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CRISPR prime editing with ribonucleoprotein complexes in zebrafish and primary human cells

Nature Biotechnology volume 40, pages 189–193 (2022)Cite this article

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An Author Correction to this article was published on 13 May 2021

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Abstract

Prime editors have been delivered using DNA or RNA vectors. Here we demonstrate prime editing with purified ribonucleoprotein complexes. We introduced somatic mutations in zebrafish embryos with frequencies as high as 30% and demonstrate germline transmission. We also observed unintended insertions, deletions and prime editing guide RNA (pegRNA) scaffold incorporations. In HEK293T and primary human T cells, prime editing with purified ribonucleoprotein complexes introduced desired edits with frequencies of up to 21 and 7.5%, respectively.

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Fig. 1: Ribonucleoprotein-mediated PE in zebrafish.
Fig. 2: Germline transmission of PE-mediated edits corresponding to human pathogenic variants in zebrafish and RNP-mediated PE in human cells.

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Data availability

Deep sequencing data will be deposited in the NCBI Sequence Read Archive (project no: PRJNA713914). Uncropped gel images in Fig. 1b (left) and Supplementary Fig. 2e can be found in Supplementary information. Source data are provided with this paper.

Code availability

The authors will make available all custom computer code used in this work upon request.

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References

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

    Article  CAS  Google Scholar 

  2. 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  Google Scholar 

  3. Lin, Q. et al. Prime genome editing in rice and wheat. Nat. Biotechnol. 38, 582–585 (2020).

    Article  CAS  Google Scholar 

  4. Liu, Y. et al. Efficient generation of mouse models with the prime editing system. Cell Discov. 6, 27 (2020).

    Article  CAS  Google Scholar 

  5. Surun, D. et al. Efficient generation and correction of mutations in human iPS cells utilizing mRNAs of CRISPR base editors and prime editors. Genes (Basel) 11, 511 (2020).

  6. Bosch, J. A., Birchak, G. & Perrimon, N. Precise genome engineering in Drosophila using prime editing. Proc. Natl Acad. Sci. USA 118, e2021996118 (2021).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  8. Hwang, W. Y. et al. Efficient genome editing in zebrafish using a CRISPR–Cas system. Nat. Biotechnol. 31, 227–229 (2013).

    Article  CAS  Google Scholar 

  9. Chen, B. et al. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell 155, 1479–1491 (2013).

    Article  CAS  Google Scholar 

  10. Gronskov, K., Ek, J. & Brondum-Nielsen, K. Oculocutaneous albinism. Orphanet J. Rare Dis. 2, 43 (2007).

    Article  Google Scholar 

  11. Zhang, Y. et al. Programmable base editing of zebrafish genome using a modified CRISPR–Cas9 system. Nat. Commun. 8, 118 (2017).

    Article  CAS  Google Scholar 

  12. Munoz-Maldonado, C., Zimmer, Y. & Medova, M. A comparative analysis of individual RAS mutations in cancer biology. Front. Oncol. 9, 1088 (2019).

    Article  Google Scholar 

  13. Jao, L. E., Wente, S. R. & Chen, W. Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system. Proc. Natl Acad. Sci. USA 110, 13904–13909 (2013).

    Article  CAS  Google Scholar 

  14. Levy, J. M. et al. Cytosine and adenine base editing of the brain, liver, retina, heart and skeletal muscle of mice via adeno-associated viruses. Nat. Biomed. Eng. 4, 97–110 (2020).

    Article  CAS  Google Scholar 

  15. Kim, S., Kim, D., Cho, S. W., Kim, J. & Kim, J. S. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 24, 1012–1019 (2014).

    Article  CAS  Google Scholar 

  16. Gagnon, J. A. et al. Efficient mutagenesis by Cas9 protein-mediated oligonucleotide insertion and large-scale assessment of single-guide RNAs. PLoS ONE 9, e98186 (2014).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  18. Davidson, W. F. & Parish, C. R. A procedure for removing red cells and dead cells from lymphoid cell suspensions. J. Immunol. Methods 7, 291–300 (1975).

    Article  CAS  Google Scholar 

  19. Bae, S., Park, J. & Kim, J. S. Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30, 1473–1475 (2014).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the Hassenfeld Scholar Award (to J.-R.J.Y.), NIH no. R01 GM134069 (to J.-R.J.Y.), NIH no. RM1 HG009490 (to J.K.J. and L.P.), NIH no. R35 GM118158 (to J.K.J.) and National Human Genome Research Institute Genomic Innovator Award no. R35 HG010717 (to L.P.). The authors received funding from Defense Advanced Research Projects Agency Safe Genes program (HR0011-17-2-0042) for this research. The views, opinions and/or findings expressed should not be interpreted as representing the official views or policies of the Department of Defense or the U.S. Government. K.P. was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – Projektnummer 417577129. J.M. received support from the China Scholarship Council (no. 201808210354). A.S. received support from a John Hansen Research Grant from DKMS (no. DKMS-SLS-JHRG-2020-04). We thank K. K. Lam for technical assistance and K. K. Lam and J. Grünewald for discussions and technical advice. We thank L. Paul-Pottenplackel for help with revision of the manuscript.

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Author notes

  1. These authors contributed equally: Karl Petri, Weiting Zhang, Junyan Ma, Andrea Schmidts.

  2. These authors jointly supervised this work: J. Keith Joung, Jing-Ruey Joanna Yeh.

Authors and Affiliations

  1. Molecular Pathology Unit and Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA, USA

    Karl Petri, Hyunho Lee, Joy E. Horng, Daniel Y. Kim, Ibrahim C. Kurt, Kendell Clement, Jonathan Y. Hsu, Luca Pinello & J. Keith Joung

  2. Department of Pathology, Harvard Medical School, Boston, MA, USA

    Karl Petri, Hyunho Lee, Joy E. Horng, Daniel Y. Kim, Ibrahim C. Kurt, Kendell Clement, Jonathan Y. Hsu, Luca Pinello & J. Keith Joung

  3. Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, MA, USA

    Weiting Zhang, Junyan Ma & Jing-Ruey Joanna Yeh

  4. Department of Medicine, Harvard Medical School, Boston, MA, USA

    Weiting Zhang, Junyan Ma, Andrea Schmidts, Marcela V. Maus & Jing-Ruey Joanna Yeh

  5. Medical College, Dalian University, Dalian, China

    Junyan Ma

  6. Cellular Immunotherapy Program, Massachusetts General Hospital Cancer Center, Charlestown, MA, USA

    Andrea Schmidts & Marcela V. Maus

Authors
  1. Karl Petri
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Contributions

K.P., W.Z., J.M., H.L., A.S., J.E.H., I.C.K., J.K.J. and J.-R.J.Y. designed the project. K.P., W.Z., J.M., H.L., A.S., J.E.H. and D.Y.K. performed the experiments. K.P., W.Z., J.M., H.L., A.S., J.E.H., I.C.K. and J.Y.H. developed the methods. K.P., H.L., K.C. and L.P. performed informatic analysis. M.V.M., J.K.J. and J.-R.J.Y. provided resources and overseeing. K.P., W.Z., J.M., A.S., H.L., J.K.J. and J.-R.J.Y. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to J. Keith Joung or Jing-Ruey Joanna Yeh.

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Competing interests

J.K.J. has financial interests in Beam Therapeutics, Chroma Medicine (formerly known as YKY, Inc.), Editas Medicine, Excelsior Genomics, Pairwise Plants, Poseida Therapeutics, SeQure Dx, Transposagen Biopharmaceuticals and Verve Therapeutics (formerly known as Endcadia). K.P. has a financial interests in SeQure Dx, Inc.. L.P. has financial interests in Edilytics, SeQure Dx, Inc. and Excelsior Genomics. K.P. and D.Y.K. are paid consultants to Verve Therapeutics. K.C. is an employee, shareholder and officer of Edilytics, Inc. The interests of J.K.J., L.P., K.C., K.P. and D.Y.K. were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies. M.V.M. is an inventor on patents related to adoptive cell therapies, held by Massachusetts General Hospital and University of Pennsylvania (some licensed to Novartis). M.V.M. holds equity in TCR2 and Century Therapeutics, and has served as a consultant for multiple companies involved in cell therapies. The remaining authors declare no competing interests.

Additional information

Peer review information Nature Biotechnology thanks Jia Chen, Ayal Hendel 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. 1–14, Supplementary Notes 1–5 and Supplementary Data 1.

Reporting Summary

Supplementary Data

Uncropped gel image for Supplementary Fig. 2e.

Supplementary Tables

Supplementary Tables 1–16.

Source data

Source Data Fig. 1

Uncropped gel image for Fig. 1b, left panel.

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Petri, K., Zhang, W., Ma, J. et al. CRISPR prime editing with ribonucleoprotein complexes in zebrafish and primary human cells. Nat Biotechnol 40, 189–193 (2022). https://doi.org/10.1038/s41587-021-00901-y

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