D., & Lee, S. Y. (2016) · EcoSal Plus · doi:10.1128/ecosalplus.esp-0010-2015 · Cited by 53
From elements to modules: regulatory evolution in Ascomycota fungi
Wohlbach, D. J., Thompson, D. A., Gasch, A. P., & Regev, A. (2009) · Current Opinion in Genetics & Development · doi:10.1016/j.gde.2009.09.007 · Cited by 52
“cerevisiae, experimental evolution and direct engineering [66,67] can highlight the effect of well-defined selective pressures on regulatory evolution.”
BackgroundCustom selenoprotein production enabled by laboratory evolution of recoded bacterial strains
Thyer, R. T., Shroff, R., Klein, D. R., d’Oelsnitz, S., Cotham, V. C., Byrom, M., Brodbelt, J. S., & Ellington, A. D. (2018) · Nature Biotechnology · doi:10.1038/nbt.4154 · Cited by 50
Characterizing posttranslational modifications in prokaryotic metabolism using a multiscale workflow
Brunk, E., Chang, R. L., Xia, J., Hefzi, H., Yurkovich, J. T., Kim, D., Buckmiller, E., Wang, H. H., Cho, B., Yang, C., et al. (2018) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1811971115 · Cited by 51
“Oligonucleotide Design for MAGE.”
MethodInfluentialSelf-citationA novel conjugal donor strain for improved DNA transfer into Clostridium spp.
Woods, C., Humphreys, C. M., Rodrigues, R., Ingle, P., Rowe, P., Henstra, A. M., Köpke, M., Simpson, S. D., Winzer, K., & Minton, N. P. (2019) · Anaerobe · doi:10.1016/j.anaerobe.2019.06.020 · Cited by 51
“However, if sufficiently high frequencies of transfer could be obtained in this organism then it would be an important step towards implementation of several high throughput techniques such as Multiplex Automated Genomic Engineering (MAGE) [26] or Transposon Directed Insertion Sequencing (TraDIS) [27].”
BackgroundEngineering Life: A Review of Synthetic Biology
Hanczyc, M. M. (2020) · Artificial Life · doi:10.1162/artl_a_00318 · Cited by 51
“exogenous ssDNA fragments to outcompete the endogenous Okazaki fragments [54].”
BackgroundRetron-mediated multiplex genome editing and continuous evolution in Escherichia coli
Liu, W., Zuo, S., Shao, Y., Bi, K., Zhao, J., Huang, L., Xu, Z., & Lian, J. (2023) · Nucleic Acids Research · doi:10.1093/nar/gkad607 · Cited by 53
“Based on previous studies, the combination of idi , dxs and ytjC engineering or the combination of dxs , idi , dxr , rpoS and ytjC engineering were determined to be the most effecti v e in increasing lycopene production ( 24 ).”
InfluentialEngineering improved ethanol production in Escherichia coli with a genome-wide approach
Woodruff, L. B. A., Boyle, N. R., & Gill, R. T. (2013) · Metabolic Engineering · doi:10.1016/j.ymben.2013.01.006 · Cited by 49
“Future work could utilize the genetic targets identified here along with the additional genome engineering approaches (Lynch et al., 2007; Wang et al., 2009; Warner et al., 2010) to engineer further improvements to the production of ethanol (or possibly other alcohols), such as selecting for improved MIC or combinator…”
MethodTALE nucleases and next generation GM crops
Mahfouz, M. M., & Li, L. (2011) · GM Crops · doi:10.4161/gmcr.2.2.17254 · Cited by 50
“Multiplex Automated Genome Engineering (MAGE) technique was used to generate genomic variants in bacteria.(35) The MAGE technique employed oligomediated allelic replacements and was successfully applied to generate genomic variants with increased production of lycopene.”
MethodWu, T., Ye, L., Zhao, D., Li, S., Li, Q., Zhang, B., & Bi, C. (2018) · 3 Biotech · doi:10.1007/s13205-018-1298-8 · Cited by 50
The Synthetic Biology Toolkit for Photosynthetic Microorganisms
Vavitsas, K., Crozet, P., Vinde, M. H., Davies, F. K., Lemaire, S. D., & Vickers, C. E. (2019) · PLANT PHYSIOLOGY · doi:10.1104/pp.19.00345 · Cited by 57
“For example, multiplex automated genome engineering (Wang et al., 2009) and its counterpart yeast (Saccharomyces cerevisiae) oligo-mediated genome engineering (DiCarlo et al.”
BackgroundShang, Y., & Huang, S. (2019) · Plant Communications · doi:10.1016/j.xplc.2019.100012 · Cited by 51
“Protein engineering approaches, especially those developed based on directed evolution, require the capacity to generate a large number of sequences, and this is usually achieved by performing ep-PCR (Cadwell and Joyce, 1992), site-directed mutagenesis (Wang and Malcolm, 1999), multiplex automated genome engineering (…”
BackgroundGenetic toolkits for engineering Rhodococcus species with versatile applications
Liang, Y., & Yu, H. (2021) · Biotechnology Advances · doi:10.1016/j.biotechadv.2021.107748 · Cited by 50
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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