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

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

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.

Access options

Subscribe to Journal

Get full journal access for 1 year

$59.00

only $4.92 per issue

All prices are NET prices.

VAT will be added later in the checkout.

Tax calculation will be finalised during checkout.

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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.

References

  1. 1.

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

    CAS 
    Article 

    Google Scholar
     

  2. 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).

    CAS 
    Article 

    Google Scholar
     

  3. 3.

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

    CAS 
    Article 

    Google Scholar
     

  4. 4.

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

    CAS 
    Article 

    Google Scholar
     

  5. 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. 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 

    Google Scholar
     

  7. 7.

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

    CAS 
    Article 

    Google Scholar
     

  8. 8.

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

    CAS 
    Article 

    Google Scholar
     

  9. 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).

    CAS 
    Article 

    Google Scholar
     

  10. 10.

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

    Article 

    Google Scholar
     

  11. 11.

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

    CAS 
    Article 

    Google Scholar
     

  12. 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. 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).

    CAS 
    Article 

    Google Scholar
     

  14. 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).

    CAS 
    Article 

    Google Scholar
     

  15. 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).

    CAS 
    Article 

    Google Scholar
     

  16. 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. 17.

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

    CAS 
    Article 

    Google Scholar
     

  18. 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).

    CAS 
    Article 

    Google Scholar
     

  19. 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).

    CAS 
    Article 

    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.), the Defense Advanced Research Projects Agency Safe Genes Program grant no. HR0011-17-2-0042 (to J.K.J.) and National Human Genome Research Institute Genomic Innovator Award no. R35 HG010717 (to L.P.). 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.

Author information

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.

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

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.

Ethics declarations

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.

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

Supplementary information

About this article

Verify currency and authenticity via CrossMark

Cite this article

Petri, K., Zhang, W., Ma, J. et al. CRISPR prime editing with ribonucleoprotein complexes in zebrafish and primary human cells.
Nat Biotechnol (2021). https://doi.org/10.1038/s41587-021-00901-y

Download citation

Read More

Leave a Reply

Your email address will not be published. Required fields are marked *