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.

  • Article
  • Published:

Targeted DNA integration in human cells without double-strand breaks using CRISPR-associated transposases

Nature Biotechnology volume 42, pages 87–98 (2024)Cite this article

Subjects

Abstract

Conventional genome engineering with CRISPR–Cas9 creates double-strand breaks (DSBs) that lead to undesirable byproducts and reduce product purity. Here we report an approach for programmable integration of large DNA sequences in human cells that avoids the generation of DSBs by using Type I-F CRISPR-associated transposases (CASTs). We optimized DNA targeting by the QCascade complex through protein design and developed potent transcriptional activators by exploiting the multi-valent recruitment of the AAA+ ATPase TnsC to genomic sites targeted by QCascade. After initial detection of plasmid-based integration, we screened 15 additional CAST systems from a wide range of bacterial hosts, identified a homolog from Pseudoalteromonas that exhibits improved activity and further increased integration efficiencies. Finally, we discovered that bacterial ClpX enhances genomic integration by multiple orders of magnitude, likely by promoting active disassembly of the post-integration CAST complex, akin to its known role in Mu transposition. Our work highlights the ability to reconstitute complex, multi-component machineries in human cells and establishes a strong foundation to exploit CRISPR-associated transposases for eukaryotic genome engineering.

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: Reconstitution of protein–RNA CAST components in human cells.
Fig. 2: Development of QCascade-based and TnsC-based transcriptional activators to monitor DNA targeting.
Fig. 3: Potent genomic transcriptional activation via RNA-guided recruitment of the AAA+ ATPase, TnsC.
Fig. 4: Plasmid-based RNA-guided DNA integration in human cells using diverse CASTs.
Fig. 5: ClpX-mediated enhancement of genomic DNA integration with eCAST-3.

Similar content being viewed by others

Data availability

Sequencing data have been deposited in the National Center for Biotechnology Informationʼs Sequence Read Archive under Gene Expression Omnibus accession number GSE223174 (ref. 92). Source data are provided with this paper.

References

  1. 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 

  2. Knott, G. J. & Doudna, J. A. CRISPR–Cas guides the future of genetic engineering. Science 361, 866–869 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Anzalone, A. V., Koblan, L. W. & Liu, D. R. Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors. Nat. Biotechnol. 38, 824–844 (2020).

    Article  CAS  PubMed  Google Scholar 

  4. Maruyama, T. et al. Inhibition of non-homologous end joining increases the efficiency of CRISPR/Cas9-mediated precise genome editing. Nature 33, 538–542 (2015).

    CAS  Google Scholar 

  5. Nakade, S. et al. Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9. Nat. Commun. 5, 5560 (2014).

    Article  CAS  PubMed  Google Scholar 

  6. Chu, V. T. et al. Increasing the efficiency of homology-directed repair for CRISPR–Cas9-induced precise gene editing in mammalian cells. Nat. Biotechnol. 33, 543–548 (2015).

    Article  CAS  PubMed  Google Scholar 

  7. Yeh, C. D., Richardson, C. D. & Corn, J. E. Advances in genome editing through control of DNA repair pathways. Nat. Cell Biol. 21, 1468–1478 (2019).

    Article  CAS  PubMed  Google Scholar 

  8. Heyer, W.-D., Ehmesn, K. T. & Liu, J. Regulation of homologous recombination in eukaryotes. Annu. Rev. Genet. 44, 113–139 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Moynahan, M. E. & Jasin, M. Mitotic homologous recombination maintains genomic stability and suppresses tumorigenesis. Nat. Rev. Mol. Cell Biol. 11, 196–207 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Lin, S., Staahl, B. T., Alla, R. K. & Doudna, J. A. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. eLife 3, e04766 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  11. Zuccaro, M. V. et al. Allele-specific chromosome removal after Cas9 cleavage in human embryos. Cell 183, 1650–1654 (2020).

    Article  CAS  PubMed  Google Scholar 

  12. Adikusuma, F. et al. Large deletions induced by Cas9 cleavage. Nature 560, E8–E9 (2018).

    Article  CAS  PubMed  Google Scholar 

  13. Kosicki, M., Tomberg, K. & Bradley, A. Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements. Nat. Biotechnol. 36, 765–771 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Leibowitz, M. L. et al. Chromothripsis as an on-target consequence of CRISPR–Cas9 genome editing. Nat. Genet. 53, 895–905 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Kim, Y. B. et al. Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions. Nat. Biotechnol. 35, 371–376 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. 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 

  17. Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Anzalone, A. V. et al. Programmable deletion, replacement, integration and inversion of large DNA sequences with twin prime editing. Nat. Biotechnol. 40, 731–740 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  19. Yarnall, M. T. N. et al. Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases. Nat. Biotechnol. https://doi.org/10.1038/s41587-022-01527-4 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  20. Naldini, L., Trono, D. & Verma, I. M. Lentiviral vectors, two decades later. Science 353, 1101–1102 (2016).

    Article  CAS  PubMed  Google Scholar 

  21. Querques, I. et al. A highly soluble Sleeping Beauty transposase improves control of gene insertion. Nat. Biotechnol. 37, 1502–1512 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Yusa, K., Zhou, L., Li, M. A., Bradley, A. & Craig, N. L. A hyperactive piggyBac transposase for mammalian applications. Proc. Natl Acad. Sci. USA 108, 1531–1536 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Tipanee, J., Vandendriessche, T. & Chuah, M. K. Transposons: moving forward from preclinical studies to clinical trials. Hum. Gene Ther. 28, 1087–1104 (2017).

    Article  CAS  PubMed  Google Scholar 

  24. Gaidukov, L. et al. A multi-landing pad DNA integration platform for mammalian cell engineering. Nucleic Acids Res. 46, 4072–4086 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Durrant, M. G. et al. Systematic discovery of recombinases for efficient integration of large DNA sequences into the human genome. Nat. Biotechnol. https://doi.org/10.1038/s41587-022-01494-w (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  26. Hew, B. E., Sato, R., Mauro, D., Stoytchev, I. & Owens, J. B. RNA-guided piggyBac transposition in human cells. Synth. Biol. 4, ysz018 (2019).

    Article  CAS  Google Scholar 

  27. Kovač, A. et al. RNA-guided retargeting of Sleeping Beauty transposition in human cells. eLife 9, e53868 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  28. Luo, W. et al. Comparative analysis of chimeric ZFP-, TALE- and Cas9-piggyBac transposases for integration into a single locus in human cells. Nucleic Acids Res. 45, 8411–8422 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Chen, S. P. & Wang, H. H. An engineered Cas-Transposon system for programmable and site-directed DNA transpositions. CRISPR J. 2, 376–394 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. 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  PubMed  Google Scholar 

  31. 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  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Cameron, P. et al. Harnessing type I CRISPR–Cas systems for genome engineering in human cells. Nat. Biotechnol. 37, 1471–1477 (2019).

    Article  CAS  PubMed  Google Scholar 

  34. Chen, Y. et al. Repurposing type I-F CRISPR–Cas system as a transcriptional activation tool in human cells. Nat. Commun. 11, 3136 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Pickar-Oliver, A. et al. Targeted transcriptional modulation with type I CRISPR–Cas systems in human cells. Nat. Biotechnol. 37, 1493–1501 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Dolan, A. E. et al. Introducing a spectrum of long-range genomic deletions in human embryonic stem cells using type I CRISPR–Cas. Mol. Cell 74, 936–950 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Young, J. K. et al. The repurposing of type I-E CRISPR-Cascade for gene activation in plants. Commun. Biol. 2, 383 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  38. Strecker, J. et al. RNA-guided DNA insertion with CRISPR-associated transposases. Science 364, 48–53 (2019).

    Article  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. 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  PubMed  PubMed Central  Google Scholar 

  41. Halpin-Healy, T. S., Klompe, S. E., Sternberg, S. H. & Fernández, I. S. Structural basis of DNA targeting by a transposon-encoded CRISPR–Cas system. Nature 577, 271–274 (2020).

    Article  CAS  PubMed  Google Scholar 

  42. Peters, J. E. Tn7. Microbiol. Spectr. https://doi.org/10.1128/microbiolspec.MDNA3-0010-2014 (2014).

  43. Hoffmann, F. T. et al. Selective TnsC recruitment enhances the fidelity of RNA-guided transposition. Nature 609, 384–393 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Behler, J. & Hess, W. R. Approaches to study CRISPR RNA biogenesis and the key players involved. Methods 172, 12–26 (2020).

    Article  CAS  PubMed  Google Scholar 

  45. Szczelkun, M. D., Tikhomirova, M. S., Sinkunas, T., Gasiunas, G. & Karvelis, T. Direct observation of R-loop formation by single RNA-guided Cas9 and Cascade effector complexes. Proc. Natl Acad. Sci. USA 111, 9798–9803 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Gilbert, L. A. et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442–451 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  47. Mali, P. et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat. Biotechnol. 31, 833–838 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Perez-pinera, P. et al. RNA-guided gene activation by CRISPR–Cas9-based transcription factors. Nat. Methods 10, 973–976 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Tanenbaum, M. E., Gilbert, L. A., Qi, L. S., Weissman, J. S. & Vale, R. D. A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell 159, 635–646 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Konermann, S. et al. Genome-scale transcriptional activation by an engineered CRISPR–Cas9 complex. Nature 517, 583–588 (2015).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Querques, I., Schmitz, M., Oberli, S., Chanez, C. & Jinek, M. Target site selection and remodelling by type V CRISPR-transposon systems. Nature 599, 49–502 (2021).

    Article  Google Scholar 

  53. Chavez, A. et al. Highly-efficient Cas9-mediated transcriptional programming. Nat. Methods 12, 326–3228 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Thakore, P. I. et al. Highly specific epigenome editing by CRISPR–Cas9 repressors for silencing of distal regulatory elements. Nat. Methods 12, 1143–1149 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Wang, T., Larcher, L. M., Ma, L. & Veedu, R. N. Systematic screening of commonly used commercial transfection reagents towards efficient transfection of single-stranded oligonucleotides. Molecules 23, 2564 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  56. Walker, M. W. G., Klompe, S. E., Zhang, D. J. & Sternberg, S. H. Transposon mutagenesis libraries reveal novel molecular requirements during CRISPR RNA-guided DNA integration. Preprint at https://www.biorxiv.org/content/10.1101/2023.01.19.524723v1 (2023).

  57. Sarnovsky, R. J., May, E. W. & Craig, N. L. The Tn7 transposase is a heteromeric complex in which DNA breakage and joining activities are distributed between different gene products. EMBO J. 15, 6348–6361 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. North, S. H. & Nakai, H. Host factors that promote transpososome disassembly and the PriA-PriC pathway for restart primosome assembly. Mol. Microbiol. 56, 1601–1616 (2005).

    Article  CAS  PubMed  Google Scholar 

  59. Adeyemi, R. O. et al. The Protexin complex counters resection on stalled forks to promote homologous recombination and crosslink repair. Mol. Cell 81, 4440–4456 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Ciccia, A. & Elledge, S. J. The DNA damage response: making it safe to play with knives. Mol. Cell 40, 179–204 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Holder, J. W. & Craig, N. L. Architecture of the Tn7 posttransposition complex: an elaborate nucleoprotein structure. J. Mol. Biol. 401, 167–181 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Levchenko, I., Luo, L. & Baker, T. A. Disassembly of the Mu transposase tetramer by the ClpX chaperone. Genes Dev. 9, 2399–2408 (1995).

    Article  CAS  PubMed  Google Scholar 

  63. Kruklitis, R., Welty, D. J. & Nakai, H. ClpX protein of Escherichia coli activates bacteriophage Mu transposase in the strand transfer complex for initiation of Mu DNA synthesis. EMBO J. 15, 935–944 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Mhammedi-Alaoul, A., Pato, M. & Gama, M.-J. & Toussaint, A. A new component of bacteriophage Mu replicative transposition machinery: the Escherichia coli ClpX protein. Mol. Microbiol. 11, 1109–1116 (1994).

    Article  Google Scholar 

  65. Abdelhakim, A. H., Oakes, E. C., Sauer, R. T. & Baker, T. A. Unique contacts direct high-priority recognition of the tetrameric Mu transposase-DNA complex by the AAA+ unfoldase ClpX. Mol. Cell 30, 39–50 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Sauer, R. T. & Baker, T. A. AAA+ proteases: ATP-fueled machines of protein destruction. Annu. Rev. Biochem. 80, 587–612 (2011).

    Article  CAS  PubMed  Google Scholar 

  67. Levchenko, I., Yamauchi, M. & Baker, T. A. ClpX and MuB interact with overlapping regions of Mu transposase: implications for control of the transposition pathway. Genes Dev. 11, 1561–1572 (1997).

    Article  CAS  PubMed  Google Scholar 

  68. Baker, T. A. & Sauer, R. T. ClpXP, an ATP-powered unfolding and protein-degradation machine. Biochim. Biophys. Acta Mol. Cell Res. 1823, 15–28 (2012).

    Article  CAS  Google Scholar 

  69. Hersch, G. L., Burton, R. E., Bolon, D. N., Baker, T. A. & Sauer, R. T. Asymmetric interactions of ATP with the AAA+ ClpX6 unfoldase: allosteric control of a protein machine. Cell 121, 1017–1027 (2005).

    Article  CAS  PubMed  Google Scholar 

  70. Joshi, S. A., Hersch, G. L., Baker, T. A. & Sauer, R. T. Communication between ClpX and ClpP during substrate processing and degradation. Nat. Struct. Mol. Biol. 11, 404–411 (2004).

    Article  CAS  PubMed  Google Scholar 

  71. Siddiqui, S. M., Sauer, R. T. & Baker, T. A. Role of the processing pore of the ClpX AAA+ ATPase in the recognition and engagement of specific protein substrates. Genes Dev. 18, 369–374 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Strecker, J., Zhang, F. & Ladha, A. CRISPR-associated transposase systems and methods of use thereof. https://patents.google.com/patent/WO2020131862A1/en (2020).

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

    Article  PubMed  Google Scholar 

  74. Özcan, A. et al. Programmable RNA targeting with the single-protein CRISPR effector Cas7-11. Nature 597, 720–725 (2021).

    Article  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  76. Gu, B. et al. Transcription-coupled changes in nuclear mobility of mammalian cis-regulatory elements. Science 359, 1050–1055 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Chen, B., Guan, J. & Huang, B. Imaging specific genomic DNA in living cells. Annu. Rev. Biophys. 45, 1–23 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Schmitz, M., Querques, I., Oberli, S., Chanez, C. & Jinek, M. Structural basis for the assembly of the type V CRISPR-associated transposon complex. Cell 185, 4999–5010 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  79. Fricke, T. et al. Targeted RNA knockdown by a type III CRISPR–Cas complex in zebrafish. CRISPR J. 3, 299–313 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Petassi, M. T., Hsieh, S. & Peters, J. E. Guide RNA categorization enables target site choice in Tn7-CRISPR-Cas transposons. Cell 183, 1757–1771 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Yeo, N. C. et al. An enhanced CRISPR repressor for targeted mammalian gene regulation. Nat. Methods 15, 611–616 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  82. Lee, T. I., Johnstone, S. E. & Young, R. A. Chromatin immunoprecipitation and microarray-based analysis of protein location. Nat. Protoc. 1, 729–748 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  83. Weinberg, D. N. et al. The histone mark H3K36me2 recruits DNMT3A and shapes the intergenic DNA methylation landscape. Nature 573, 281–286 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Qiu, X. et al. CoBRA: Containerized Bioinformatics Workflow for Reproducible ChIP/ATAC-seq Analysis. Genomics Proteomics Bioinformatics 19, 652–661 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  85. Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  86. Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  87. Robinson, J. T. et al. Integrative genomics viewer. Nat. Biotechnol. 29, 24–26 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  88. Quinlan, A. R. & Hall, I. M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  89. Amemiya, H. M., Kundaje, A. & Boyle, A. P. The ENCODE blacklist: identification of problematic regions of the genome. Sci. Rep. 9, 9354 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  90. Stark, R. & Brown, G. DiffBind: differential binding analysis of ChIP-Seq peak data. http://bioconductor.org/packages/release/bioc/vignettes/DiffBind/inst/doc/DiffBind.pdf 1–29 (2011).

  91. Ramírez, F. et al. deepTools2: a next generation web server for deep-sequencing data analysis. Nucleic Acids Res. 44, W160–W165 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  92. Lampe, G. D. et al. Integration in human cells without double-strand breaks using CRISPR RNA-guided transposases. Gene Expression Omnibus https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE223174 (2023).

Download references

Acknowledgements

We thank N. Jaber and S. R. Pesari for laboratory support, L. F. Landweber for qPCR instrument access, A. Santiago-Frangos for helpful ClpX discussions, the Columbia University Institute for Genomic Medicine for ddPCR instrument access, W. Frankel for thermocycler instrument access, the Columbia Stem Cell Initiative Flow Cytometry Core, M. Kissner and R. Gordon-Schneider for assistance in cell sorting and flow cytometry analysis, and the JP Sulzberger Columbia Genome Center for NGS support. We thank C. Lu and V. Sahu for cell sonicator instrument access and valuable discussions on ChIP-seq analysis. R.T.K. is supported by NIH grant 1F31HL167530-01 from the National Heart, Lung, and Blood Institute. A.C. is supported by a Career Award for Medical Scientists from the Burroughs Wellcome Fund and NIH grant HG011855-01 from the National Human Genome Research Institute. S.H.S. is supported by NIH grant DP2HG011650, a Pew Biomedical Scholarship, a Sloan Research Fellowship, an Irma T. Hirschl Career Scientist Award, and a generous startup package from the Columbia University Irving Medical Center Dean’s Office and the Vagelos Precision Medicine Fund.

Author information

Author notes

  1. Tyler S. Halpin-Healy

    Present address: Regeneron Pharmaceuticals, Inc., Tarrytown, NY, USA

  2. Sanne E. Klompe

    Present address: Department of Genomes and Genetics, Institut Pasteur, Paris, France

  3. Marcus I. Hogan

    Present address: Helen Wills Neuroscience Institute, University of California, Berkeley, Berkeley, CA, USA

  4. Phuc Leo H. Vo

    Present address: Vertex Pharmaceuticals, Inc., Boston, MA, USA

  5. Alejandro Chavez

    Present address: Department of Pediatrics, University of California, San Diego, La Jolla, CA, USA

  6. These authors contributed equally: George D. Lampe, Rebeca T. King.

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA

    George D. Lampe, Rebeca T. King, Tyler S. Halpin-Healy, Sanne E. Klompe, Marcus I. Hogan, Stephen Tang & Samuel H. Sternberg

  2. Department of Molecular Pharmacology and Therapeutics, Columbia University, New York, NY, USA

    Phuc Leo H. Vo

  3. Department of Pathology and Cell Biology, Columbia University, New York, NY, USA

    Alejandro Chavez

Authors
  1. George D. Lampe
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rebeca T. King
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tyler S. Halpin-Healy
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sanne E. Klompe
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Marcus I. Hogan
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Phuc Leo H. Vo
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Stephen Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Alejandro Chavez
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Samuel H. Sternberg
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

G.D.L. performed western blots, plasmid-based reporter assays and experiments screening CAST homologs and host proteins. R.T.K. performed genomic activation assays, ChIP experiments and ddPCR analyses. G.D.L. and R.T.K. performed integration assays and quantified integration efficiency by qPCR and amplicon sequencing. T.S.H.-H. performed initial human cell experiments and, together with M.I.H., tested tagged constructs for activity in E. coli. S.E.K. tested tagged constructs in E. coli and assisted with initial NGS library preparation and data analysis. P.L.H.V. designed and cloned constructs for initial I-E activation assays. S.T. performed ChIP-seq analyses. A.C. provided expert support and helped S.H.S. supervise the project. S.H.S., G.D.L., R.T.K. and A.C. discussed the data and wrote the manuscript, with input from all authors.

Corresponding author

Correspondence to Samuel H. Sternberg.

Ethics declarations

Competing interests

Columbia University has filed a patent application related to this work. S.E.K., P.L.H.V., A.C. and S.H.S. are inventors on other patents and patent applications related to CRISPR–Cas systems and uses thereof. A.C. is a consultant for Vor Biopharma and an equity holder and scientific advisor for Cellgorithmics. S.H.S. is a co-founder of and scientific advisor to Dahlia Biosciences, a scientific advisor to Prime Medicine and CrisprBits and an equity holder in Dahlia Biosciences and CrisprBits. The remaining authors declare no competing interests.

Peer review

Peer review information

Nature Biotechnology thanks the anonymous reviewers for their contribution to the peer review of this work.

Additional information

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

Supplementary information

Supplementary Information

Supplementary Figs. 1–15.

Reporting Summary

Supplementary Tables 1–4

Supplementary Tables.

Source data

Source Data Fig. 1

Unprocessed western blots.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lampe, G.D., King, R.T., Halpin-Healy, T.S. et al. Targeted DNA integration in human cells without double-strand breaks using CRISPR-associated transposases. Nat Biotechnol 42, 87–98 (2024). https://doi.org/10.1038/s41587-023-01748-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41587-023-01748-1

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research