Continuous targeted hypermutation with a tunable mutation window

Posted on
continuous-targeted-hypermutation-with-a-tunable-mutation-window
Continuous targeted hypermutation with a tunable mutation window

References

  1. Ohta, T. Mechanisms of molecular evolution. Philos. Trans. R. Soc. Lond. B Biol. Sci. 355, 1623–1626 (2000).

  2. Arnold, F. H. Design by directed evolution. Acc. Chem. Res. 31, 125–131 (1998).

    Google Scholar 

  3. Packer, M. S. & Liu, D. R. Methods for the directed evolution of proteins. Nat. Rev. Genet. 16, 379–394 (2015).

    Google Scholar 

  4. Morrison, M. S., Podracky, C. J. & Liu, D. R. The developing toolkit of continuous directed evolution. Nat. Chem. Biol. 16, 610–619 (2020).

    Google Scholar 

  5. Molina, R. S. et al. In vivo hypermutation and continuous evolution. Nat. Rev. Methods Prim. 2, 36 (2022).

    Google Scholar 

  6. Rix, G. & Liu, C. C. Systems for in vivo hypermutation: a quest for scale and depth in directed evolution. Curr. Opin. Chem. Biol. 64, 20–26 (2021).

    Google Scholar 

  7. Di Noia, J. M. & Neuberger, M. S. Molecular mechanisms of antibody somatic hypermutation. Annu. Rev. Biochem. 76, 1–22 (2007).

    Google Scholar 

  8. Wang, Y., Meng, F.-L. & Yeap, L.-S. DNA flexibility can shape the preferential hypermutation of antibody genes. Trends Immunol. 45, 167–176(2024).

  9. Bothwell, A. L. et al. Heavy chain variable region contribution to the NPb family of antibodies: somatic mutation evident in a γ2a variable region. Cell 24, 625–637 (1981).

    Google Scholar 

  10. Betz, A. G., Rada, C., Pannell, R., Milstein, C. & Neuberger, M. S. Passenger transgenes reveal intrinsic specificity of the antibody hypermutation mechanism: clustering, polarity, and specific hot spots. Proc. Natl. Acad. Sci. USA 90, 2385–2388 (1993).

    Google Scholar 

  11. Xu, L. et al. Average gene length is highly conserved in prokaryotes and eukaryotes and diverges only between the two kingdoms. Mol. Biol. Evol. 23, 1107–1108 (2006).

    Google Scholar 

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

    Google Scholar 

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

    Google Scholar 

  14. Álvarez, B., Mencía, M., De Lorenzo, V. & Fernández, L. Á In vivo diversification of target genomic sites using processive base deaminase fusions blocked by dCas9. Nat. Commun. 11, 6436 (2020).

    Google Scholar 

  15. Cravens, A., Jamil, O. K., Kong, D., Sockolosky, J. T. & Smolke, C. D. Polymerase-guided base editing enables in vivo mutagenesis and rapid protein engineering. Nat. Commun. 12, 1579 (2021).

    Google Scholar 

  16. Park, H. & Kim, S. Gene-specific mutagenesis enables rapid continuous evolution of enzymes in vivo. Nucleic Acids Res. 49, e32–e32 (2021).

    Google Scholar 

  17. Ravikumar, A., Arzumanyan, G. A., Obadi, M. K., Javanpour, A. A. & Liu, C. C. Scalable, continuous evolution of genes at mutation rates above genomic error thresholds. Cell 175, 1946–1957. e1913 (2018).

    Google Scholar 

  18. Rix, G. et al. Continuous evolution of user-defined genes at 1 million times the genomic mutation rate. Science 386, eadm9073 (2024).

    Google Scholar 

  19. Tian, R. et al. Establishing a synthetic orthogonal replication system enables accelerated evolution in E. coli. Science 383, 421–426 (2024).

    Google Scholar 

  20. Diercks, C. S. et al. An orthogonal T7 replisome for continuous hypermutation and accelerated evolution in E. coli. Science 389, 618–622 (2025).

    Google Scholar 

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

    Google Scholar 

  22. Halperin, S. O. et al. CRISPR-guided DNA polymerases enable diversification of all nucleotides in a tunable window. Nature 560, 248–252 (2018).

    Google Scholar 

  23. Chen, X. D. et al. Helicase-assisted continuous editing for programmable mutagenesis of endogenous genomes. Science 386, eadn5876 (2024).

    Google Scholar 

  24. Hurtado, J. E. et al. Nickase fidelity drives EvolvR-mediated diversification in mammalian cells. Nat. Commun. 16, 3723 (2025).

    Google Scholar 

  25. Kuznedelov, K. et al. Altered stoichiometry Escherichia coli Cascade complexes with shortened CRISPR RNA spacers are capable of interference and primed adaptation. Nucleic Acids Res. 44, 10849–10861 (2016).

  26. Brouns, S. J. et al. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321, 960–964 (2008).

    Google Scholar 

  27. Xue, C. & Sashital, D. G. Mechanisms of type IE and IF CRISPR-Cas systems in Enterobacteriaceae. EcoSal 8, ESP-0008–ESP-2018 (2019).

  28. Luo, M. L. et al. The CRISPR RNA-guided surveillance complex in Escherichia coli accommodates extended RNA spacers. Nucleic Acids Res. 44, 7385–7394 (2016).

  29. Songailiene, I. et al. Decision-making in Cascade complexes harboring crRNAs of altered length. Cell Rep. 28, 3157–3166. e3154 (2019).

    Google Scholar 

  30. 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).

    Google Scholar 

  31. Nishida, K. et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science 353, aaf8729 (2016).

    Google Scholar 

  32. Gu, S., Bodai, Z., Cowan, Q. T. & Komor, A. C. Base editors: expanding the types of DNA damage products harnessed for genome editing. Gene Genome Ed. 1, 100005 (2021).

    Google Scholar 

  33. Schellenberger, V. et al. A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner. Nat. Biotechnol. 27, 1186–1190 (2009).

    Google Scholar 

  34. Hayes, R. P. et al. Structural basis for promiscuous PAM recognition in type I–E Cascade from E. coli. Nature 530, 499–503 (2016).

    Google Scholar 

  35. Van Erp, P. B. et al. Mechanism of CRISPR-RNA guided recognition of DNA targets in Escherichia coli. Nucleic Acids Res. 43, 8381–8391 (2015).

    Google Scholar 

  36. Mol, C. D. et al. Crystal structure of human uracil-DNA glycosylase in complex with a protein inhibitor: protein mimicry of DNA. Cell 82, 701–708 (1995).

    Google Scholar 

  37. Kast, P. & Hennecke, H. Amino acid substrate specificity of Escherichia coli phenylalanyl-tRNA synthetase altered by distinct mutations. J. Mol. Biol. 222, 99–124 (1991).

    Google Scholar 

  38. Seo, D., Koh, B., Eom, G. -e, Kim, H. W. & Kim, S. A dual gene-specific mutator system installs all transition mutations at similar frequencies in vivo. Nucleic Acids Res. 51, e59–e59 (2023).

    Google Scholar 

  39. Dillard, K. E. et al. Assembly and translocation of a CRISPR-Cas primed acquisition complex. Cell 175, 934–946. e915 (2018).

    Google Scholar 

  40. Komor, A. C. et al. Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C: G-to-T: A base editors with higher efficiency and product purity. Sci. Adv. 3, eaao4774 (2017).

    Google Scholar 

  41. 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).

    Google Scholar 

  42. Neugebauer, M. E. et al. Evolution of an adenine base editor into a small, efficient cytosine base editor with low off-target activity. Nat. Biotechnol. 41, 673–685 (2023).

    Google Scholar 

  43. Wittig, S., Songailiene, I. & Schmidt, C. Formation and stoichiometry of CRISPR-Cascade complexes with varying spacer lengths revealed by native mass spectrometry. J. Am. Soc. Mass Spectrom. 31, 538–546 (2020).

    Google Scholar 

  44. Tuminauskaite, D. et al. DNA interference is controlled by R-loop length in a type I-F1 CRISPR-Cas system. BMC Biol. 18, 1–16 (2020).

    Google Scholar 

  45. Semenova, E. et al. Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence. Proc. Natl. Acad. Sci. USA 108, 10098–10103 (2011).

    Google Scholar 

  46. Cooper, L. A., Stringer, A. M. & Wade, J. T. Determining the specificity of cascade binding, interference, and primed adaptation in vivo in the Escherichia coli type IE CRISPR-Cas system. MBio 9, 02100–02117 (2018).

    Google Scholar 

  47. Rutkauskas, M. et al. Directional R-loop formation by the CRISPR-Cas surveillance complex cascade provides efficient off-target site rejection. Cell Rep. 10, 1534–1543 (2015).

    Google Scholar 

  48. Krivoy, A. et al. Primed CRISPR adaptation in Escherichia coli cells does not depend on conformational changes in the Cascade effector complex detected in Vitro. Nucleic Acids Res. 46, 4087–4098 (2018).

    Google Scholar 

  49. Luo, M. L., Mullis, A. S., Leenay, R. T. & Beisel, C. L. Repurposing endogenous type I CRISPR-Cas systems for programmable gene repression. Nucleic Acids Res. 43, 674–681 (2015).

    Google Scholar 

  50. Schnell, J. R., Dyson, H. J. & Wright, P. E. Structure, dynamics, and catalytic function of dihydrofolate reductase. Annu. Rev. Biophys. Biomol. Struct. 33, 119–140 (2004).

    Google Scholar 

  51. Tamer, Y. T. et al. High-order epistasis in catalytic power of dihydrofolate reductase gives rise to a rugged fitness landscape in the presence of trimethoprim selection. Mol. Biol. evol. 36, 1533–1550 (2019).

    Google Scholar 

  52. Manna, M. S. et al. A trimethoprim derivative impedes antibiotic resistance evolution. Nat. Commun. 12, 2949 (2021).

    Google Scholar 

  53. Cadwell, R. C. & Joyce, G. F. Randomization of genes by PCR mutagenesis. Genome Res. 2, 28–33 (1992).

    Google Scholar 

  54. Wang, H. H. et al. Programming cells by multiplex genome engineering and accelerated evolution. Nature 460, 894–898 (2009).

    Google Scholar 

  55. Findlay, G. M., Boyle, E. A., Hause, R. J., Klein, J. C. & Shendure, J. Saturation editing of genomic regions by multiplex homology-directed repair. Nature 513, 120–123 (2014).

    Google Scholar 

  56. Huang, T. P., Newby, G. A. & Liu, D. R. Precision genome editing using cytosine and adenine base editors in mammalian cells. Nat. Protoc. 16, 1089–1128 (2021).

    Google Scholar 

  57. Zimmermann, A. et al. A Cas3-base editing tool for targetable in vivo mutagenesis. Nat. Commun. 14, 3389 (2023).

    Google Scholar 

  58. Li, J. et al. Precise large-fragment deletions in mammalian cells and mice generated by dCas9-controlled CRISPR/Cas3. Sci. Adv. 10, eadk8052 (2024).

    Google Scholar 

  59. Xu Hua Fu, B., Wainberg, M., Kundaje, A. & Fire, A. Z. High-throughput characterization of Cascade type IE CRISPR guide efficacy reveals unexpected PAM diversity and target sequence preferences. Genetics 206, 1727–1738 (2017).

    Google Scholar 

  60. Musharova, O. et al. Systematic analysis of Type I-E Escherichia coli CRISPR-Cas PAM sequences ability to promote interference and primed adaptation. Mol. Microbiol. 111, 1558–1570 (2019).

    Google Scholar 

  61. García-Nafría, J., Watson, J. F. & Greger, I. H. IVA cloning: A single-tube universal cloning system exploiting bacterial In Vivo Assembly. Sci. Rep. 6, 27459 (2016).

    Google Scholar 

  62. Reikofski, J. & Tao, B. Y. Polymerase chain reaction (PCR) techniques for site-directed mutagenesis. Biotechnol. Adv. 10, 535–547 (1992).

    Google Scholar 

  63. Panayotatos, N. & Truong, K. Cleavage within an RNase III site can control mRNA stability and protein synthesis in vivo. Nucleic Acids Res. 13, 2227–2240 (1985).

    Google Scholar 

  64. Lou, C., Stanton, B., Chen, Y.-J., Munsky, B. & Voigt, C. A. Ribozyme-based insulator parts buffer synthetic circuits from genetic context. Nat. Biotechnol. 30, 1137–1142 (2012).

    Google Scholar 

  65. Otto, M. et al. Targeting 16S rDNA for stable recombinant gene expression in Pseudomonas. ACS Synth. Biol. 8, 1901–1912 (2019).

    Google Scholar 

  66. Nielsen, A. A. et al. Genetic circuit design automation. Science 352, aac7341 (2016).

    Google Scholar 

  67. Neves, D., Vos, S., Blank, L. M. & Ebert, B. E. Pseudomonas mRNA 2.0: boosting gene expression through enhanced mRNA stability and translational efficiency. Front. Bioeng. Biotechnol. 7, 458 (2020).

    Google Scholar 

  68. Masella, A. P., Bartram, A. K., Truszkowski, J. M., Brown, D. G. & Neufeld, J. D. PANDAseq: paired-end assembler for illumina sequences. BMC Bioinforma. 13, 1–7 (2012).

    Google Scholar 

  69. Li, H. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. Preprint at arXiv https://doi.org/10.48550/arXiv.1303.3997 (2013).

  70. Li, H. A statistical framework for SNP calling, mutation discovery, association mapping and population genetical parameter estimation from sequencing data. Bioinformatics 27, 2987–2993 (2011).

    Google Scholar 

  71. Li, H. et al. The sequence alignment/map format and SAMtools. bioinformatics 25, 2078–2079 (2009).

    Google Scholar 

  72. 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).

    Google Scholar 

  73. Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014).

    Google Scholar 

  74. Brunner, E. & Munzel, U. The nonparametric Behrens-Fisher problem: asymptotic theory and a small-sample approximation. Biom. J. J. Math. Methods Biosci. 42, 17–25 (2000).

    Google Scholar 

  75. Neubert, K. & Brunner, E. A studentized permutation test for the non-parametric Behrens–Fisher problem. Comput. Stat. Data Anal. 51, 5192–5204 (2007).

    Google Scholar 

  76. Lee, C. REPSECTevo_data_and_code_v1.01. Zenodo, https://doi.org/10.5281/zenodo.17470273 (2025).

Download references