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Designing and executing prime editing experiments in mammalian cells

Abstract

Prime editing (PE) is a precision gene editing technology that enables the programmable installation of substitutions, insertions and deletions in cells and animals without requiring double-strand DNA breaks (DSBs). The mechanism of PE makes it less dependent on cellular replication and endogenous DNA repair than homology-directed repair-based approaches, and its ability to precisely install edits without creating DSBs minimizes indels and other undesired outcomes. The capabilities of PE have also expanded since its original publication. Enhanced PE systems, PE4 and PE5, manipulate DNA repair pathways to increase PE efficiency and reduce indels. Other advances that improve PE efficiency include engineered pegRNAs (epegRNAs), which include a structured RNA motif to stabilize and protect pegRNA 3′ ends, and the PEmax architecture, which improves editor expression and nuclear localization. New applications such as twin PE (twinPE) can precisely insert or delete hundreds of base pairs of DNA and can be used in tandem with recombinases to achieve gene-sized (>5 kb) insertions and inversions. Achieving optimal PE requires careful experimental design, and the large number of parameters that influence PE outcomes can be daunting. This protocol describes current best practices for conducting PE and twinPE experiments and describes the design and optimization of pegRNAs. We also offer guidelines for how to select the proper PE system (PE1 to PE5 and twinPE) for a given application. Finally, we provide detailed instructions on how to perform PE in mammalian cells. Compared with other procedures for editing human cells, PE offers greater precision and versatility, and can be completed within 2–4 weeks.

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Fig. 1: Mechanism of PE.
Fig. 2: Architecture of an epegRNA.
Fig. 3: Experimental design of epegRNAs.
Fig. 4: Experimental design for twinPE.
Fig. 5: Design of a PE3b/PE5b nicking sgRNA.
Fig. 6: Experimental workflow for PE optimization.
Fig. 7: Example results.

Data availability

Sequencing data used to generate Fig. 7 are deposited at the NCBI Sequence Read Archive database under PRJNA817825.

Code availability

The code used for HTS processing and analysis is accessible at https://github.com/pinellolab/CRISPResso2.

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Acknowledgements

We thank D. Gao and T. Huang for helpful discussions and A. Vieira for feedback on this manuscript. We thank A. Anzalone and other Liu Laboratory members who advanced PE technology. Figures were created with BioRender.com. This work was supported by US NIH U01AI142756, RM1HG009490, R01EB031172 and R35GM118062, the Howard Hughes Medical Institute and the Bill & Melinda Gates Foundation. J.L.D., A.A.S., P.B.R. and P.J.C. are supported by the NSF Graduate Research Fellowship program. J.L.D. is supported by a Fannie and John Hertz Foundation Fellowship.

Author information

Authors and Affiliations

Authors

Contributions

J.L.D. and A.A.S. contributed equally and wrote elements of the introduction, protocol and figures. P.B.R. assisted with figure creation and provided advice on pegRNA optimization and design. P.J.C. performed optimization experiments and made figures. D.R.L. supervised the research and wrote parts of the manuscript. All authors edited the manuscript.

Corresponding author

Correspondence to David R. Liu.

Ethics declarations

Competing interests

J.L.D., A.A.S., P.B.R., P.J.C. and D.R.L. have filed patent applications on PE technologies and applications. P.J.C. is currently an employee of Prime Medicine. D.R.L. is a consultant and equity holder of Prime Medicine, Beam Therapeutics, Pairwise Plants and Chroma Medicine, companies that use genome editing or genome engineering.

Peer review

Peer review information

Nature Protocols thanks Sangsu Bae, Hyongbum H. Kim, Myungjae Song, Goosang Yu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Related links

Key references using this protocol:

Anzalone, A. V. et al. Nature 576, 149–157 (2019): https://doi.org/10.1038/s41586-019-1711-4

Nelson, J. W. et al. Nat. Biotechnol. 40, 402–410 (2022): https://doi.org/10.1038/s41587-021-01039-7

Chen, P. J. et al. Cell 184, 5635–5652 (2021): https://doi.org/10.1016/j.cell.2021.09.018

Anzalone, A. V. et al. Nat. Biotechnol. 40, 731–740 (2022): https://doi.org/10.1038/s41587-021-01133-w

Supplementary information

Reporting Summary

Supplementary Table 1

pegRNA and nicking sgRNA sequences used in Fig. 7.

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Doman, J.L., Sousa, A.A., Randolph, P.B. et al. Designing and executing prime editing experiments in mammalian cells. Nat Protoc (2022). https://doi.org/10.1038/s41596-022-00724-4

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