Optimizing complex phenotypes through model-guided multiplex genome engineering

Kuznetsov, Gleb; Goodman, Daniel B.; Filsinger, Gabriel T.; Landon, Matthieu; Rohland, Nadin; Aach, John; Lajoie, Marc J.; Church, George M.

doi:10.1186/s13059-017-1217-z

Title: Optimizing complex phenotypes through model-guided multiplex genome engineering

Journal Article · Thu May 25 00:00:00 EDT 2017 · Genome Biology (Online)

DOI:https://doi.org/10.1186/s13059-017-1217-z· OSTI ID:1618923

; Goodman, Daniel B.; Filsinger, Gabriel T.; Landon, Matthieu; Rohland, Nadin; Aach, John; Lajoie, Marc J.; Church, George M.

We present a method for identifying genomic modifications that optimize a complex phenotype through multiplex genome engineering and predictive modeling. We apply our method to identify six single nucleotide mutations that recover 59% of the fitness defect exhibited by the 63-codon E. coli strain C321.ΔA. By introducing targeted combinations of changes in multiplex we generate rich genotypic and phenotypic diversity and characterize clones using whole-genome sequencing and doubling time measurements. Regularized multivariate linear regression accurately quantifies individual allelic effects and overcomes bias from hitchhiking mutations and context-dependence of genome editing efficiency that would confound other strategies.

View Journal Article

Cite

Export

Save

Research Organization:: Harvard Univ., Boston, MA (United States). Harvard Medical School; Harvard Univ., Cambridge, MA (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER)

Contributing Organization:: AWS Cloud Credits for Research Program

Grant/Contract Number:: FG02-02ER63445

OSTI ID:: 1618923

Alternate ID(s):: OSTI ID: 1371697; OSTI ID: 1389296

Journal Information:: Genome Biology (Online), Journal Name: Genome Biology (Online) Vol. 18 Journal Issue: 1; ISSN 1474-760X

Publisher:: Springer Science + Business MediaCopyright Statement

Country of Publication:: United Kingdom

Language:: English

Citation Metrics:

Cited by: 15 works

Citation information provided by
Web of Science

References (41)

Design, synthesis, and testing toward a 57-codon genome Ostrov, N.; Landon, M.; Guell, M. Science, Vol. 353, Issue 6301 https://doi.org/10.1126/science.aaf3639	journal	August 2016
Genomic Recoding Broadly Obstructs the Propagation of Horizontally Transferred Genetic Elements Ma, Natalie Jing; Isaacs, Farren J. Cell Systems, Vol. 3, Issue 2 https://doi.org/10.1016/j.cels.2016.06.009	journal	August 2016
Diminishing Returns Epistasis Among Beneficial Mutations Decelerates Adaptation Chou, H. -H.; Chiu, H. -C.; Delaney, N. F. Science, Vol. 332, Issue 6034 https://doi.org/10.1126/science.1203799	journal	June 2011
Multiplexed tracking of combinatorial genomic mutations in engineered cell populations Zeitoun, Ramsey I.; Garst, Andrew D.; Degen, George D. Nature Biotechnology, Vol. 33, Issue 6, p. 631-637 https://doi.org/10.1038/nbt.3177	journal	March 2015
Enhanced levels of λ Red-mediated recombinants in mismatch repair mutants Costantino, N.; Court, D. L. Proceedings of the National Academy of Sciences, Vol. 100, Issue 26, p. 15748-15753 https://doi.org/10.1073/pnas.2434959100	journal	December 2003
Biocontainment of genetically modified organisms by synthetic protein design Mandell, Daniel J.; Lajoie, Marc J.; Mee, Michael T. Nature, Vol. 518, Issue 7537 https://doi.org/10.1038/nature14121	journal	January 2015
Rational optimization of tolC as a powerful dual selectable marker for genome engineering Gregg, Christopher J.; Lajoie, Marc J.; Napolitano, Michael G. Nucleic Acids Research, Vol. 42, Issue 7 https://doi.org/10.1093/nar/gkt1374	journal	January 2014
Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants the Keio collection Baba, Tomoya; Ara, Takeshi; Hasegawa, Miki Molecular Systems Biology, Vol. 2, Article No. 2006.0008 https://doi.org/10.1038/msb4100050	journal	February 2006
Precise Manipulation of Chromosomes in Vivo Enables Genome-Wide Codon Replacement Isaacs, Farren J.; Carr, Peter A.; Wang, Harris H. Science, Vol. 333, Issue 6040, p. 348-353 https://doi.org/10.1126/science.1205822	journal	July 2011
Programming cells by multiplex genome engineering and accelerated evolution Wang, Harris H.; Isaacs, Farren J.; Carr, Peter A. Nature, Vol. 460, Issue 7257, p. 894-898 https://doi.org/10.1038/nature08187	journal	July 2009
Direct Mutagenesis of Thousands of Genomic Targets Using Microarray-Derived Oligonucleotides Bonde, Mads T.; Kosuri, Sriram; Genee, Hans J. ACS Synthetic Biology, Vol. 4, Issue 1 https://doi.org/10.1021/sb5001565	journal	June 2014
The Escherichia coli Cpx Envelope Stress Response Regulates Genes of Diverse Function That Impact Antibiotic Resistance and Membrane Integrity Raivio, T. L.; Leblanc, S. K. D.; Price, N. L. Journal of Bacteriology, Vol. 195, Issue 12 https://doi.org/10.1128/JB.00105-13	journal	April 2013
EcoCyc: a comprehensive database of Escherichia coli biology Keseler, I. M.; Collado-Vides, J.; Santos-Zavaleta, A. Nucleic Acids Research, Vol. 39, Issue Database https://doi.org/10.1093/nar/gkq1143	journal	November 2010
Regularization and variable selection via the elastic net Zou, Hui; Hastie, Trevor Journal of the Royal Statistical Society: Series B (Statistical Methodology), Vol. 67, Issue 2 https://doi.org/10.1111/j.1467-9868.2005.00503.x	journal	April 2005
Adaptive laboratory evolution – principles and applications for biotechnology Dragosits, Martin; Mattanovich, Diethard Microbial Cell Factories, Vol. 12, Issue 1 https://doi.org/10.1186/1475-2859-12-64	journal	January 2013
Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells Shalem, Ophir; Sanjana, Neville E.; Hartenian, Ella Science, Vol. 343, Issue 6166, p. 84-87 https://doi.org/10.1126/science.1247005	journal	December 2013
Synthetic chromosome arms function in yeast and generate phenotypic diversity by design Dymond, Jessica S.; Richardson, Sarah M.; Coombes, Candice E. Nature, Vol. 477, Issue 7365 https://doi.org/10.1038/nature10403	journal	September 2011
Negative Epistasis Between Beneficial Mutations in an Evolving Bacterial Population Khan, A. I.; Dinh, D. M.; Schneider, D. Science, Vol. 332, Issue 6034 https://doi.org/10.1126/science.1203801	journal	June 2011
Trackable Multiplex Recombineering for Gene-Trait Mapping in E. coli Mansell, Thomas J.; Warner, Joseph R.; Gill, Ryan T. Methods in Molecular Biology https://doi.org/10.1007/978-1-62703-299-5_12	book	January 2013
Gene Ontology: tool for the unification of biology Ashburner, Michael; Ball, Catherine A.; Blake, Judith A. Nature Genetics, Vol. 25, Issue 1 https://doi.org/10.1038/75556	journal	May 2000
Codon Bias as a Means to Fine-Tune Gene Expression Quax, Tessa E. F.; Claassens, Nico J.; Söll, Dieter Molecular Cell, Vol. 59, Issue 2 https://doi.org/10.1016/j.molcel.2015.05.035	journal	July 2015
Use of Adaptive Laboratory Evolution To Discover Key Mutations Enabling Rapid Growth of Escherichia coli K-12 MG1655 on Glucose Minimal Medium LaCroix, Ryan A.; Sandberg, Troy E.; O'Brien, Edward J. Applied and Environmental Microbiology, Vol. 81, Issue 1 https://doi.org/10.1128/AEM.02246-14	journal	October 2014
Genome engineering Escherichia coli for L-DOPA overproduction from glucose Wei, Tao; Cheng, Bi-Yan; Liu, Jian-Zhong Scientific Reports, Vol. 6, Issue 1 https://doi.org/10.1038/srep30080	journal	July 2016
Emergent rules for codon choice elucidated by editing rare arginine codons in Escherichia coli Napolitano, Michael G.; Landon, Matthieu; Gregg, Christopher J. Proceedings of the National Academy of Sciences, Vol. 113, Issue 38 https://doi.org/10.1073/pnas.1605856113	journal	September 2016
Adenylate Cyclase and the Cyclic AMP Receptor Protein Modulate Stress Resistance and Virulence Capacity of Uropathogenic Escherichia coli Donovan, Grant T.; Norton, J. Paul; Bower, Jean M. Infection and Immunity, Vol. 81, Issue 1 https://doi.org/10.1128/IAI.00796-12	journal	October 2012
Inexpensive Multiplexed Library Preparation for Megabase-Sized Genomes Baym, Michael; Kryazhimskiy, Sergey; Lieberman, Tami D. PLOS ONE, Vol. 10, Issue 5 https://doi.org/10.1371/journal.pone.0128036	journal	May 2015
Evolution-guided optimization of biosynthetic pathways Raman, Srivatsan; Rogers, Jameson K.; Taylor, Noah D. Proceedings of the National Academy of Sciences, Vol. 111, Issue 50, p. 17803-17808 https://doi.org/10.1073/pnas.1409523111	journal	December 2014
Design and synthesis of a minimal bacterial genome Hutchison, C. A.; Chuang, R. -Y.; Noskov, V. N. Science, Vol. 351, Issue 6280 https://doi.org/10.1126/science.aad6253	journal	March 2016
Evolution of Escherichia coli to 42 °C and Subsequent Genetic Engineering Reveals Adaptive Mechanisms and Novel Mutations Sandberg, Troy E.; Pedersen, Margit; LaCroix, Ryan A. Molecular Biology and Evolution, Vol. 31, Issue 10 https://doi.org/10.1093/molbev/msu209	journal	July 2014
Emergent Properties of Reduced-Genome Escherichia coli Posfai, G.; Plunkett III, Guy; Fehér, Tamás Science, Vol. 312, Issue 5776, p. 1044-1046 https://doi.org/10.1126/science.1126439	journal	May 2006
RegulonDB version 9.0: high-level integration of gene regulation, coexpression, motif clustering and beyond Gama-Castro, Socorro; Salgado, Heladia; Santos-Zavaleta, Alberto Nucleic Acids Research, Vol. 44, Issue D1 https://doi.org/10.1093/nar/gkv1156	journal	November 2015
Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector Kabadi, Ami M.; Ousterout, David G.; Hilton, Isaac B. Nucleic Acids Research, Vol. 42, Issue 19 https://doi.org/10.1093/nar/gku749	journal	August 2014
Recoded organisms engineered to depend on synthetic amino acids Rovner, Alexis J.; Haimovich, Adrian D.; Katz, Spencer R. Nature, Vol. 518, Issue 7537 https://doi.org/10.1038/nature14095	journal	January 2015
Genomically Recoded Organisms Expand Biological Functions Lajoie, M. J.; Rovner, A. J.; Goodman, D. B. Science, Vol. 342, Issue 6156, p. 357-360 https://doi.org/10.1126/science.1241459	journal	October 2013
Yeast Oligo-Mediated Genome Engineering (YOGE) DiCarlo, James E.; Conley, Andrew J.; Penttilä, Merja ACS Synthetic Biology, Vol. 2, Issue 12 https://doi.org/10.1021/sb400117c	journal	November 2013
Millstone: software for multiplex microbial genome analysis and engineering Goodman, Daniel B.; Kuznetsov, Gleb; Lajoie, Marc J. Genome Biology, Vol. 18, Issue 1 https://doi.org/10.1186/s13059-017-1223-1	journal	May 2017
Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements Lutz, R. Nucleic Acids Research, Vol. 25, Issue 6, p. 1203-1210 https://doi.org/10.1093/nar/25.6.1203	journal	March 1997
Cost-effective, high-throughput DNA sequencing libraries for multiplexed target capture Rohland, N.; Reich, D. Genome Research, Vol. 22, Issue 5 https://doi.org/10.1101/gr.128124.111	journal	January 2012
Refactoring bacteriophage T7 Chan, Leon Y.; Kosuri, Sriram; Endy, Drew Molecular Systems Biology, Vol. 1, Issue 1 https://doi.org/10.1038/msb4100025	journal	January 2005
Genetic Screens in Human Cells Using the CRISPR-Cas9 System Wang, Tim; Wei, Jenny J.; Sabatini, David M. Science, Vol. 343, Issue 6166 https://doi.org/10.1126/science.1246981	journal	December 2013
A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w Cingolani, Pablo; Platts, Adrian; Wang, Le Lily Fly, Vol. 6, Issue 2 https://doi.org/10.4161/fly.19695	journal	April 2012

Figures / Tables (5)

Similar Records

Adaptive evolution of genomically recoded Escherichia coli

Journal Article · Tue Feb 13 00:00:00 EST 2018 · Proceedings of the National Academy of Sciences of the United States of America · OSTI ID:1618923

Wannier, Timothy M.; Kunjapur, Aditya M.; Rice, Daniel P.; +3 more

Galactosemia: A strategy to identify new biochemical phenotypes and molecular genotypes

Journal Article · Wed Mar 01 00:00:00 EST 1995 · American Journal of Human Genetics · OSTI ID:1618923

Elsas, L J; Langley, S; Steele, E; +7 more

Improved bacterial recombineering by parallelized protein discovery

Journal Article · Thu May 28 00:00:00 EDT 2020 · Proceedings of the National Academy of Sciences of the United States of America · OSTI ID:1618923

Wannier, Timothy M.; Nyerges, Akos; Kuchwara, Helene M.; +9 more

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
60 APPLIED LIFE SCIENCES
Genome engineering
Predictive modeling
Synthetic organisms
Genome engineering
Predictive modeling
Synthetic organisms

Title: Optimizing complex phenotypes through model-guided multiplex genome engineering

Citation Formats

References (41)

Figures / Tables (5)

Similar Records

Related Subjects