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.

  • Protocol
  • Published:

Precise kilobase-scale genomic insertions in mammalian cells using PASTE

Nature Protocols volume 20, pages 1546–1583 (2025)Cite this article

Subjects

Abstract

Programmable gene integration technologies are an emerging modality with exciting applications in both basic research and therapeutic development. Programmable addition via site-specific targeting elements (PASTE) is a programmable gene integration approach for precise and efficient programmable integration of large DNA sequences into the genome. PASTE offers improved editing efficiency, purity and programmability compared with previous methods for long insertions into the mammalian genome. By combining the specificity and cargo size capabilities of site-specific integrases with the programmability of prime editing, PASTE can precisely insert cargoes of at least 36 kb with efficiencies of up to 60%. Here we outline best practices for design, execution and analysis of PASTE experiments, with protocols for integration of EGFP at the human NOLC1 and ACTB genomic loci and for readout by next generation sequencing and droplet digital PCR. We provide guidelines for designing and optimizing a custom PASTE experiment for integration of desired payloads at alternative genomic loci, as well as example applications for in-frame protein tagging and multiplexed insertions. To facilitate experimental setup, we include the necessary sequences and plasmids for the delivery of PASTE components to cells via plasmid transfection or in vitro transcribed RNA. Most experiments in this protocol can be performed in as little as 2 weeks, allowing for precise and versatile programmable gene insertion.

Key points

  • Programmable addition via site-specific targeting elements (PASTE) combines the specificity, efficiency and cargo size advantages of site-specific integrases with the programmability of prime editing for precise and efficient integration of large DNA sequences into mammalian genomes.

  • PASTE offers improved editing efficiency, purity and reprogrammability compared with previous methods for long insertions into the mammalian genome.

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

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to the full article PDF.

USD 39.95

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

Fig. 1: Overview of workflow for implementing PASTE for a new application.
Fig. 2: Comparison of PASTE to other technologies for programmable DNA insertion.
Fig. 3: Sequence-level view of PASTE editing at an example locus.
Fig. 4: Modifying the frame of the PASTE insert and generation of NGS barcodes or atgRNA crosses.
Fig. 5: Overview of design for NGS and ddPCR assays.
Fig. 6: Expected outcomes from PASTE editing.

Similar content being viewed by others

Data availability

Sequencing data used in Fig. 6 are deposited at the NCBI Sequence Read Archive (SRA) database under accession PRJNA1101023.

References

  1. Sheridan, C. The world’s first CRISPR therapy is approved: who will receive it? Nat. Biotechnol. 42, 3–4 (2024).

    Article  CAS  PubMed  Google Scholar 

  2. Veit, G. et al. From CFTR biology toward combinatorial pharmacotherapy: expanded classification of cystic fibrosis mutations. Mol. Biol. Cell 27, 424–433 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Ausländer, S. & Fussenegger, M. Engineering gene circuits for mammalian cell-based applications. Cold Spring Harb. Perspect. Biol. 8, a023895 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  4. Tou, C. J. & Kleinstiver, B. P. Recent advances in double-strand break-free kilobase-scale genome editing technologies. Biochemistry 62, 3493–3499 (2023).

    Article  CAS  PubMed  Google Scholar 

  5. Suzuki, K. et al. In vivo genome editing via CRISPR–Cas9-mediated homology-independent targeted integration. Nature 540, 144–149 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Nami, F. et al. Strategies for in vivo genome editing in nondividing cells. Trends Biotechnol. 36, 770–786 (2018).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Hsu, P. D., Lander, E. S. & Zhang, F. Development and applications of CRISPR–Cas9 for genome engineering. Cell 157, 1262–1278 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Wright, A. V., Nuñez, J. K. & Doudna, J. A. Biology and applications of CRISPR systems: harnessing nature’s toolbox for genome engineering. Cell 164, 29–44 (2016).

    Article  CAS  PubMed  Google Scholar 

  11. Chapman, J. R., Taylor, M. R. G. & Boulton, S. J. Playing the end game: DNA double-strand break repair pathway choice. Mol. Cell 47, 497–510 (2012).

    Article  CAS  PubMed  Google Scholar 

  12. Geisinger, J. M. & Stearns, T. CRISPR–Cas9 treatment causes extended TP53-dependent cell cycle arrest in human cells. Nucleic Acids Res. 48, 9067–9081 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Wang, H. et al. Development of a self-restricting CRISPR–Cas9 system to reduce off-target effects. Mol. Ther. Methods Clin. Dev. 18, 390–401 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Ihry, R. J. et al. p53 inhibits CRISPR–Cas9 engineering in human pluripotent stem cells. Nat. Med. 24, 939–946 (2018).

    Article  CAS  PubMed  Google Scholar 

  15. 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 

  16. 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 

  17. 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 

  18. 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 

  19. Ferreira da Silva, J. et al. Prime editing efficiency and fidelity are enhanced in the absence of mismatch repair. Nat. Commun. 13, 760 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Chen, P. J. et al. Enhanced prime editing systems by manipulating cellular determinants of editing outcomes. Cell 184, 5635–5652.e29 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Yan, J. et al. Improving prime editing with an endogenous small RNA-binding protein. Nature https://doi.org/10.1038/s41586-024-07259-6 (2024).

  22. Nelson, J. W. et al. Engineered pegRNAs improve prime editing efficiency. Nat. Biotechnol. 40, 402–410 (2022).

    Article  CAS  PubMed  Google Scholar 

  23. 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  PubMed  Google Scholar 

  24. Wang, J. et al. Efficient targeted insertion of large DNA fragments without DNA donors. Nat. Methods 19, 331–340 (2022).

    Article  CAS  PubMed  Google Scholar 

  25. Zheng, C. et al. Template-jumping prime editing enables large insertion and exon rewriting in vivo. Nat. Commun. 14, 3369 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Jiang, T., Zhang, X.-O., Weng, Z. & Xue, W. Deletion and replacement of long genomic sequences using prime editing. Nat. Biotechnol. 40, 227–234 (2022).

    Article  CAS  PubMed  Google Scholar 

  27. 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).

  28. Smith, M. C. M., Brown, W. R. A., McEwan, A. R. & Rowley, P. A. Site-specific recombination by phiC31 integrase and other large serine recombinases. Biochem. Soc. Trans. 38, 388–394 (2010).

    Article  CAS  PubMed  Google Scholar 

  29. Doman, J. L. et al. Phage-assisted evolution and protein engineering yield compact, efficient prime editors. Cell 186, 3983–4002.e26 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Merrick, C. A., Zhao, J. & Rosser, S. J. Serine integrases: advancing synthetic biology. ACS Synth. Biol. 7, 299–310 (2018).

    Article  CAS  PubMed  Google Scholar 

  31. Meinke, G., Bohm, A., Hauber, J., Pisabarro, M. T. & Buchholz, F. Cre recombinase and other tyrosine recombinases. Chem. Rev. 116, 12785–12820 (2016).

    Article  CAS  PubMed  Google Scholar 

  32. Smith, M. C. M. Phage-encoded serine integrases and other large serine recombinases. Microbiol. Spectr. https://doi.org/10.1128/microbiolspec.MDNA3-0059-2014 (2015).

  33. Choi, J. et al. Precise genomic deletions using paired prime editing. Nat. Biotechnol. 40, 218–226 (2022).

    Article  CAS  PubMed  Google Scholar 

  34. Zhuang, Y. et al. Increasing the efficiency and precision of prime editing with guide RNA pairs. Nat. Chem. Biol. 18, 29–37 (2022).

    Article  CAS  PubMed  Google Scholar 

  35. Tao, R. et al. Bi-PE: bi-directional priming improves CRISPR–Cas9 prime editing in mammalian cells. Nucleic Acids Res. 50, 6423–6434 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Strecker, J. et al. RNA-guided DNA insertion with CRISPR-associated transposases. Science https://doi.org/10.1126/science.aax9181 (2019).

  37. 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 

  38. Tou, C. J., Orr, B. & Kleinstiver, B. P. Precise cut-and-paste DNA insertion using engineered type V-K CRISPR-associated transposases. Nat. Biotechnol. 41, 968–979 (2023).

    Article  CAS  PubMed  Google Scholar 

  39. Lampe, G. D. et al. Targeted DNA integration in human cells without double-strand breaks using CRISPR-associated transposases. Nat. Biotechnol. 42, 87–98 (2024).

    Article  CAS  PubMed  Google Scholar 

  40. Durrant, M. G. et al. Systematic discovery of recombinases for efficient integration of large DNA sequences into the human genome. Nat. Biotechnol. 41, 488–499 (2023).

    Article  CAS  PubMed  Google Scholar 

  41. Ghosh, P., Kim, A. I. & Hatfull, G. F. The orientation of mycobacteriophage Bxb1 integration is solely dependent on the central dinucleotide of attP and attB. Mol. Cell 12, 1101–1111 (2003).

    Article  CAS  PubMed  Google Scholar 

  42. Bednarski, C., Tomczak, K., Vom Hövel, B., Weber, W.-M. & Cathomen, T. Targeted integration of a super-exon into the CFTR locus leads to functional correction of a cystic fibrosis cell line model. PLoS ONE 11, e0161072 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  43. Sun, C. et al. Precise integration of large DNA sequences in plant genomes using PrimeRoot editors. Nat. Biotechnol. 42, 316–327 (2024).

    Article  CAS  PubMed  Google Scholar 

  44. Kim, H. K. et al. Predicting the efficiency of prime editing guide RNAs in human cells. Nat. Biotechnol. 39, 198–206 (2021).

    Article  CAS  PubMed  Google Scholar 

  45. Koeppel, J. et al. Prediction of prime editing insertion efficiencies using sequence features and DNA repair determinants. Nat. Biotechnol. 41, 1446–1456 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Ferrari, S. et al. Choice of template delivery mitigates the genotoxic risk and adverse impact of editing in human hematopoietic stem cells. Cell Stem Cell 29, 1428–1444.e9 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Hsu, J. Y. et al. PrimeDesign software for rapid and simplified design of prime editing guide RNAs. Nat. Commun. 12, 1034 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Hwang, G.-H. et al. PE-Designer and PE-Analyzer: web-based design and analysis tools for CRISPR prime editing. Nucleic Acids Res. 49, W499–W504 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Anderson, M. V., Haldrup, J., Thomsen, E. A., Wolff, J. H. & Mikkelsen, J. G. pegIT—a web-based design tool for prime editing. Nucleic Acids Res. 49, W505–W509 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Chow, R. D., Chen, J. S., Shen, J. & Chen, S. A web tool for the design of prime-editing guide RNAs. Nat. Biomed. Eng. 5, 190–194 (2021).

    Article  CAS  PubMed  Google Scholar 

  51. Doman, J. L., Sousa, A. A., Randolph, P. B., Chen, P. J. & Liu, D. R. Designing and executing prime editing experiments in mammalian cells. Nat. Protoc. 17, 2431–2468 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. 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. 41, 500–512 (2023).

    Article  CAS  PubMed  Google Scholar 

  53. Park, S.-J. et al. Targeted mutagenesis in mouse cells and embryos using an enhanced prime editor. Genome Biol. 22, 170 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  54. Oscorbin, I. P., Wong, P. F., Boyarskikh, U. A., Khrapov, E. A. & Filipenko, M. L. The attachment of a DNA-binding Sso7d-like protein improves processivity and resistance to inhibitors of M-MuLV reverse transcriptase. FEBS Lett. 594, 4338–4356 (2020).

    Article  CAS  PubMed  Google Scholar 

  55. Segura, M. M., Alba, R., Bosch, A. & Chillón, M. Advances in helper-dependent adenoviral vector research. Curr. Gene Ther. 8, 222–235 (2008).

    Article  CAS  PubMed  Google Scholar 

  56. Brunetti-Pierri, N. & Ng, P. Progress towards liver and lung-directed gene therapy with helper-dependent adenoviral vectors. Curr. Gene Ther. 9, 329–340 (2009).

    Article  CAS  PubMed  Google Scholar 

  57. Kay, M. A., He, C.-Y. & Chen, Z.-Y. A robust system for production of minicircle DNA vectors. Nat. Biotechnol. 28, 1287–1289 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Hendel, A. et al. Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nat. Biotechnol. 33, 985–989 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Hu, J. et al. Detecting DNA double-stranded breaks in mammalian genomes by linear amplification-mediated high-throughput genome-wide translocation sequencing. Nat. Protoc. 11, 853–871 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Giannoukos, G. et al. UDiTaS, a genome editing detection method for indels and genome rearrangements. BMC Genomics 19, 212 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  61. Tsai, S. Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR–Cas nucleases. Nat. Biotechnol. 33, 187–197 (2015).

    Article  CAS  PubMed  Google Scholar 

  62. Lazzarotto, C. R. et al. CHANGE-seq reveals genetic and epigenetic effects on CRISPR–Cas9 genome-wide activity. Nat. Biotechnol. 38, 1317–1327 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Clement, K. et al. CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat. Biotechnol. 37, 224–226 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Luo, J. et al. A protocol for rapid generation of recombinant adenoviruses using the AdEasy system. Nat. Protoc. 2, 1236–1247 (2007).

    Article  CAS  PubMed  Google Scholar 

  65. Suleman, S. et al. Rapid and inexpensive purification of adenovirus vectors using an optimised aqueous two-phase technology. J. Virol. Methods 299, 114305 (2022).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

C.W.F. is supported by a grant from the Simons Foundation International to the Simons Center for the Social Brain at MIT. C.S.-U. is supported by a Friends of the McGovern fellowship. J.S.G. and O.O.A. are supported by NIH grants 1R21-AI149694, R01-EB031957, 1R01GM148745, R56-HG011857 and R01AG074932; The McGovern Institute Neurotechnology program; the K. Lisa Yang and Hock E. Tan Center for Molecular Therapeutics in Neuroscience; Impetus Grants; the Cystic Fibrosis Foundation Pioneer Grant; Google Ventures; Pivotal Life Sciences; MGB Gene and Cell Therapy Institute; the Yosemite Fund; Harvey Family Foundation; Termeer Foundation; and Winston Fu. We thank the members of the Abudayyeh-Gootenberg labs for support and advice.

Author information

Author notes

  1. These authors contributed equally: Christopher W. Fell, Cian Schmitt-Ulms.

  2. These authors jointly supervised this work: Jonathan S. Gootenberg, Omar O. Abudayyeh.

Authors and Affiliations

  1. Harvard Medical School, Harvard University, Boston, MA, USA

    Christopher W. Fell, Cian Schmitt-Ulms, Dario V. Tagliaferri, Jonathan S. Gootenberg & Omar O. Abudayyeh

  2. Division of Engineering in Medicine, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Cambridge, MA, USA

    Christopher W. Fell, Cian Schmitt-Ulms, Dario V. Tagliaferri, Jonathan S. Gootenberg & Omar O. Abudayyeh

  3. Gene and Cell Therapy Institute, Mass General Brigham, Cambridge, MA, USA

    Christopher W. Fell, Cian Schmitt-Ulms, Dario V. Tagliaferri, Jonathan S. Gootenberg & Omar O. Abudayyeh

  4. Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Boston, MA, USA

    Christopher W. Fell, Cian Schmitt-Ulms, Dario V. Tagliaferri, Jonathan S. Gootenberg & Omar O. Abudayyeh

  5. Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA, USA

    Cian Schmitt-Ulms

  6. Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA

    Jonathan S. Gootenberg & Omar O. Abudayyeh

Authors
  1. Christopher W. Fell
    View author publications

    Search author on:PubMed Google Scholar

  2. Cian Schmitt-Ulms
    View author publications

    Search author on:PubMed Google Scholar

  3. Dario V. Tagliaferri
    View author publications

    Search author on:PubMed Google Scholar

  4. Jonathan S. Gootenberg
    View author publications

    Search author on:PubMed Google Scholar

  5. Omar O. Abudayyeh
    View author publications

    Search author on:PubMed Google Scholar

Contributions

C.W.F. and C.S.-U. equally contributed to writing the introduction and protocol and generating all figures and performing experiments. D.V.T. performed experiments and assisted with protocol writing. O.O.A. and J.S.G supervised research and contributed to writing the manuscript and drafting of the figures. All authors edited the manuscript.

Corresponding authors

Correspondence to Jonathan S. Gootenberg or Omar O. Abudayyeh.

Ethics declarations

Competing interests

C.W.F., C.S.-U., J.S.G. and O.O.A. are inventors on patent applications related to CRISPR technologies. O.O.A. and J.S.G. are co-founders of Sherlock Biosciences, Doppler Biosciences, Circle Labs and Tome Biosciences.

Peer review

Peer review information

Nature Protocols thanks Rasmus Bak, Shahid Mansoor, Yiping Qi and the other, anonymous, reviewer(s) 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.

Key reference

Yarnall, M. T. N. et al. Nat. Biotechnol. 41, 500–512 (2023): https://doi.org/10.1038/s41587-022-01527-4

Supplementary information

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

Fell, C.W., Schmitt-Ulms, C., Tagliaferri, D.V. et al. Precise kilobase-scale genomic insertions in mammalian cells using PASTE. Nat Protoc 20, 1546–1583 (2025). https://doi.org/10.1038/s41596-024-01090-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Version of record:

  • Issue date:

  • DOI: https://doi.org/10.1038/s41596-024-01090-z

This article is cited by

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