CoChat
Sign Up
Cited by 7Evolution and Genetic DynamicsCRISPR and Genetic EngineeringRNA and protein synthesis mechanisms

The DOI matches its Crossref record; 3 of the 3 references it cites were found in a registry or an index.

A chimeric viral platform for directed evolution in mammalian cells

Open access
Cole, Alexander J., Denes, Christopher E., Moreno, Cesar L., Hunault, Lise, Dobson, Thomas, Hesselson, Daniel, Neely, G. Gregory·2025·Nature Communications
DOI 10.1038/s41467-025-59438-2PMC PMC12059018

Published version of a preprint

Open this paper in CoChat — free accountOpen PDF

Abstract

Directed evolution is a process of mutation and artificial selection to breed biomolecules with new or improved activity. Directed evolution platforms are primarily prokaryotic or yeast-based, and stable mammalian systems have been challenging to establish and apply. To this end, we develop PROTein Evolution Using Selection (PROTEUS), a platform that uses chimeric virus-like vesicles to enable extended mammalian directed evolution campaigns without loss of system integrity. This platform is stable and can generate sufficient diversity for directed evolution in mammalian systems. Using PROTEUS, we alter the doxycycline responsiveness of tetracycline-controlled transactivators, generating a more sensitive TetON-4G tool for gene regulation with mammalian-specific adaptations. PROTEUS is also compatible with intracellular nanobody evolution, and we use it to evolve a DNA damage-responsive anti-p53 nanobody. Overall, PROTEUS is an efficient and stable platform to direct evolution of biomolecules within mammalian cells.

Overview

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.1038/s41467-025-59438-2

Cites

3 of 3 references verified0 citation errors · 0 not checkable

Statistics

Verification

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Citations

Cites

Flagged first, then by citations

Research works printed but not linked: 50

  1. Romero, P. A. & Arnold, F. H. Exploring protein fitness landscapes by directed evolution. Nat. Rev. Mol. Cell Biol. 10, 866–876 (2009). · DOI
  2. Packer, M. S. & Liu, D. R. Methods for the directed evolution of proteins. Nat. Rev. Genet. 16, 379–394 (2015). · DOI
  3. Eigen, M. & Gardiner, W. Evolutionary molecular engineering based on RNA replication. J. Macromol. Sci. Part A Pure Appl. Chem. 56, 967–978 (1984). · DOI
  4. Liao, H., McKenzie, T. & Hageman, R. Isolation of a thermostable enzyme variant by cloning and selection in a thermophile. Proc. Natl Acad. Sci. USA. 83, 576–580 (1986). · DOI
  5. Chen, K. Q. & Arnold, F. H. Enzyme engineering for nonaqueous solvents: random mutagenesis to enhance activity of subtilisin E in polar organic media. Biotechnology 9, 1073–1077 (1991). · DOI
  6. Stemmer, W. P. Rapid evolution of a protein in vitro by DNA shuffling. Nature 370, 389–391 (1994). · DOI
  7. Esvelt, K. M., Carlson, J. C. & Liu, D. R. A system for the continuous directed evolution of biomolecules. Nature 472, 499–503 (2011). · DOI
  8. Ravikumar, A., Arrieta, A. & Liu, C. C. An orthogonal DNA replication system in yeast. Nat. Chem. Biol. 10, 175–177 (2014). · DOI
  9. Hendel, S. J. & Shoulders, M. D. Directed evolution in mammalian cells. Nat. Methods 18, 346–357 (2021). · DOI
  10. Azam, M., Latek, R. R. & Daley, G. Q. Mechanisms of autoinhibition and STI-571/imatinib resistance revealed by mutagenesis of BCR-ABL. Cell 112, 831–843 (2003). · DOI
  11. Park, H. & Kim, S. Gene-specific mutagenesis enables rapid continuous evolution of enzymes in vivo. Nucleic Acids Res. 49, e32 (2021). · DOI
  12. Hess, G. T. et al. Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells. Nat. Methods 13, 1036–1042 (2016). · DOI
  13. Ma, L. & Lin, Y. Orthogonal RNA replication enables directed evolution and Darwinian adaptation in mammalian cells. Nat. Chem. Biol. 21, 451–463 (2025). · DOI
  14. Molina, R. S. et al. In vivo hypermutation and continuous evolution. Nat. Rev. Methods Primers 2, 36 (2022). · DOI
  15. Das, A. T. et al. Viral evolution as a tool to improve the tetracycline-regulated gene expression system. J. Biol. Chem. 279, 18776–18782 (2004). · DOI · Paper page
  16. Berman, C. M. et al. An adaptable platform for directed evolution in human cells. J. Am. Chem. Soc. 140, 18093–18103 (2018). · DOI
  17. Jewel, D. et al. Virus-assisted directed evolution of enhanced suppressor tRNAs in mammalian cells. Nat. Methods 20, 95–103 (2023). · DOI
  18. Klenk, C. et al. A Vaccinia-based system for directed evolution of GPCRs in mammalian cells. Nat. Commun. 14, 1770 (2023). · DOI
  19. English, J. G. et al. VEGAS as a platform for facile directed evolution in mammalian. Cells Cell 178, 748–761.e17 (2019). · DOI
  20. Denes, C. E. et al. The VEGAS platform is unsuitable for mammalian directed evolution. ACS Synth. Biol. 11, 3544–3549 (2022). · DOI
  21. Brown, R. S., Anastasakis, D. G., Hafner, M. & Kielian, M. Multiple capsid protein binding sites mediate selective packaging of the alphavirus genomic RNA. Nat. Commun. 11, 4693 (2020). · DOI
  22. Ruiz-Guillen, M. et al. Capsid-deficient alphaviruses generate propagative infectious microvesicles at the plasma membrane. Cell. Mol. Life Sci. 73, 3897–3916 (2016). · DOI
  23. Rolls, M. M., Webster, P., Balba, N. H. & Rose, J. K. Novel infectious particles generated by expression of the vesicular stomatitis virus glycoprotein from a self-replicating RNA. Cell 79, 497–506 (1994). · DOI
  24. Rose, N. F. et al. In vitro evolution of high-titer, virus-like vesicles containing a single structural protein. Proc. Natl Acad. Sci. USA. 111, 16866–16871 (2014). · DOI
  25. Akhrymuk, I., Lukash, T., Frolov, I. & Frolova, E. I. Novel mutations in nsP2 abolish chikungunya virus-induced transcriptional shutoff and make the virus less cytopathic without affecting its replication rates. J. Virol. 93, e02062-18 (2019). · DOI
  26. Belshaw, R., Pybus, O. G. & Rambaut, A. The evolution of genome compression and genomic novelty in RNA viruses. Genome Res 17, 1496–1504 (2007). · DOI
  27. Patterson, E. I. et al. Measuring alphavirus fidelity using non-infectious virus particles. Viruses 12, 546 (2020). · DOI
  28. O’Hara, P. J., Nichol, S. T., Horodyski, F. M. & Holland, J. J. Vesicular stomatitis virus defective interfering particles can contain extensive genomic sequence rearrangements and base substitutions. Cell 36, 915–924 (1984). · DOI
  29. Drewett, V. et al. Serum response factor cleavage by caspases 3 and 7 linked to apoptosis in human BJAB cells. J. Biol. Chem. 276, 33444–33451 (2001). · DOI
  30. Hecht, B., Müller, G. & Hillen, W. Noninducible Tet repressor mutations map from the operator binding motif to the C terminus. J. Bacteriol. 175, 1206–1210 (1993). · DOI
  31. Müller, G. et al. Characterization of non-inducible Tet repressor mutants suggests conformational changes necessary for induction. Nat. Struct. Biol. 2, 693–703 (1995). · DOI
  32. Van Winkle, A. P., Gates, I. D. & Kallos, M. S. Mass transfer limitations in embryoid bodies during human embryonic stem cell differentiation. Cells Tissues Organs 196, 34–47 (2012). · DOI
  33. Dingus, J. G., Tang, J. C. Y., Amamoto, R., Wallick, G. K. & Cepko, C. L. A general approach for stabilizing nanobodies for intracellular expression. Elife 11, e68253 (2022). · DOI
  34. Bethuyne, J. et al. A nanobody modulates the p53 transcriptional program without perturbing its functional architecture. Nucleic Acids Res. 42, 12928–12938 (2014). · DOI
  35. Denes, C. E. & Neely, G. G. REPLACE-ing RNA through evolution. Nat. Chem. Biol. 3, 322–323 (2025). · DOI
  36. Urakova, N. et al. Β-d-N 4-hydroxycytidine is a potent anti-alphavirus compound that induces a high level of mutations in the viral genome. J. Virol. 92, e01965–17 (2018). · DOI
  37. Vignuzzi, M. & López, C. B. Defective viral genomes are key drivers of the virus-host interaction. Nat. Microbiol 4, 1075–1087 (2019). · DOI
  38. Bass, B. L. RNA editing by adenosine deaminases that act on RNA. Annu. Rev. Biochem. 71, 817–846 (2002). · DOI
  39. Tian, R. et al. Establishing a synthetic orthogonal replication system enables accelerated evolution in. Science 383, 421–426 (2024). · DOI
  40. Otsuki, K., Maeda, J., Yamamoto, H. & Tsubokura, M. Studies on avian infectious bronchitis virus (IBV). III. Interferon induction by and sensitivity to interferon of IBV. Arch. Virol. 60, 249–255 (1979). · DOI
  41. Liljeström, P. & Garoff, H. A new generation of animal cell expression vectors based on the Semliki Forest virus replicon. Biotechnology 9, 1356–1361 (1991). · DOI
  42. Meshram, C. D. et al. Lack of nsP2-specific nuclear functions attenuates chikungunya virus replication both in vitro and in vivo. Virology 534, 14–24 (2019). · DOI
  43. Conant, D. et al. Inference of CRISPR edits from Sanger trace. Data. CRISPR J. 5, 123–130 (2022). · DOI
  44. Loo, L. et al. Fibroblast-expressed LRRC15 is a receptor for SARS-CoV-2 spike and controls antiviral and antifibrotic transcriptional programs. PLoS Biol. 21, e3001967 (2023). · DOI
  45. Fritzell, K., Xu, L.-D., Otrocka, M., Andréasson, C. & Öhman, M. Sensitive ADAR editing reporter in cancer cells enables high-throughput screening of small molecule libraries. Nucleic Acids Res 47, e22 (2019). · DOI
  46. Koboldt, D. C. et al. VarScan 2: somatic mutation and copy number alteration discovery in cancer by exome sequencing. Genome Res 22, 568–576 (2012). · DOI
  47. Baumschlager, A., Rullan, M. & Khammash, M. Exploiting natural chemical photosensitivity of anhydrotetracycline and tetracycline for dynamic and setpoint chemo-optogenetic control. Nat. Commun. 11, 3834 (2020). · DOI
  48. Giandomenico, S. L., Sutcliffe, M. & Lancaster, M. A. Generation and long-term culture of advanced cerebral organoids for studying later stages of neural development. Nat. Protoc. 16, 579–602 (2021). · DOI
  49. Pettersen, E. F. et al. UCSF Chimera–a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004). · DOI
  50. Luo, Y., Batalao, A., Zhou, H. & Zhu, L. Mammalian two-hybrid system: a complementary approach to the yeast two-hybrid system. Biotechniques 22, 350–352 (1997). · DOI

Metadata

Authors
Cole, A. J., Denes, C. E., Moreno, C. L., Hunault, L., Dobson, T., Hesselson, D., & Neely, G. G.
Affiliations
The University of Sydney (AU)
Corresponding author
  • Hesselson, Daniel
  • Neely, G. Gregory
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2025-05-07
Volume
16
Issue
1
Pages
4250-4250
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-025-59438-2
PMC
PMC12059018
OpenAlex
W4410139679
Cited by
7
References
3
Citations by year
2026: 5 · 2025: 2
FWCI
4.12
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
Department of Health | National Health and Medical Research Council (GNT1185002, GNT1107514, GNT1158164, GNT1158165)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Evolution and Genetic Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
Keywords
directed evolution, mammalian cells, tetracycline-controlled transactivator, DNA damage response, gene expression regulation, protein evolution
MeSH terms
Animals, Humans, Tumor Suppressor Protein p53, Directed Molecular Evolution, HEK293 Cells, Single-Domain Antibodies

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review