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.

  • Brief Communication
  • Published:

Engineered CRISPR prime editors with compact, untethered reverse transcriptases

Nature Biotechnology volume 41, pages 337–343 (2023)Cite this article

Subjects

Abstract

The CRISPR prime editor PE2 consists of a Streptococcus pyogenes Cas9 nickase (nSpCas9) fused at its C-terminus to a Moloney murine leukemia virus reverse transcriptase (MMLV-RT). Here we show that separated nSpCas9 and MMLV-RT proteins function as efficiently as intact PE2 in human cells. We use this Split-PE system to rapidly identify and engineer more compact prime editor architectures that also broaden the types of RTs used for prime editing.

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: Split and intact prime editors function with similar efficiencies in human HEK293T cells.
Fig. 2: Rapid screening of variant RT domains using the Split-PE platform in HEK293T cells.
Fig. 3: Comparison of Split-PEΔRH with a split-intein PE system in HEK293T cells and dual AAV delivery of Split-PEΔRH to U2OS cells.

Similar content being viewed by others

Data availability

Plasmids encoding constructs used in this study have been deposited at Addgene and will be available at https://www.addgene.org/Keith_Joung (Addgene plasmid nos. 190104–190112). All targeted amplicon sequencing data have been deposited at the National Center of Biotechnology Information’s Sequence Read Archive and can be accessed via http://www.ncbi.nlm.nih.gov/bioproject/861237 (ref. 40).

Code availability

Custom Python scripts that were generated for CRISPResso analyses are provided in the Supplementary Information (Supplementary Note 2).

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  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Li, X. et al. Highly efficient prime editing by introducing same-sense mutations in pegRNA or stabilizing its structure. Nat. Commun. 13, 1669 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Xu, W. et al. A design optimized prime editor with expanded scope and capability in plants. Nat Plants 8, 45–52 (2022).

    Article  CAS  PubMed  Google Scholar 

  5. Wang, Y. et al. BE-PIGS: a base-editing tool with deaminases inlaid into Cas9 PI domain significantly expanded the editing scope. Signal Transduct. Target Ther. 4, 36 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  6. Petri, K. et al. CRISPR prime editing with ribonucleoprotein complexes in zebrafish and primary human cells. Nat. Biotechnol. 40, 189–193 (2022).

    Article  CAS  PubMed  Google Scholar 

  7. Kleinstiver, B. P. et al. Broadening the targeting range of Staphylococcus aureus CRISPR–Cas9 by modifying PAM recognition. Nat. Biotechnol. 33, 1293–1298 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. 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 

  9. Kleinstiver, B. P. et al. High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529, 490–495 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Gu, J. et al. Substitution of Asp114 or Arg116 in the fingers domain of Moloney murine leukemia virus reverse transcriptase affects interactions with the template-primer resulting in decreased processivity. J. Mol. Biol. 305, 341–359 (2001).

    Article  CAS  PubMed  Google Scholar 

  11. Das, D. et al. A directed approach to improving the solubility of Moloney murine leukemia virus reverse transcriptase. Protein Sci. 10, 1936–1941 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Katano, Y. et al. Generation of thermostable Moloney murine leukemia virus reverse transcriptase variants using site saturation mutagenesis library and cell-free protein expression system. Biosci. Biotechnol. Biochem. 81, 2339–2345 (2017).

    Article  CAS  PubMed  Google Scholar 

  13. Cote, M. L. et al. Murine leukemia virus reverse transcriptase: structural comparison with HIV-1 reverse transcriptase. Virus Res. 134, 186–202 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Das, D. et al. The crystal structure of the monomeric reverse transcriptase from Moloney murine leukemia virus. Structure 12, 819–829 (2004).

    Article  CAS  PubMed  Google Scholar 

  15. Bock, D. et al. In vivo prime editing of a metabolic liver disease in mice. Sci. Transl. Med. 14, eabl9238 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Ann Ran, F. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–191 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  17. Yu, S. F. et al. Human foamy virus replication: a pathway distinct from that of retroviruses and hepadnaviruses. Science 271, 1579–1582 (1996).

    Article  CAS  PubMed  Google Scholar 

  18. Wohrl, B. M. Structural and functional aspects of foamy virus protease-reverse transcriptase. Viruses 11, 598 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  19. Berkhout, B. et al. Identification of an active reverse transcriptase enzyme encoded by a human endogenous HERV-K retrovirus. J. Virol. 73, 2365–2375 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Lee, Y. N. et al. Reconstitution of an infectious human endogenous retrovirus. PLoS Pathog. 3, e10 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  21. Mills, D. A. et al. Splicing of a group II intron involved in the conjugative transfer of pRS01 in lactococci. J. Bacteriol. 178, 3531–3538 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Mohr, S. et al. Thermostable group II intron reverse transcriptase fusion proteins and their use in cDNA synthesis and next-generation RNA sequencing. RNA 19, 958–970 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Dai, L. et al. ORF-less and reverse-transcriptase-encoding group II introns in archaebacteria, with a pattern of homing into related group II intron ORFs. RNA 9, 14–19 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Blocker, F. J. et al. Domain structure and three-dimensional model of a group II intron-encoded reverse transcriptase. RNA 11, 14–28 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Stamos, J. L. et al. Structure of a thermostable group II intron reverse transcriptase with template-primer and its functional and evolutionary implications. Mol. Cell 68, 926–939 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Zhao, C. et al. Crystal structures of a group II intron maturase reveal a missing link in spliceosome evolution. Nat. Struct. Mol. Biol. 23, 558–565 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Zhao, C. et al. An ultraprocessive, accurate reverse transcriptase encoded by a metazoan group II intron. RNA 24, 183–195 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Kelley, L. A. et al. The Phyre2 web portal for protein modeling, prediction and analysis. Nat. Protoc. 10, 845–858 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Liu, B. et al. A split prime editor with untethered reverse transcriptase and circular RNA template. Nat. Biotechnol. 40, 779–786 (2022).

    Article  CAS  PubMed  Google Scholar 

  31. Truong, D. J. et al. Development of an intein-mediated split–Cas9 system for gene therapy. Nucleic Acids Res. 43, 6450–6458 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. 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  PubMed  PubMed Central  Google Scholar 

  33. Toro, N. et al. Multiple origins of reverse transcriptases linked to CRISPR–Cas systems. RNA Biol. 16, 1486–1493 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  34. Hopp, T. P. et al. A short polypeptide marker sequence useful for recombinant protein identification and purification. Bio/Technology 6, 1204–1210 (1988).

    Article  CAS  Google Scholar 

  35. Rohland, N. et al. Cost-effective, high-throughput DNA sequencing libraries for multiplexed target capture. Genome Res. 22, 939–946 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).

    Article  CAS  PubMed  Google Scholar 

  37. Kleinstiver, B. P. et al. Engineered CRISPR–Cas9 nucleases with altered PAM specificities. Nature 523, 481–485 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  38. Gramlich, M. et al. Antisense‐mediated exon skipping: a therapeutic strategy for titin‐based dilated cardiomyopathy. EMBO Mol. Med. 7, 562–576 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. 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 

  40. Grünewald, J. et al. Datasets. Sequence Read Archive (SRA). http://www.ncbi.nlm.nih.gov/bioproject/861237 (2022).

Download references

Acknowledgements

Support for this work was provided by the National Institutes of Health (RM1 HG009490 and R35 GM118158 to J.K.J.). J.K.J. is additionally supported by the Desmond and Ann Heathwood MGH Research Scholar Award and the Robert B. Colvin, M.D., Endowed Chair in Pathology. J.G. was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation), Projektnummer 416375182. The project described was supported by a Career Development Award from the American Society of Gene & Cell Therapy (awarded to J.G.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the American Society of Gene & Cell Therapy. K.P. was funded by the DFG, Projektnummer 417577129. We thank M. K. Clement for technical advice; R. Zhou, J. Y. Hsu, A. Schmidts, J. F. Angstman and P. Exconde for discussions and technical advice; and L. P. Pottenplackel for assistance with editing the manuscript.

Author information

Author notes

  1. Julian Grünewald

    Present address: First Department of Medicine, Cardiology, Angiology, Pneumology, Klinikum rechts der Isar, Technical University of Munich, TUM School of Medicine and Health, Munich, Germany

  2. Julian Grünewald

    Present address: Center for Organoid Systems and Tissue Engineering (COS), Garching, Germany

  3. Julian Grünewald

    Present address: TranslaTUM - Organoid Hub, Munich, Germany

  4. Julian Grünewald

    Present address: DZHK (German Center of Cardiovascular Research), Munich Heart Alliance, Munich, Germany

  5. These authors contributed equally: Julian Grünewald, Bret R. Miller, Regan N. Szalay.

  6. These authors jointly supervised this work: Julian Grünewald, J. Keith Joung.

Authors and Affiliations

  1. Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA, USA

    Julian Grünewald, Bret R. Miller, Regan N. Szalay, Peter K. Cabeceiras, Christopher J. Woodilla, Eliza Jane B. Holtz, Karl Petri & J. Keith Joung

  2. Center for Cancer Research and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, MA, USA

    Julian Grünewald, Bret R. Miller, Regan N. Szalay, Peter K. Cabeceiras, Christopher J. Woodilla, Eliza Jane B. Holtz, Karl Petri & J. Keith Joung

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

    Julian Grünewald, Karl Petri & J. Keith Joung

Authors
  1. Julian Grünewald
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Bret R. Miller
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Regan N. Szalay
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Peter K. Cabeceiras
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Christopher J. Woodilla
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Eliza Jane B. Holtz
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Karl Petri
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. J. Keith Joung
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

B.R.M., R.N.S., C.J.W., P.K.C., E.J.B.H. and J.G. performed the laboratory experiments. J.G., R.S. and K.P. performed the computational analyses. J.G., B.R.M, R.N.S. and J.K.J. conceived of and designed the study. J.K.J. and J.G. supervised the work. J.G., R.N.S. and J.K.J. wrote the initial manuscript draft, with the help of L. P. Pottenplackel, and all authors contributed to the writing of the final manuscript.

Corresponding authors

Correspondence to Julian Grünewald or J. Keith Joung.

Ethics declarations

Competing interests

J.K.J. and two other investigators who work on the National Institutes of Health award supporting this research, but are not authors on this publication, are co-founders of and have a financial interest in SeQure Dx, Inc., a company developing technologies for gene editing target profiling. J.K.J. also has, or had during the course of this research, financial interests in several companies developing gene editing technology: Beam Therapeutics, Blink Therapeutics, Chroma Medicine, Editas Medicine, EpiLogic Therapeutics, Excelsior Genomics, Hera Biolabs, Monitor Biotechnologies, Nvelop Therapeutics (f/k/a ETx, Inc.), Pairwise Plants, Poseida Therapeutics and Verve Therapeutics. J.K.J.ʼs interests were reviewed and are managed by Massachusetts General Hospital and Mass General Brigham in accordance with their conflict of interest policies. J.K.J. is a co-inventor on various patents and patent applications that describe gene editing and epigenetic editing technologies. K.P. has a financial interest in SeQure Dx, Inc. K.P.ʼs interests and relationships have been disclosed to Massachusetts General Hospital and Mass General Brigham in accordance with their conflict of interest policies. P.K.C. is a paid consultant to and has financial interests in Nvelop Therapeutics (f/k/a ETx, Inc.). J.G., B.R.M. and J.K.J. are co-inventors on a patent application that has been filed by Mass General Brigham/Massachusetts General Hospital on engineered bipartite PE architectures, reduced size PEs and non-MMLV-RT PE architectures.

Peer review

Peer review information

Nature Biotechnology thanks Jia Chen 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.

Supplementary information

Supplementary Information

Supplementary Figs. 1–10 and Supplementary Notes 1 and 2

Reporting Summary

Supplementary Table 1

Editing frequencies in comparison experiments between intact and split PE architectures.

Supplementary Table 2

Editing frequencies and PBS/RTT lengths for a subset of comparison experiments between intact and split PE architectures.

Supplementary Table 3

Constructs used in this study.

Supplementary Table 4

gRNA and amplicon sequences, next-generation sequencing barcodes and read depth of individual experiments.

Rights and permissions

Springer Nature or its licensor 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

Grünewald, J., Miller, B.R., Szalay, R.N. et al. Engineered CRISPR prime editors with compact, untethered reverse transcriptases. Nat Biotechnol 41, 337–343 (2023). https://doi.org/10.1038/s41587-022-01473-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41587-022-01473-1

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