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Deletion and replacement of long genomic sequences using prime editing

Abstract

Genomic insertions, duplications and insertion/deletions (indels), which account for ~14% of human pathogenic mutations, cannot be accurately or efficiently corrected by current gene-editing methods, especially those that involve larger alterations (>100 base pairs (bp)). Here, we optimize prime editing (PE) tools for creating precise genomic deletions and direct the replacement of a genomic fragment ranging from ~1 kilobases (kb) to ~10 kb with a desired sequence (up to 60 bp) in the absence of an exogenous DNA template. By conjugating Cas9 nuclease to reverse transcriptase (PE-Cas9) and combining it with two PE guide RNAs (pegRNAs) targeting complementary DNA strands, we achieve precise and specific deletion and repair of target sequences via using this PE-Cas9-based deletion and repair (PEDAR) method. PEDAR outperformed other genome-editing methods in a reporter system and at endogenous loci, efficiently creating large and precise genomic alterations. In a mouse model of tyrosinemia, PEDAR removed a 1.38-kb pathogenic insertion within the Fah gene and precisely repaired the deletion junction to restore FAH expression in liver.

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Fig. 1: PEDAR mediates large target deletion and simultaneous insertion at an endogenous genomic locus.
Fig. 2: Flexibility of PEDAR in programming a larger deletion and insertion in HEK293T cells.
Fig. 3: PEDAR generates an in-frame deletion to restore mCherry expression in TLR cells.
Fig. 4: PEDAR corrects the pathogenic insertion in a tyrosinemia I mouse model.

Data availability

A Reporting Summary for this article is available as a Supplementary Information file. The raw gel images underlying Figs. 1c,d, 2b,c,f,g, 3d and 4f and Supplementary Figs. 2d,e, 3a,b,e,f and 4a,b are provided as a Source Data file and an additional Supplementary Data file, respectively. The NCBI ClinVar database is accessible at https://www.ncbi.nlm.nih.gov/clinvar/. The raw DNA sequencing data are available at the NCBI Sequence Read Archive database under accession numbers PRJNA746292 and PRJNA746489Source data are provided with this paper.

References

  1. Landrum, M. J. et al. ClinVar: public archive of relationships among sequence variation and human phenotype. Nucleic Acids Res. 42, D980–D985 (2014).

    CAS  PubMed  Google Scholar 

  2. Cordaux, R. & Batzer, M. A. The impact of retrotransposons on human genome evolution. Nat. Rev. Genet. 10, 691–703 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Chen, J. M., Stenson, P. D., Cooper, D. N. & Ferec, C. A systematic analysis of LINE-1 endonuclease-dependent retrotranspositional events causing human genetic disease. Hum. Genet. 117, 411–427 (2005).

    CAS  PubMed  Google Scholar 

  4. Hancks, D. C. & Kazazian, H. H. Roles for retrotransposon insertions in human disease. Mob. DNA 7, 9 (2016).

    PubMed  PubMed Central  Google Scholar 

  5. Wang, L., Norris, E. T. & Jordan, I. K. Human retrotransposon insertion polymorphisms are associated with health and disease via gene regulatory phenotypes. Front. Microbiol. 8, 1418 (2017).

    PubMed  PubMed Central  Google Scholar 

  6. Hancks, D. C. & Kazazian, H. H. Jr. Active human retrotransposons: variation and disease. Curr. Opin. Genet. Dev. 22, 191–203 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Qian, Y. et al. Identification of pathogenic retrotransposon insertions in cancer predisposition genes. Cancer Genet. 216-217, 159–169 (2017).

    CAS  PubMed  Google Scholar 

  8. Ran, F. A. et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154, 1380–1389 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Kato, T. et al. Creation of mutant mice with megabase-sized deletions containing custom-designed breakpoints by means of the CRISPR/Cas9 system. Sci. Rep. 7, 59 (2017).

    PubMed  PubMed Central  Google Scholar 

  11. Hara, S. et al. Microinjection-based generation of mutant mice with a double mutation and a 0.5 Mb deletion in their genome by the CRISPR/Cas9 system. J. Reprod. Dev. 62, 531–536 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Wang, L. et al. Large genomic fragment deletion and functional gene cassette knock-in via Cas9 protein mediated genome editing in one-cell rodent embryos. Sci. Rep. 5, 17517 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 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).

    CAS  PubMed  Google Scholar 

  14. Zheng, Q. et al. Precise gene deletion and replacement using the CRISPR/Cas9 system in human cells. Biotechniques 57, 115–124 (2014).

    CAS  PubMed  Google Scholar 

  15. Cox, D. B., Platt, R. J. & Zhang, F. Therapeutic genome editing: prospects and challenges. Nat. Med. 21, 121–131 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Liu, M. et al. Methodologies for improving HDR efficiency. Front. Genet. 9, 691 (2018).

    CAS  PubMed  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).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Matsoukas, I. G. Prime editing: genome editing for rare genetic diseases without double-strand breaks or donor DNA. Front. Genet. 11, 528 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Liu, P. et al. Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice. Nat. Commun. 12, 2121 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Jang, H. et al. Prime editing enables precise genome editing in mouse liver and retina. Preprint at bioRxiv https://doi.org/10.1101/2021.01.08.425835 (2021).

  21. Schene, I. F. et al. Prime editing for functional repair in patient-derived disease models. Nat. Commun. 11, 5352 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Jiang, Y. Y. et al. Prime editing efficiently generates W542L and S621I double mutations in two ALS genes in maize. Genome Biol. 21, 257 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Song, X., Huang, H., Xiong, Z., Ai, L. & Yang, S. CRISPR–Cas9D10A nickase-assisted genome editing in Lactobacillus casei. Appl. Environ. Microbiol. 83, 1259–1275 (2017).

    Google Scholar 

  24. Cho, S. W. et al. Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Genome Res. 24, 132–141 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Sfeir, A. & Symington, L. S. Microhomology-mediated end joining: a back-up survival mechanism or dedicated pathway? Trends Biochem. Sci 40, 701–714 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Bhargava, R., Onyango, D. O. & Stark, J. M. Regulation of single-strand annealing and its role in genome maintenance. Trends Genet. 32, 566–575 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  28. Mir, A. et al. Heavily and fully modified RNAs guide efficient SpyCas9-mediated genome editing. Nat. Commun. 9, 2641 (2018).

    PubMed  PubMed Central  Google Scholar 

  29. Certo, M. T. et al. Tracking genome engineering outcome at individual DNA breakpoints. Nat. Methods 8, 671–676 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Zhan, H., Li, A., Cai, Z., Huang, W. & Liu, Y. Improving transgene expression and CRISPR–Cas9 efficiency with molecular engineering-based molecules. Clin. Transl Med. 10, e194 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Chen, R. et al. Enrichment of transiently transfected mesangial cells by cell sorting after cotransfection with GFP. Am. J. Physiol. 276, F777–F785 (1999).

    CAS  PubMed  Google Scholar 

  32. Homann, S. et al. A novel rapid and reproducible flow cytometric method for optimization of transfection efficiency in cells. PLoS ONE 12, e0182941 (2017).

    PubMed  PubMed Central  Google Scholar 

  33. Pham, C. T., MacIvor, D. M., Hug, B. A., Heusel, J. W. & Ley, T. J. Long-range disruption of gene expression by a selectable marker cassette. Proc. Natl Acad. Sci. USA 93, 13090–13095 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Grompe, M. et al. Loss of fumarylacetoacetate hydrolase is responsible for the neonatal hepatic dysfunction phenotype of lethal albino mice. Genes Dev. 7, 2298–2307 (1993).

    CAS  PubMed  Google Scholar 

  35. Paulk, N. K. et al. Adeno-associated virus gene repair corrects a mouse model of hereditary tyrosinemia in vivo. Hepatology 51, 1200–1208 (2010).

    CAS  PubMed  Google Scholar 

  36. Choi, J. et al. Precise genomic deletions using paired prime editing. Nat. Biotechnol. https://doi.org/10.1038/s41587-021-01025-z (in the press).

  37. VanLith, C. J. et al. Ex vivo hepatocyte reprograming promotes homology-directed DNA repair to correct metabolic disease in mice after transplantation. Hepatol. Commun. 3, 558–573 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Dutta, A. et al. Microhomology-mediated end joining is activated in irradiated human cells due to phosphorylation-dependent formation of the XRCC1 repair complex. Nucleic Acids Res. 45, 2585–2599 (2016).

    PubMed Central  Google Scholar 

  39. Aida, T. et al. Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ. BMC Genomics 17, 979 (2016).

    PubMed  PubMed Central  Google Scholar 

  40. Warby, S. C. et al. CAG expansion in the Huntington disease gene is associated with a specific and targetable predisposing haplogroup. Am. J. Hum. Genet. 84, 351–366 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Wang, Y. et al. Identification of a Xist silencing domain by Tiling CRISPR. Sci. Rep. 9, 2408 (2019).

    PubMed  PubMed Central  Google Scholar 

  42. He, W. et al. De novo identification of essential protein domains from CRISPR–Cas9 tiling-sgRNA knockout screens. Nat. Commun. 10, 4541 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Xue, W. et al. Response and resistance to NF-κB inhibitors in mouse models of lung adenocarcinoma. Cancer Discov. 1, 236–247 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Magoc, T. & Salzberg, S. L. FLASH: fast length adjustment of short reads to improve genome assemblies. Bioinformatics 27, 2957–2963 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank C. Mello, P. Zamore, S. Wolfe, T. Flotte and E. Sontheimer for discussions and E. Haberlin for editing the manuscript. We thank E. Sontheimer (UMass Medical School) for providing the HEK293T-TLR cell line and M. Grompe (Oregon Health & Science University) for providing the FahΔExon5 mice. We thank Y. Liu, Y. Gu and E. Kittler in the UMass Morphology, Flow Cytometry and Deep Sequencing Cores for support. W.X. was supported by grants from the National Institutes of Health (DP2HL137167, P01HL131471 and UG3HL147367), American Cancer Society (129056-RSG-16-093), the Lung Cancer Research Foundation and the Cystic Fibrosis Foundation. T.J. was supported by grants from National Institutes of Health (K99HL153940).

Author information

Authors and Affiliations

Authors

Contributions

T.J. and W.X. designed the study. T.J. performed experiments. T.J., X.-O.Z. and Z.W. analyzed the data. T.J. and W.X. wrote the manuscript with comments from all authors.

Corresponding author

Correspondence to Wen Xue.

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Competing interests

UMass has filed a patent application on this work. W.X. is a consultant for the Cystic Fibrosis Foundation Therapeutics Lab. The other authors declare no competing interests.

Additional information

Peer review information Nature Biotechnology thanks Daesik Kim and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–8 and Tables 1–3.

Reporting Summary

Supplementary Data

Unprocessed gel and blot images for Supplementary Figs. 2d,e, 3a,b,e,f and 4a,b.

Source data

Source Data Fig. 1

Unprocessed gels and blots for Fig. 1c,d.

Source Data Fig. 2

Unprocessed gels and blots for Fig. 2b,c,f,g.

Source Data Fig. 3

Unprocessed gels and blots for Fig. 3d.

Source Data Fig. 4

Unprocessed gels and blots for Fig. 4f.

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Jiang, T., Zhang, XO., Weng, Z. et al. Deletion and replacement of long genomic sequences using prime editing. Nat Biotechnol 40, 227–234 (2022). https://doi.org/10.1038/s41587-021-01026-y

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