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Engineered CRISPR prime editors with compact, untethered reverse transcriptases

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.

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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.

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).

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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

Authors and Affiliations

Authors

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.

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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.

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Grünewald, J., Miller, B.R., Szalay, R.N. et al. Engineered CRISPR prime editors with compact, untethered reverse transcriptases. Nat Biotechnol (2022). https://doi.org/10.1038/s41587-022-01473-1

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