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Precise cut-and-paste DNA insertion using engineered type V-K CRISPR-associated transposases

Abstract

CRISPR-associated transposases (CASTs) enable recombination-independent, multi-kilobase DNA insertions at RNA-programmed genomic locations. However, the utility of type V-K CASTs is hindered by high off-target integration and a transposition mechanism that results in a mixture of desired simple cargo insertions and undesired plasmid cointegrate products. Here we overcome both limitations by engineering new CASTs with improved integration product purity and genome-wide specificity. To do so, we engineered a nicking homing endonuclease fusion to TnsB (named HELIX) to restore the 5′ nicking capability needed for cargo excision on the DNA donor. HELIX enables cut-and-paste DNA insertion with up to 99.4% simple insertion product purity, while retaining robust integration efficiencies on genomic targets. HELIX has substantially higher on-target specificity than canonical CASTs, and we identify several novel factors that further regulate targeted and genome-wide integration. Finally, we extend HELIX to other type V-K orthologs and demonstrate the feasibility of HELIX-mediated integration in human cell contexts.

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Fig. 1: Development and characterization of HELIX.
Fig. 2: Characterization of DNA insertions on genomic targets using HELIX.
Fig. 3: Extension of HELIX to type V-K CAST orthologs.
Fig. 4: Specificity profiling of ShCAST and ShHELIX systems.
Fig. 5: HELIX-mediated DNA insertion in human cell lysates and human cells.

Data availability

Sequencing data has been deposited with the NCBI Sequence Read Archive (SRA) under BioProject ID PRJNA889059. All other primary datasets are available in Supplementary Table 4.

Code availability

A custom script was used to calculate on-target integration from specificity analyses and is available in Supplementary Note 6. All other analyses were performed using publicly avaliable software noted in the Methods.

References

  1. Hendrie, P. C. & Russell, D. W. Gene targeting with viral vectors. Mol. Ther. 12, 9–17 (2005).

    Article  CAS  Google Scholar 

  2. Thomas, C. E., Ehrhardt, A. & Kay, M. A. Progress and problems with the use of viral vectors for gene therapy. Nat. Rev. Genet. 4, 346–358 (2003).

    Article  CAS  Google Scholar 

  3. Tellier, M., Bouuaert, C. C. & Chalmers, R. Mariner and the ITm superfamily of transposons. Microbiol. Spectr. 3, 3.2.06 (2015).

    Article  Google Scholar 

  4. van Opijnen, T. & Camilli, A. Transposon insertion sequencing: a new tool for systems-level analysis of microorganisms. Nat. Rev. Microbiol. 11, 435–442 (2013).

    Article  Google Scholar 

  5. Haniford, D. B. & Ellis, M. J. Transposons Tn 10 and Tn 5. Microbiol. Spectr. 3, 3.1.06 (2015).

    Article  Google Scholar 

  6. Plasterk, R. H. A., Izsvák, Z. & Ivics, Z. Resident aliens: the Tc1/ mariner superfamily of transposable elements. Trends Genet. 15, 326–332 (1999).

    Article  CAS  Google Scholar 

  7. Wilson, M. H., Coates, C. J. & George, A. L. PiggyBac transposon-mediated gene transfer in human cells. Mol. Ther. 15, 139–145 (2007).

    Article  CAS  Google Scholar 

  8. Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).

    Article  CAS  Google Scholar 

  9. Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

    Article  CAS  Google Scholar 

  10. Rouet, P., Smih, F. & Jasin, M. Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells. Proc. Natl Acad. Sci. USA 91, 6064–6068 (1994).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  12. Wang, H. H. et al. Genome-scale promoter engineering by coselection MAGE. Nat. Methods 9, 591–593 (2012).

    Article  Google Scholar 

  13. Jiang, W., Bikard, D., Cox, D., Zhang, F. & Marraffini, L. A. RNA-guided editing of bacterial genomes using CRISPR–Cas systems. Nat. Biotechnol. 31, 233–239 (2013).

    Article  CAS  Google Scholar 

  14. Strecker, J. et al. RNA-guided DNA insertion with CRISPR-associated transposases. Science 365, 48–53 (2019).

    Article  CAS  Google Scholar 

  15. Klompe, S. E., Vo, P. L. H., Halpin-Healy, T. S. & Sternberg, S. H. Transposon-encoded CRISPR–Cas systems direct RNA-guided DNA integration. Nature 571, 219–225 (2019).

    Article  CAS  Google Scholar 

  16. Vo, P. L. H. et al. CRISPR RNA-guided integrases for high-efficiency, multiplexed bacterial genome engineering. Nat. Biotechnol. 39, 480–489 (2021).

    Article  CAS  Google Scholar 

  17. Vo, P. L. H., Acree, C., Smith, M. L. & Sternberg, S. H. Unbiased profiling of CRISPR RNA-guided transposition products by long-read sequencing. Mob. DNA 12, 13 (2021).

    Article  CAS  Google Scholar 

  18. Saito, M. et al. Dual modes of CRISPR-associated transposon homing. Cell 184, 2441–2453.e18 (2021).

    Article  CAS  Google Scholar 

  19. Strecker, J., Ladha, A., Makarova, K. S., Koonin, E. V. & Zhang, F. Response to comment on “RNA-guided DNA insertion with CRISPR-associated transposases”. Science 368, eabb2920 (2020).

    Article  CAS  Google Scholar 

  20. Rubin, B. E. et al. Species- and site-specific genome editing in complex bacterial communities. Nat. Microbiol. 7, 34–47 (2022).

    Article  CAS  Google Scholar 

  21. Rybarski, J. R., Hu, K., Hill, A. M., Wilke, C. O. & Finkelstein, I. J. Metagenomic discovery of CRISPR-associated transposons. Proc. Natl Acad. Sci. USA 118, e2112279118 (2021).

    Article  CAS  Google Scholar 

  22. May, E. W. & Craig, N. L. Switching from cut-and-paste to replicative Tn7 transposition. Science 272, 401–404 (1996).

    Article  CAS  Google Scholar 

  23. Kholodii, G. Y. et al. Four genes, two ends, and a res region are involved in transposition of Tn5053: a paradigm for a novel family of transposons carrying either a mer operon or an integron. Mol. Microbiol. 17, 1189–1200 (1995).

    Article  CAS  Google Scholar 

  24. Hickman, A. B. et al. Unexpected structural diversity in DNA recombination. Mol. Cell 5, 1025–1034 (2000).

    Article  CAS  Google Scholar 

  25. Xu, S. Sequence-specific DNA nicking endonucleases. Biomol. Concepts 6, 253–267 (2015).

    Article  CAS  Google Scholar 

  26. Gasiunas, G., Barrangou, R., Horvath, P. & Siksnys, V. Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc. Natl Acad. Sci. USA 109, E2579–E2586 (2012).

    Article  CAS  Google Scholar 

  27. Jinek, M. et al. A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).

    Article  CAS  Google Scholar 

  28. Xu, S. & Gupta, Y. K. Natural zinc ribbon HNH endonucleases and engineered zinc finger nicking endonuclease. Nucleic Acids Res. 41, 378–390 (2013).

    Article  CAS  Google Scholar 

  29. McConnell Smith, A. et al. Generation of a nicking enzyme that stimulates site-specific gene conversion from the I-AniI LAGLIDADG homing endonuclease. Proc. Natl Acad. Sci. USA 106, 5099–5104 (2009).

    Article  CAS  Google Scholar 

  30. Niu, Y., Tenney, K., Li, H. & Gimble, F. S. Engineering variants of the I-SceI homing endonuclease with strand-specific and site-specific DNA-nicking activity. J. Mol. Biol. 382, 188–202 (2008).

    Article  CAS  Google Scholar 

  31. Kong, S., Liu, X., Fu, L., Yu, X. & An, C. I-PfoP3I: a novel nicking HNH homing endonuclease encoded in the group I intron of the DNA polymerase gene in Phormidium foveolarum phage Pf-WMP3. PLoS ONE 7, e43738 (2012).

    Article  CAS  Google Scholar 

  32. Landthaler, M. & Shub, D. A. The nicking homing endonuclease I-BasI is encoded by a group I intron in the DNA polymerase gene of the Bacillus thuringiensis phage Bastille. Nucleic Acids Res. 31, 3071–3077 (2003).

    Article  CAS  Google Scholar 

  33. Shen, Y. et al. Structural basis for DNA targeting by the Tn7 transposon. Nat. Struct. Mol. Biol. 29, 143–151 (2022).

    Article  CAS  Google Scholar 

  34. Stoddard, B. L. Homing endonucleases from mobile group I introns: discovery to genome engineering. Mob. DNA 5, 7 (2014).

    Article  Google Scholar 

  35. Takeuchi, R., Certo, M., Caprara, M. G., Scharenberg, A. M. & Stoddard, B. L. Optimization of in vivo activity of a bifunctional homing endonuclease and maturase reverses evolutionary degradation. Nucleic Acids Res. 37, 877–890 (2009).

    Article  CAS  Google Scholar 

  36. Querques, I., Schmitz, M., Oberli, S., Chanez, C. & Jinek, M. Target site selection and remodelling by type V CRISPR–transposon systems. Nature 599, 497–502 (2021).

    Article  CAS  Google Scholar 

  37. Park, J.-U., Tsai, A. W.-L., Chen, T. H., Peters, J. E. & Kellogg, E. H. Mechanistic details of CRISPR-associated transposon recruitment and integration revealed by cryo-EM. Proc. Natl. Acad. Sci. USA 119, e2202590119 (2022).

    Article  CAS  Google Scholar 

  38. Tenjo-Castaño, F. et al. Structure of the TnsB transposase-DNA complex of type V-K CRISPR-associated transposon. Nat. Commun. 13, 5792 (2022).

    Article  Google Scholar 

  39. Liu, R., Qiu, J., Finger, L. D., Zheng, L. & Shen, B. The DNA-protein interaction modes of FEN-1 with gap substrates and their implication in preventing duplication mutations. Nucleic Acids Res. 34, 1772–1784 (2006).

    Article  CAS  Google Scholar 

  40. Scalley-Kim, M., McConnell-Smith, A. & Stoddard, B. L. Coevolution of a homing endonuclease and its host target sequence. J. Mol. Biol. 372, 1305–1319 (2007).

    Article  CAS  Google Scholar 

  41. Gilpatrick, T. et al. Targeted nanopore sequencing with Cas9-guided adapter ligation. Nat. Biotechnol. 38, 433–438 (2020).

    Article  CAS  Google Scholar 

  42. Park, J.-U. et al. Structural basis for target site selection in RNA-guided DNA transposition systems. Science 373, 768–774 (2021).

    Article  CAS  Google Scholar 

  43. Schmitz, M., Querques, I., Oberli, S., Chanez, C. & Jinek, M. Structural basis for RNA-mediated assembly of type V CRISPR-associated transposons. Preprint at BioRxiv https://doi.org/10.1101/2022.06.17.496590 (2022).

  44. Mizuno, N. et al. MuB is an AAA+ ATPase that forms helical filaments to control target selection for DNA transposition. Proc. Natl Acad. Sci. USA 110, E2441–E2450 (2013).

    Article  CAS  Google Scholar 

  45. Skelding, Z., Queen-Baker, J. & Craig, N. L. Alternative interactions between the Tn7 transposase and the Tn7 target DNA binding protein regulate target immunity and transposition. EMBO J. 22, 5904–5917 (2003).

    Article  CAS  Google Scholar 

  46. Stellwagen, A. E. & Craig, N. L. Avoiding self: two Tn7-encoded proteins mediate target immunity in Tn7 transposition. EMBO J. 16, 6823–6834 (1997).

    Article  CAS  Google Scholar 

  47. Kolter, R., Inuzuka, M. & Helinski, D. R. Trans-complementation-dependent replication of a low molecular weight origin fragment from plasmid R6K. Cell 15, 1199–1208 (1978).

    Article  CAS  Google Scholar 

  48. Metcalf, W. W., Jiang, W. & Wanner, B. L. Use of the rep technique for allele replacement to construct new Escherichia coli hosts for maintenance of R6K gamma origin plasmids at different copy numbers. Gene 138, 1–7 (1994).

    Article  CAS  Google Scholar 

  49. Klompe, S. E. et al. Evolutionary and mechanistic diversity of Type I-F CRISPR-associated transposons. Mol. Cell 82, 616–628.e5 (2022).

    Article  CAS  Google Scholar 

  50. Jonathan Strecker, Feng Zhang, Alim Ladha. CRISPR-associated transposase systems and methods of use thereof. International patent WO2020131862A1 (2020).

  51. Wu, Z. & Chaconas, G. Flanking host sequences can exert an inhibitory effect on the cleavage step of the in vitro mu DNA strand transfer reaction. J. Biol. Chem. 267, 9552–9558 (1992).

    Article  CAS  Google Scholar 

  52. Krüger, R. & Filutowicz, M. Dimers of pi protein bind the A+T-rich region of the R6K gamma origin near the leading-strand synthesis start sites: regulatory implications. J. Bacteriol. 182, 2461–2467 (2000).

    Article  Google Scholar 

  53. Harshey R. M, Transposable phage Mu. Microbiol. Spectr. 2, 2.5.31 (2014).

    Article  Google Scholar 

  54. Chalmers, R., Guhathakurta, A., Benjamin, H. & Kleckner, N. IHF modulation of Tn10 transposition: sensory transduction of supercoiling status via a proposed protein/DNA molecular spring. Cell 93, 897–908 (1998).

    Article  CAS  Google Scholar 

  55. Swingle, B., O’Carroll, M., Haniford, D. & Derbyshire, K. M. The effect of host-encoded nucleoid proteins on transposition: H-NS influences targeting of both IS903 and Tn10. Mol. Microbiol. 52, 1055–1067 (2004).

    Article  CAS  Google Scholar 

  56. Zayed, H., Izsvák, Z., Khare, D., Heinemann, U. & Ivics, Z. The DNA-bending protein HMGB1 is a cellular cofactor of Sleeping Beauty transposition. Nucleic Acids Res. 31, 2313–2322 (2003).

    Article  CAS  Google Scholar 

  57. Filutowicz, M. & Appelt, K. The integration host factor of Escherichia coli binds to multiple sites at plasmid R6K gamma origin and is essential for replication. Nucleic Acids Res. 16, 3829–3843 (1988).

    Article  CAS  Google Scholar 

  58. Sharpe, P. L. & Craig, N. L. Host proteins can stimulate Tn7 transposition: a novel role for the ribosomal protein L29 and the acyl carrier protein. EMBO J. 17, 5822–5831 (1998).

    Article  CAS  Google Scholar 

  59. Parks, A. R. et al. Transposition into replicating DNA occurs through interaction with the processivity factor. Cell 138, 685–695 (2009).

    Article  CAS  Google Scholar 

  60. Strecker, J. et al. Engineering of CRISPR–Cas12b for human genome editing. Nat. Commun. 10, 212 (2019).

    Article  CAS  Google Scholar 

  61. Xu, X. et al. Engineered miniature CRISPR–Cas system for mammalian genome regulation and editing. Mol. Cell 81, 4333–4345 (2021).

    Article  CAS  Google Scholar 

  62. Kim, D. Y. et al. Efficient CRISPR editing with a hypercompact Cas12f1 and engineered guide RNAs delivered by adeno-associated virus. Nat. Biotechnol. 40, 94–102 (2022).

    Article  CAS  Google Scholar 

  63. Anzalone, A. V. et al. Programmable deletion, replacement, integration and inversion of large DNA sequences with twin prime editing. Nat. Biotechnol. 40, 731–740 (2022).

    Article  CAS  Google Scholar 

  64. Ioannidi, E. I. et al. Drag-and-drop genome insertion without DNA cleavage with CRISPR-directed integrases. Preprint at BioRxiv https://doi.org/10.1101/2021.11.01.466786 (2021).

  65. Li, H. Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics 34, 3094–3100 (2018).

    Article  CAS  Google Scholar 

  66. Rohland, N. & Reich, D. Cost-effective, high-throughput DNA sequencing libraries for multiplexed target capture. Genome Res 22, 939–946 (2012).

    Article  CAS  Google Scholar 

  67. Kleinstiver, B. P. et al. Engineered CRISPR–Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing. Nat. Biotechnol. 37, 276–282 (2019).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank L. T. Hille, C. R. R. Alves, and R. A. Silverstein for suggestions about the manuscript, P. M. Boone for assistance with ddPCR, and B. L. Stoddard for advice. C.J.T. was supported by a National Science Foundation Graduate Research Fellowship grant number 2020295403. B.P.K. was supported by a Mass General Hospital Howard M. Goodman Fellowship.

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Contributions

C.J.T conceived of the idea, designed and performed experiments, and wrote the manuscript draft. B.O. conducted experiments for ShoHELIX and cargo size comparisons. B.P.K supervised the study and contributed to experimental design. C.J.T and B.P.K wrote and revised the manuscript with input from B.O.

Corresponding author

Correspondence to Benjamin P. Kleinstiver.

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Competing interests

C.J.T. and B.P.K are inventors on patents and/or patent applications filed by Massachusetts General Brigham that describe genome-engineering technologies. B.P.K. is a consultant for EcoR1 capital, and is an advisor to Acrigen Biosciences, Life Edit Therapeutics, and Prime Medicine. B.O. declares no competing interests.

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Tou, C.J., Orr, B. & Kleinstiver, B.P. Precise cut-and-paste DNA insertion using engineered type V-K CRISPR-associated transposases. Nat Biotechnol (2023). https://doi.org/10.1038/s41587-022-01574-x

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