Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Perspective
  • Published:

Directed evolution in mammalian cells

Nature Methods volume 18, pages 346–357 (2021)Cite this article

Subjects

Abstract

Directed evolution experiments are typically carried out using in vitro systems, bacteria, or yeast—even when the goal is to probe or modulate mammalian biology. Performing directed evolution in systems that do not match the intended mammalian environment severely constrains the scope and functionality of the targets that can be evolved. We review new platforms that are now making it possible to use the mammalian cell itself as the setting for directed evolution and present an overview of frontier challenges and high-impact targets for this approach.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Directed evolution for function in mammalian cells.
Fig. 2: Directed evolution in mammalian cells.
Fig. 3: Select examples of directed evolution experiments using ex mammalia mutagenesis.
Fig. 4: Targeted in mammalia mutagenesis.
Fig. 5: mPACE-style directed evolution platforms.
Fig. 6: Select examples of high-impact targets for mammalian cell-based directed evolution.

Similar content being viewed by others

References

  1. Drummond, D. A. & Bloom, J. D. A Nobel Prize for evolution. Evolution 73, 630–631 (2019).

    Article  Google Scholar 

  2. Visintin, M., Tse, E., Axelson, H., Rabbitts, T. H. & Cattaneo, A. Selection of antibodies for intracellular function using a two-hybrid in vivo system. Proc. Natl Acad. Sci. USA 96, 11723–11728 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Herwig, L. et al. Directed evolution of a bright near-infrared fluorescent rhodopsin using a synthetic chromophore. Cell Chem. Biol. 24, 415–425 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Buskirk, A. R., Ong, Y.-C., Gartner, Z. J. & Liu, D. R. Directed evolution of ligand dependence: small-molecule-activated protein splicing. Proc. Natl Acad. Sci. USA 101, 10505–10510 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Peck, S. H., Chen, I. & Liu, D. R. Directed evolution of a small-molecule-triggered intein with improved splicing properties in mammalian cells. Chem. Biol. 18, 619–630 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Italia, J. S. et al. Genetically encoded protein sulfation in mammalian cells. Nat. Chem. Biol. 16, 379–382 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Armbruster, B. N., Li, X., Pausch, M. H., Herlitze, S. & Roth, B. L. Evolving the lock to fit the key to create a family of G protein-coupled receptors potently activated by an inert ligand. Proc. Natl Acad. Sci. USA 104, 5163–5168 (2007).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  8. Pourmir, A. & Johannes, T. W. Directed evolution: selection of the host organism. Comput. Struct. Biotechnol. J. 2, e201209012 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  9. Kachroo, A. H. et al. Systematic humanization of yeast genes reveals conserved functions and genetic modularity. Science 348, 921–925 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Cirino, P. C., Mayer, K. M. & Umeno, D. Generating mutant libraries using error-prone PCR. Methods Mol. Biol. 231, 3–9 (2003).

    CAS  PubMed  Google Scholar 

  11. Muteeb, G. & Sen, R. Random mutagenesis using a mutator strain. Methods Mol. Biol. 634, 411–419 (2010).

    Article  CAS  PubMed  Google Scholar 

  12. Badran, A. H. & Liu, D. R. Development of potent in vivo mutagenesis plasmids with broad mutational spectra. Nat. Commun. 6, 8425 (2015).

    Article  CAS  PubMed  Google Scholar 

  13. Villette, V. et al. Ultrafast two-photon imaging of a high-gain voltage indicator in awake behaving mice. Cell 179, 1590–1608 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Odegard, V. H. & Schatz, D. G. Targeting of somatic hypermutation. Nat. Rev. Immunol. 6, 573–583 (2006).

    Article  CAS  PubMed  Google Scholar 

  15. Pickar-Oliver, A. & Gersbach, C. A. The next generation of CRISPR–Cas technologies and applications. Nat. Rev. Mol. Cell Biol. 20, 490–507 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Moore, C. L., Papa, L. J. 3rd & Shoulders, M. D. A processive protein chimera introduces mutations across defined DNA regions in vivo. J. Am. Chem. Soc. 140, 11560–11564 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Chen, H. et al. Efficient, continuous mutagenesis in human cells using a pseudo-random DNA editor. Nat. Biotechnol. 38, 165–168 (2020).

    Article  CAS  PubMed  Google Scholar 

  18. Sansbury, B. M., Hewes, A. M. & Kmiec, E. B. Understanding the diversity of genetic outcomes from CRISPR–Cas generated homology-directed repair. Commun. Biol. 2, 458 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Erdogan, M., Fabritius, A., Basquin, J. & Griesbeck, O. Targeted in situ protein diversification and intra-organelle validation in mammalian cells. Cell Chem. Biol. 27, 610–621 (2020).

    Article  CAS  PubMed  Google Scholar 

  20. Emery, C. M. et al. MEK1 mutations confer resistance to MEK and B-RAF inhibition. Proc. Natl Acad. Sci. USA 106, 20411–20416 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. 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).

    Article  CAS  PubMed  Google Scholar 

  22. McDermott, M. et al. In vitro development of chemotherapy and targeted therapy drug-resistant cancer cell lines: a practical guide with case studies. Front. Oncol. 4, 40 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  23. Booth, L. et al. The afatinib resistance of in vivo generated H1975 lung cancer cell clones is mediated by SRC/ERBB3/c-KIT/c-MET compensatory survival signaling. Oncotarget 7, 19620–19630 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  24. Forment, J. V. et al. Genome-wide genetic screening with chemically mutagenized haploid embryonic stem cells. Nat. Chem. Biol. 13, 12–14 (2017).

    Article  CAS  PubMed  Google Scholar 

  25. Berman, C. M. et al. An adaptable platform for directed evolution in human cells. J. Am. Chem. Soc. 140, 18093–18103 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. English, J. G. et al. VEGAS as a platform for facile directed evolution in mammalian cells. Cell 178, 748–761 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Banaszynski, L. A., Chen, L.-C., Maynard-Smith, L. A., Ooi, A. G. L. & Wandless, T. J. A rapid, reversible, and tunable method to regulate protein function in living cells using synthetic small molecules. Cell 126, 995–1004 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Iwamoto, M., Björklund, T., Lundberg, C., Kirik, D. & Wandless, T. J. A general chemical method to regulate protein stability in the mammalian central nervous system. Chem. Biol. 17, 981–988 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Miyazaki, Y., Imoto, H., Chen, L.-C. & Wandless, T. J. Destabilizing domains derived from the human estrogen receptor. J. Am. Chem. Soc. 134, 3942–3945 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Piatkevich, K. D. et al. A robotic multidimensional directed evolution approach applied to fluorescent voltage reporters. Nat. Chem. Biol. 14, 352–360 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Raz, T., Nardi, V., Azam, M., Cortes, J. & Daley, G. Q. Farnesyl transferase inhibitor resistance probed by target mutagenesis. Blood 110, 2102–2109 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Maji, B. et al. Multidimensional chemical control of CRISPR–Cas9. Nat. Chem. Biol. 13, 9–11 (2017).

    Article  CAS  PubMed  Google Scholar 

  33. Dietz, O. et al. Characterization of the small exported Plasmodium falciparum membrane protein SEMP1. PLoS ONE 9, e103272 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  34. Hochbaum, D. R. et al. All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins. Nat. Methods 11, 825–833 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. St-Pierre, F. et al. High-fidelity optical reporting of neuronal electrical activity with an ultrafast fluorescent voltage sensor. Nat. Neurosci. 17, 884–889 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Capdeville, R., Buchdunger, E., Zimmermann, J. & Matter, A. Glivec (STI571, imatinib), a rationally developed, targeted anticancer drug. Nat. Rev. Drug Discov. 1, 493–502 (2002).

    Article  CAS  PubMed  Google Scholar 

  37. Soverini, S. et al. BCR-ABL kinase domain mutation analysis in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors: recommendations from an expert panel on behalf of European LeukemiaNet. Blood 118, 1208–1215 (2011).

    Article  CAS  PubMed  Google Scholar 

  38. Mahon, M. J. Vectors bicistronically linking a gene of interest to the SV40 large T antigen in combination with the SV40 origin of replication enhance transient protein expression and luciferase reporter activity. Biotechniques 51, 119–128 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Brown, D. M. & Glass, J. I. Technology used to build and transfer mammalian chromosomes. Exp. Cell Res. 388, 111851 (2020).

    Article  CAS  PubMed  Google Scholar 

  40. Ravikumar, A., Arzumanyan, G. A., Obadi, M. K. A., Javanpour, A. A. & Liu, C. C. Scalable, continuous evolution of genes at mutation rates above genomic error thresholds. Cell 175, 1946–1957 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Pavri, R. & Nussenzweig, M. C. AID targeting in antibody diversity. Adv. Immunol. 110, 1–26 (2011).

    Article  CAS  PubMed  Google Scholar 

  42. Bowers, P. M. et al. Coupling mammalian cell surface display with somatic hypermutation for the discovery and maturation of human antibodies. Proc. Natl Acad. Sci. USA 108, 20455–20460 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Wang, L., Jackson, W. C., Steinbach, P. A. & Tsien, R. Y. Evolution of new nonantibody proteins via iterative somatic hypermutation. Proc. Natl Acad. Sci. USA 101, 16745–16749 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Al-Qaisi, T. S., Su, Y.-C. & Roffler, S. R. Transient AID expression for in situ mutagenesis with improved cellular fitness. Sci. Rep. 8, 9413 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  45. Wang, C. L., Harper, R. A. & Wabl, M. Genome-wide somatic hypermutation. Proc. Natl Acad. Sci. USA 101, 7352–7356 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Donovan, K. F. et al. Creation of novel protein variants with CRISPR/Cas9-mediated mutagenesis: turning a screening by-product into a discovery tool. PLoS ONE 12, e0170445 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  47. Ipsaro, J. J. et al. Rapid generation of drug-resistance alleles at endogenous loci using CRISPR–Cas9 indel mutagenesis. PLoS ONE 12, e0172177 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  48. Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A. & Liu, D. R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Ma, Y. et al. Targeted AID-mediated mutagenesis (TAM) enables efficient genomic diversification in mammalian cells. Nat. Methods 13, 1029–1035 (2016).

    Article  CAS  PubMed  Google Scholar 

  50. Hess, G. T. et al. Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells. Nat. Methods 13, 1036–1042 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Devilder, M. C. et al. Ex vivo evolution of human antibodies by CRISPR-X: from a naive B cell repertoire to affinity matured antibodies. BMC Biotechnol. 19, 14 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  52. Gaudelli, N. M. et al. Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage. Nature 551, 464–471 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Gaudelli, N. M. et al. Directed evolution of adenine base editors with increased activity and therapeutic application. Nat. Biotechnol. 38, 892–900 (2020).

  54. Thiel, V., Herold, J., Schelle, B. & Siddell, S. G. Infectious RNA transcribed in vitro from a cDNA copy of the human coronavirus genome cloned in vaccinia virus. J. Gen. Virol. 82, 1273–1281 (2001).

    Article  CAS  PubMed  Google Scholar 

  55. Park, H. & Kim, S. Gene-specific mutagenesis enables rapid continuous evolution of enzymes in vivo. Nucleic Acids Res. https://doi.org/10.1093/nar/gkaa1231 (2021).

  56. Álvarez, B., Mencía, M., de Lorenzo, V. & Fernández, L. A. In vivo diversification of target genomic sites using processive base deaminase fusions blocked by dCas9. Nat. Commun. 11, e6436 (2020).

    Article  CAS  Google Scholar 

  57. Kurt, I. C. et al. CRISPR C-to-G base editors for inducing targeted DNA transversions in human cells. Nat. Biotechnol. 39, 41–46 (2021).

    Article  CAS  PubMed  Google Scholar 

  58. Esvelt, K. M., Carlson, J. C. & Liu, D. R. A system for the continuous directed evolution of biomolecules. Nature 472, 499–503 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Dai, X., Zhang, X., Ostrikov, K. & Abrahamyan, L. Host receptors: the key to establishing cells with broad viral tropism for vaccine production. Crit. Rev. Microbiol. 46, 147–168 (2020).

    Article  CAS  PubMed  Google Scholar 

  60. Danthinne, X. & Imperiale, M. J. Production of first generation adenovirus vectors: a review. Gene Ther. 7, 1707–1714 (2000).

    Article  CAS  PubMed  Google Scholar 

  61. Chira, S. et al. Progresses towards safe and efficient gene therapy vectors. Oncotarget 6, 30675–30703 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  62. Mullick, A. et al. The cumate gene-switch: a system for regulated expression in mammalian cells. BMC Biotechnol. 6, 43 (2006).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  63. Uil, T. G. et al. Directed adenovirus evolution using engineered mutator viral polymerases. Nucleic Acids Res. 39, e30 (2011).

    Article  CAS  PubMed  Google Scholar 

  64. 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).

    Article  CAS  PubMed  Google Scholar 

  65. Richter, M. F. et al. Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity. Nat. Biotechnol. 38, 883–891 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Wang, T., Badran, A. H., Huang, T. P. & Liu, D. R. Continuous directed evolution of proteins with improved soluble expression. Nat. Chem. Biol. 14, 972–980 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Dickinson, B. C., Packer, M. S., Badran, A. H. & Liu, D. R. A system for the continuous directed evolution of proteases rapidly reveals drug-resistance mutations. Nat. Commun. 5, 5352 (2014).

    Article  CAS  PubMed  Google Scholar 

  68. Miller, S. M. et al. Continuous evolution of SpCas9 variants compatible with non-G PAMs. Nat. Biotechnol. 38, 471–481 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Mac Sweeney, A. et al. Discovery and structure-based optimization of adenain inhibitors. ACS Med. Chem. Lett. 5, 937–941 (2014).

    Article  CAS  Google Scholar 

  70. Havenga, M. J. E. et al. Exploiting the natural diversity in adenovirus tropism for therapy and prevention of disease. J. Virol. 76, 4612–4620 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Ellgaard, L., Molinari, M. & Helenius, A. Setting the standards: quality control in the secretory pathway. Science 286, 1882–1888 (1999).

    Article  CAS  PubMed  Google Scholar 

  72. Warren, L. & Lin, C. mRNA-based genetic reprogramming. Mol. Ther. 27, 729–734 (2019).

    Article  CAS  PubMed  Google Scholar 

  73. Trepotec, Z., Lichtenegger, E., Plank, C., Aneja, M. K. & Rudolph, C. Delivery of mRNA therapeutics for the treatment of hepatic diseases. Mol. Ther. 27, 794–802 (2019).

    Article  CAS  PubMed  Google Scholar 

  74. Chen, A. & Koehler, A. N. Transcription factor inhibition: lessons learned and emerging targets. Trends Mol. Med. 26, 508–518 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Conaway, R. C. & Conaway, J. W. Origins and activity of the Mediator complex. Semin. Cell Dev. Biol. 22, 729–734 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Conaway, J. W. et al. The mammalian Mediator complex. FEBS Lett. 579, 904–908 (2005).

    Article  CAS  PubMed  Google Scholar 

  77. Kennedy, B. K. Mammalian transcription factors in yeast: strangers in a familiar land. Nat. Rev. Mol. Cell Biol. 3, 41–49 (2002).

    Article  CAS  PubMed  Google Scholar 

  78. Wang, L., Xie, J. & Schultz, P. G. Expanding the genetic code. Annu. Rev. Biophys. Biomol. Struct. 35, 225–249 (2006).

    Article  PubMed  CAS  Google Scholar 

  79. Chin, J. W. Expanding and reprogramming the genetic code of cells and animals. Annu. Rev. Biochem. 83, 379–408 (2014).

    Article  CAS  PubMed  Google Scholar 

  80. Italia, J. S. et al. Expanding the genetic code of mammalian cells. Biochem. Soc. Trans. 45, 555–562 (2017).

    Article  CAS  PubMed  Google Scholar 

  81. Hammerling, M. J. et al. In vitro ribosome synthesis and evolution through ribosome display. Nat. Commun. 11, 1108 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  82. Sebastian, R. M. & Shoulders, M. D. Chemical biology framework to illuminate proteostasis. Annu. Rev. Biochem. 89, 529–555 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  83. Marinko, J. T. et al. Folding and misfolding of human membrane proteins in health and disease: from single molecules to cellular proteostasis. Chem. Rev. 119, 5537–5606 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Sachsenhauser, V. & Bardwell, J. C. A. Directed evolution to improve protein folding in vivo. Curr. Opin. Struct. Biol. 48, 117–123 (2018).

    Article  CAS  PubMed  Google Scholar 

  85. Chuh, K. N., Batt, A. R. & Pratt, M. R. Chemical methods for encoding and decoding of posttranslational modifications. Cell Chem. Biol. 23, 86–107 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  86. Christians, F. C., Scapozza, L., Crameri, A., Folkers, G. & Stemmer, W. P. C. Directed evolution of thymidine kinase for AZT phosphorylation using DNA family shuffling. Nat. Biotechnol. 17, 259–264 (1999).

    Article  CAS  PubMed  Google Scholar 

  87. Xu, H.-F., Zhang, X.-E., Zhang, Z.-P., Zhang, Y.-M. & Cass, A. E. G. Directed evolution of E. coli alkaline phosphatase towards higher catalytic activity. Biocatal. Biotransform. 21, 41–47 (2009).

    Article  Google Scholar 

  88. Hu, D., Tateno, H., Kuno, A., Yabe, R. & Hirabayashi, J. Directed evolution of lectins with sugar-binding specificity for 6-sulfo-galactose. J. Biol. Chem. 287, 20313–20320 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  89. Yang, G. et al. Fluorescence activated cell sorting as a general ultra-high-throughput screening method for directed evolution of glycosyltransferases. J. Am. Chem. Soc. 132, 10570–10577 (2010).

    Article  CAS  PubMed  Google Scholar 

  90. Atlasi, Y. & Stunnenberg, H. G. The interplay of epigenetic marks during stem cell differentiation and development. Nat. Rev. Genet. 18, 643–658 (2017).

    Article  CAS  PubMed  Google Scholar 

  91. Iwafuchi-Doi, M. & Zaret, K. S. Pioneer transcription factors in cell reprogramming. Genes Dev. 28, 2679–2692 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  92. Park, M., Patel, N., Keung, A. J. & Khalil, A. S. Engineering epigenetic regulation using synthetic read–write modules. Cell 176, 227–238 (2019).

    Article  CAS  PubMed  Google Scholar 

  93. Pulecio, J., Verma, N., Mejía-Ramírez, E., Huangfu, D. & Raya, A. CRISPR/Cas9-based engineering of the epigenome. Cell Stem Cell 21, 431–447 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  94. Julius, D. & Nathans, J. Signaling by sensory receptors. Cold Spring Harb. Perspect. Biol. 4, a005991 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  95. Subramanyam, P. & Colecraft, H. M. Ion channel engineering: perspectives and strategies. J. Mol. Biol. 427, 190–204 (2015).

    Article  CAS  PubMed  Google Scholar 

  96. Romero, P. A. & Arnold, F. H. Exploring protein fitness landscapes by directed evolution. Nat. Rev. Mol. Cell Biol. 10, 866–876 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  97. Geller, R., Pechmann, S., Acevedo, A., Andino, R. & Frydman, J. Hsp90 shapes protein and RNA evolution to balance trade-offs between protein stability and aggregation. Nat. Commun. 9, 1781 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  98. Phillips, A. M. et al. Host proteostasis modulates influenza evolution. eLife 6, e28652 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  99. Lu, R.-M. et al. Development of therapeutic antibodies for the treatment of diseases. J. Biomed. Sci. 27, 1 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  100. Guedan, S., Calderon, H., Posey, A. D. Jr. & Maus, M. V. Engineering and design of chimeric antigen receptors. Mol. Ther. Methods Clin. Dev. 12, 145–156 (2019).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

S.J.H. acknowledges support from the National Science Foundation Graduate Research Fellowship Program under grant no. 1745302. M.D.S. acknowledges support from the National Institutes of Health, National Institute of General Medical Sciences (1R35GM136354).

Author information

Authors and Affiliations

  1. Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, USA

    Samuel J. Hendel & Matthew D. Shoulders

Authors
  1. Samuel J. Hendel
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Matthew D. Shoulders
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

S.J.H. and M.D.S. wrote and revised the manuscript.

Corresponding author

Correspondence to Matthew D. Shoulders.

Ethics declarations

Competing interests

S.J.H. and M.D.S. are co-inventors on a patent application filed by MIT related to the use of adenoviruses for mammalian cell-based directed evolution. M.D.S. is a co-inventor on a patent application filed by MIT related to the MutaT7 system.

Additional information

Peer review information Nature Methods thanks the anonymous reviewers for their contribution to the peer review of this work. Rita Strack was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Hendel, S.J., Shoulders, M.D. Directed evolution in mammalian cells. Nat Methods 18, 346–357 (2021). https://doi.org/10.1038/s41592-021-01090-x

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41592-021-01090-x

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing