A swapped genetic code prevents viral infections and gene transfer

Nyerges, Akos; Vinke, Svenja; Flynn, Regan; Owen, Siân V.; Rand, Eleanor A.; Budnik, Bogdan; Keen, Eric; Narasimhan, Kamesh; Marchand, Jorge A.; Baas-Thomas, Maximilien; Liu, Min; Chen, Kangming; Chiappino-Pepe, Anush; Hu, Fangxiang; Baym, Michael; Church, George M.

doi:10.1038/s41586-023-05824-z

A swapped genetic code prevents viral infections and gene transfer

Journal Article · Wed Mar 15 00:00:00 EDT 2023 · Nature (London)

DOI:https://doi.org/10.1038/s41586-023-05824-z· OSTI ID:2228837

^[1]; Vinke, Svenja ^[2]; Flynn, Regan ^[2]; ^[2]; Rand, Eleanor A. ^[2]; Budnik, Bogdan ^[3]; Keen, Eric ^[4]; ^[2]; Marchand, Jorge A. ^[5]; ^[2]; Liu, Min ^[6]; Chen, Kangming ^[6]; ^[2]; Hu, Fangxiang ^[6]; ^[2]; ^[7]

Harvard Medical School, Boston, MA (United States); Harvard Medical School, Department of Genetics, Church Lab
Harvard Medical School, Boston, MA (United States)
Harvard University, Boston, MA (United States)
Washington University School of Medicine in St. Louis, MO (United States)
University of Washington, Seattle, WA (United States); Harvard Medical School, Boston, MA (United States)
GenScript USA Inc., Piscataway, NJ (United States)
Harvard Medical School, Boston, MA (United States); Harvard University, Boston, MA (United States)

Engineering the genetic code of an organism has been proposed to provide a firewall from natural ecosystems by preventing viral infections and gene transfer. However, numerous viruses and mobile genetic elements encode parts of the translational apparatus, potentially rendering a genetic-code-based firewall ineffective. Here we show that such mobile transfer RNAs (tRNAs) enable gene transfer and allow viral replication in Escherichia coli despite the genome-wide removal of 3 of the 64 codons and the previously essential cognate tRNA and release factor genes. We then establish a genetic firewall by discovering viral tRNAs that provide exceptionally efficient codon reassignment allowing us to develop cells bearing an amino acid-swapped genetic code that reassigns two of the six serine codons to leucine during translation. This amino acid-swapped genetic code renders cells resistant to viral infections by mistranslating viral proteomes and prevents the escape of synthetic genetic information by engineered reliance on serine codons to produce leucine-requiring proteins. As these cells may have a selective advantage over wild organisms due to virus resistance, we also repurpose a third codon to biocontain this virus-resistant host through dependence on an amino acid not found in nature. Furthermore, our results may provide the basis for a general strategy to make any organism safely resistant to all natural viruses and prevent genetic information flow into and out of genetically modified organisms.

View Accepted Manuscript (DOE)

Research Organization:: Harvard Medical School, Boston, MA (United States)

Sponsoring Organization:: National Science Foundation (NSF); USDOE Office of Science (SC), Biological and Environmental Research (BER). Biological Systems Science (BSS)

Grant/Contract Number:: FG02-02ER63445

OSTI ID:: 2228837

Journal Information:: Nature (London), Journal Name: Nature (London) Journal Issue: 7953 Vol. 615; ISSN 0028-0836

Publisher:: Nature Publishing GroupCopyright Statement

Country of Publication:: United States

Language:: English

References (63)

MassIVE MSV000089854 - Swapped genetic code block viral infections and gene transfer Church, George M. MassIVE https://doi.org/10.25345/c5ff3m41w	dataset	January 2022
How To Quantify a Genetic Firewall? A Polarity‐Based Metric for Genetic Code Engineering Schmidt, Markus; Kubyshkin, Vladimir ChemBioChem, Vol. 22, Issue 7 https://doi.org/10.1002/cbic.202000758	journal	December 2020
In VitroCodon-Reading Specificities of Unmodified tRNA Molecules with Different Anticodons on the Sequence Background ofEscherichia colitRNASer1 Takai, Kazuyuki; Takaku, Hiroshi; Yokoyama, Shigeyuki Biochemical and Biophysical Research Communications, Vol. 257, Issue 3 https://doi.org/10.1006/bbrc.1999.0538	journal	April 1999
Identification of Mutations in Laboratory-Evolved Microbes from Next-Generation Sequencing Data Using breseq Deatherage, Daniel E.; Barrick, Jeffrey E. Methods in Molecular Biology https://doi.org/10.1007/978-1-4939-0554-6_12	book	January 2014
Bacteriophage Plaques: Theory and Analysis Abedon, Stephen T.; Yin, John Methods in Molecular Biology https://doi.org/10.1007/978-1-60327-164-6_17	book	January 2009
Bacteriophage-resistant industrial fermentation strains: from the cradle to CRISPR/Cas9 Baltz, Richard H. Journal of Industrial Microbiology and Biotechnology, Vol. 45, Issue 11 https://doi.org/10.1007/s10295-018-2079-4	journal	November 2018
A single uridine modification at the wobble position of an artificial tRNA enhances wobbling in an Escherichia coli cell‐free translation system Takai, Kazuyuki; Okumura, Shuhei; Hosono, Kazumi FEBS Letters, Vol. 447, Issue 1 https://doi.org/10.1016/S0014-5793(99)00255-0	journal	March 1999
MS-READ: Quantitative measurement of amino acid incorporation Mohler, Kyle; Aerni, Hans-Rudolf; Gassaway, Brandon Biochimica et Biophysica Acta (BBA) - General Subjects, Vol. 1861, Issue 11 https://doi.org/10.1016/j.bbagen.2017.01.025	journal	November 2017
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
Degradation of host translational machinery drives tRNA acquisition in viruses Yang, Joy Y.; Fang, Wenwen; Miranda-Sanchez, Fabiola Cell Systems, Vol. 12, Issue 8 https://doi.org/10.1016/j.cels.2021.05.019	journal	August 2021
Synthetic genomes with altered genetic codes Ostrov, Nili; Nyerges, Akos; Chiappino-Pepe, Anush Current Opinion in Systems Biology, Vol. 24 https://doi.org/10.1016/j.coisb.2020.09.007	journal	December 2020
An Amino Acid-Swapped Genetic Code Fujino, Tomoshige; Tozaki, Masahiro; Murakami, Hiroshi ACS Synthetic Biology, Vol. 9, Issue 10 https://doi.org/10.1021/acssynbio.0c00196	journal	September 2020
Genome-Wide Abolishment of Mobile Genetic Elements Using Genome Shuffling and CRISPR/Cas-Assisted MAGE Allows the Efficient Stabilization of a Bacterial Chassis Umenhoffer, Kinga; Draskovits, Gábor; Nyerges, Ákos ACS Synthetic Biology, Vol. 6, Issue 8 https://doi.org/10.1021/acssynbio.6b00378	journal	April 2017
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
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
RNA-guided editing of bacterial genomes using CRISPR-Cas systems Jiang, Wenyan; Bikard, David; Cox, David Nature Biotechnology, Vol. 31, Issue 3, p. 233-239 https://doi.org/10.1038/nbt.2508	journal	January 2013
Universal sample preparation method for proteome analysis Wiśniewski, Jacek R.; Zougman, Alexandre; Nagaraj, Nagarjuna Nature Methods, Vol. 6, Issue 5 https://doi.org/10.1038/nmeth.1322	journal	April 2009
Fast gapped-read alignment with Bowtie 2 Langmead, Ben; Salzberg, Steven L. Nature Methods, Vol. 9, Issue 4 https://doi.org/10.1038/nmeth.1923	journal	March 2012
Structural basis for orthogonal tRNA specificities of tyrosyl-tRNA synthetases for genetic code expansion Kobayashi, Takatsugu; Nureki, Osamu; Ishitani, Ryuichiro Nature Structural & Molecular Biology, Vol. 10, Issue 6 https://doi.org/10.1038/nsb934	journal	May 2003
Tailed giant Tupanvirus possesses the most complete translational apparatus of the known virosphere Abrahão, Jônatas; Silva, Lorena; Silva, Ludmila Santos Nature Communications, Vol. 9, Issue 1 https://doi.org/10.1038/s41467-018-03168-1	journal	February 2018
Multiplex base editing to convert TAG into TAA codons in the human genome Chen, Yuting; Hysolli, Eriona; Chen, Anlu Nature Communications, Vol. 13, Issue 1 https://doi.org/10.1038/s41467-022-31927-8	journal	August 2022
Systematic strategies for developing phage resistant Escherichia coli strains Zou, Xuan; Xiao, Xiaohong; Mo, Ziran Nature Communications, Vol. 13, Issue 1 https://doi.org/10.1038/s41467-022-31934-9	journal	August 2022
Experimental validation that human microbiome phages use alternative genetic coding Peters, Samantha L.; Borges, Adair L.; Giannone, Richard J. Nature Communications, Vol. 13, Issue 1 https://doi.org/10.1038/s41467-022-32979-6	journal	September 2022
Widespread stop-codon recoding in bacteriophages may regulate translation of lytic genes Borges, Adair L.; Lou, Yue Clare; Sachdeva, Rohan Nature Microbiology, Vol. 7, Issue 6 https://doi.org/10.1038/s41564-022-01128-6	journal	May 2022
Total synthesis of Escherichia coli with a recoded genome Fredens, Julius; Wang, Kaihang; de la Torre, Daniel Nature, Vol. 569, Issue 7757 https://doi.org/10.1038/s41586-019-1192-5	journal	May 2019
Clades of huge phages from across Earth’s ecosystems Al-Shayeb, Basem; Sachdeva, Rohan; Chen, Lin-Xing Nature, Vol. 578, Issue 7795 https://doi.org/10.1038/s41586-020-2007-4	journal	February 2020
Viral contamination in biologic manufacture and implications for emerging therapies Barone, Paul W.; Wiebe, Michael E.; Leung, James C. Nature Biotechnology, Vol. 38, Issue 5 https://doi.org/10.1038/s41587-020-0507-2	journal	April 2020
Automated design of thousands of nonrepetitive parts for engineering stable genetic systems Hossain, Ayaan; Lopez, Eriberto; Halper, Sean M. Nature Biotechnology, Vol. 38, Issue 12 https://doi.org/10.1038/s41587-020-0584-2	journal	July 2020
Two conformations of a crystalline human tRNA synthetase–tRNA complex: implications for protein synthesis Yang, Xiang-Lei; Otero, Francella J.; Ewalt, Karla L. The EMBO Journal, Vol. 25, Issue 12 https://doi.org/10.1038/sj.emboj.7601154	journal	May 2006
Discovery of epoxyqueuosine (oQ) reductase reveals parallels between halorespiration and tRNA modification Miles, Z. D.; McCarty, R. M.; Molnar, G. Proceedings of the National Academy of Sciences, Vol. 108, Issue 18 https://doi.org/10.1073/pnas.1018636108	journal	April 2011
A cytotoxic ribonuclease which specifically cleaves four isoaccepting arginine tRNAs at their anticodon loops Tomita, K.; Ogawa, T.; Uozumi, T. Proceedings of the National Academy of Sciences, Vol. 97, Issue 15 https://doi.org/10.1073/pnas.140213797	journal	July 2000
A highly precise and portable genome engineering method allows comparison of mutational effects across bacterial species Nyerges, Ákos; Csörgő, Bálint; Nagy, István Proceedings of the National Academy of Sciences, Vol. 113, Issue 9 https://doi.org/10.1073/pnas.1520040113	journal	February 2016
Engineering posttranslational proofreading to discriminate nonstandard amino acids Kunjapur, Aditya M.; Stork, Devon A.; Kuru, Erkin Proceedings of the National Academy of Sciences, Vol. 115, Issue 3 https://doi.org/10.1073/pnas.1715137115	journal	January 2018
Adaptive evolution of genomically recoded Escherichia coli Wannier, Timothy M.; Kunjapur, Aditya M.; Rice, Daniel P. Proceedings of the National Academy of Sciences, Vol. 115, Issue 12 https://doi.org/10.1073/pnas.1715530115	journal	February 2018
Functionality of tRNAs encoded in a mobile genetic element from an acidophilic bacterium Alamos, Pamela; Tello, Mario; Bustamante, Paula RNA Biology, Vol. 15, Issue 4-5 https://doi.org/10.1080/15476286.2017.1349049	journal	August 2017
Non-parametric estimation of posterior error probabilities associated with peptides identified by tandem mass spectrometry Kall, L.; Storey, J. D.; Noble, W. S. Bioinformatics, Vol. 24, Issue 16 https://doi.org/10.1093/bioinformatics/btn294	journal	August 2008
Prokka: rapid prokaryotic genome annotation Seemann, T. Bioinformatics, Vol. 30, Issue 14 https://doi.org/10.1093/bioinformatics/btu153	journal	March 2014
Universal rules and idiosyncratic features in tRNA identity Giege, R.; Sissler, M.; Florentz, C. Nucleic Acids Research, Vol. 26, Issue 22 https://doi.org/10.1093/nar/26.22.5017	journal	November 1998
Role of a cryptic tRNA gene operon in survival under translational stress Santamaría-Gómez, Javier; Rubio, Miguel Ángel; López-Igual, Rocío Nucleic Acids Research, Vol. 49, Issue 15 https://doi.org/10.1093/nar/gkab661	journal	August 2021
tRNAscan-SE 2.0: improved detection and functional classification of transfer RNA genes Chan, Patricia P.; Lin, Brian Y.; Mak, Allysia J. Nucleic Acids Research, Vol. 49, Issue 16 https://doi.org/10.1093/nar/gkab688	journal	August 2021
Mechanistic insights into tRNA cleavage by a contact-dependent growth inhibitor protein and translation factors Wang, Jing; Yashiro, Yuka; Sakaguchi, Yuriko Nucleic Acids Research, Vol. 50, Issue 8 https://doi.org/10.1093/nar/gkac228	journal	April 2022
Phage T4 SegB protein is a homing endonuclease required for the preferred inheritance of T4 tRNA gene region occurring in co-infection with a related phage Brok-Volchanskaya, V. S.; Kadyrov, F. A.; Sivogrivov, D. E. Nucleic Acids Research, Vol. 36, Issue 6 https://doi.org/10.1093/nar/gkn053	journal	February 2008
tRNADB-CE 2011: tRNA gene database curated manually by experts Abe, T.; Ikemura, T.; Sugahara, J. Nucleic Acids Research, Vol. 39, Issue Database https://doi.org/10.1093/nar/gkq1007	journal	November 2010
tRNA recognition by a bacterial tRNA Xm32 modification enzyme from the SPOUT methyltransferase superfamily Liu, Ru-Juan; Long, Tao; Zhou, Mi Nucleic Acids Research, Vol. 43, Issue 15 https://doi.org/10.1093/nar/gkv745	journal	July 2015
PHROG: families of prokaryotic virus proteins clustered using remote homology Terzian, Paul; Olo Ndela, Eric; Galiez, Clovis NAR Genomics and Bioinformatics, Vol. 3, Issue 3 https://doi.org/10.1093/nargab/lqab067	journal	June 2021
Murine gammaherpesvirus 68 encodes tRNA-like sequences which are expressed during latency. Bowden, R. J.; Efstathiou, S.; Davis, A. J. Journal of General Virology, Vol. 78, Issue 7 https://doi.org/10.1099/0022-1317-78-7-1675	journal	July 1997
Synthetic auxotrophy remains stable after continuous evolution and in coculture with mammalian cells Kunjapur, Aditya M.; Napolitano, Michael G.; Hysolli, Eriona Science Advances, Vol. 7, Issue 27 https://doi.org/10.1126/sciadv.abf5851	journal	July 2021
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
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
The Genome Project-Write Boeke, J. D.; Church, G.; Hessel, A. Science, Vol. 353, Issue 6295 https://doi.org/10.1126/science.aaf6850	journal	June 2016
Sense codon reassignment enables viral resistance and encoded polymer synthesis Robertson, Wesley E.; Funke, Louise F. H.; de la Torre, Daniel Science, Vol. 372, Issue 6546 https://doi.org/10.1126/science.abg3029	journal	June 2021
Refactored genetic codes enable bidirectional genetic isolation Zürcher, Jérôme F.; Robertson, Wesley E.; Kappes, Tomás Science, Vol. 378, Issue 6619 https://doi.org/10.1126/science.add8943	journal	November 2022
Rapid Evolution of Reduced Susceptibility against a Balanced Dual-Targeting Antibiotic through Stepping-Stone Mutations Szili, Petra; Draskovits, Gábor; Révész, Tamás Antimicrobial Agents and Chemotherapy, Vol. 63, Issue 9 https://doi.org/10.1128/AAC.00207-19	journal	June 2019
Simple Method for Plating Escherichia coli Bacteriophages Forming Very Small Plaques or No Plaques under Standard Conditions Łoś, Joanna M.; Golec, Piotr; Węgrzyn, Grzegorz Applied and Environmental Microbiology, Vol. 74, Issue 16 https://doi.org/10.1128/AEM.00306-08	journal	August 2008
The Essential Genome of Escherichia coli K-12 Goodall, Emily C. A.; Robinson, Ashley; Johnston, Iain G. mBio, Vol. 9, Issue 1 https://doi.org/10.1128/mBio.02096-17	journal	February 2018
Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 Love, Michael I.; Huber, Wolfgang; Anders, Simon Genome Biology, Vol. 15, Issue 12 https://doi.org/10.1186/s13059-014-0550-8	journal	December 2014
Sc3.0: revamping and minimizing the yeast genome Dai, Junbiao; Boeke, Jef D.; Luo, Zhouqing Genome Biology, Vol. 21, Issue 1 https://doi.org/10.1186/s13059-020-02130-z	journal	August 2020
Systematic exploration of Escherichia coli phage–host interactions with the BASEL phage collection Maffei, Enea; Shaidullina, Aisylu; Burkolter, Marco PLOS Biology, Vol. 19, Issue 11 https://doi.org/10.1371/journal.pbio.3001424	journal	November 2021
MassIVE MSV000089854 - Swapped genetic code block viral infections and gene transfer Church, George M. MassIVE https://doi.org/10.25345/C5FF3M41W	dataset	January 2022
Global In-Silico Scenario of tRNA Genes and Their Organization in Virus Genomes Morgado, Sergio; Vicente, Ana Viruses, Vol. 11, Issue 2 https://doi.org/10.3390/v11020180	journal	February 2019
Stop codon recoding is widespread in diverse phage lineages and has the potential to regulate translation of late stage and lytic genes Borges, Adair L. Zenodo https://doi.org/10.5281/zenodo.5275335	dataset	January 2021
Resurrection of a global, metagenomically defined gokushovirus Kirchberger, Paul C.; Ochman, Howard eLife, Vol. 9 https://doi.org/10.7554/eLife.51599	journal	February 2020
Phage on tap–a quick and efficient protocol for the preparation of bacteriophage laboratory stocks Bonilla, Natasha; Rojas, Maria Isabel; Netto Flores Cruz, Giuliano PeerJ, Vol. 4 https://doi.org/10.7717/peerj.2261	journal	January 2016