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Engineered pegRNAs improve prime editing efficiency

An Author Correction to this article was published on 08 December 2021

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Abstract

Prime editing enables the installation of virtually any combination of point mutations, small insertions or small deletions in the DNA of living cells. A prime editing guide RNA (pegRNA) directs the prime editor protein to the targeted locus and also encodes the desired edit. Here we show that degradation of the 3′ region of the pegRNA that contains the reverse transcriptase template and the primer binding site can poison the activity of prime editing systems, impeding editing efficiency. We incorporated structured RNA motifs to the 3′ terminus of pegRNAs that enhance their stability and prevent degradation of the 3′ extension. The resulting engineered pegRNAs (epegRNAs) improve prime editing efficiency 3–4-fold in HeLa, U2OS and K562 cells and in primary human fibroblasts without increasing off-target editing activity. We optimized the choice of 3′ structural motif and developed pegLIT, a computational tool to identify non-interfering nucleotide linkers between pegRNAs and 3′ motifs. Finally, we showed that epegRNAs enhance the efficiency of the installation or correction of disease-relevant mutations.

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Fig. 1: Truncated pegRNAs limit PE efficiency.
Fig. 2: PE editing efficiency is enhanced by the addition of structured RNA motifs to the 3′ terminus of pegRNAs.
Fig. 3: Structural motifs increase RNA stability and efficiency of reverse transcription but reduce Cas9 binding affinity.
Fig. 4: PE-mediated editing efficiency of therapeutically relevant genome editing is improved by the use of epegRNAs.

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Data availability

High-throughput sequencing data have been deposited at the National Center of Biotechnology Information’s Sequence Read Archive database at PRJNA707486. Plasmids encoding select epegRNA Golden Gate cloning vectors have been deposited at Addgene for distribution.

Code availability

A Python implementation of pegLIT is publicly accessible at peglit.liugroup.us, and the code can be found in Supplementary Note 2 or at github.com/sshen8/peglit.

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Acknowledgements

This work was supported by U.S. National Institutes of Health grants U01Al142756, RM1HG009490, R01EB031172 and R35GM118062; the Howard Hughes Medical Institute; and the Loulou Foundation. J.W.N. and A.V.A. were supported by Jane Coffin Childs postdoctoral fellowships. P.B.R., S.P.S., K.A.E. and P.J.C. were supported by National Science Foundation graduate fellowships. G.A.N. was supported by a Helen Hay Whitney postdoctoral fellowship. We thank A. Vieira for assistance in editing this manuscript; M. O’Reilly, E. Berg and the Broad Institute Pattern team for help with figure design; S. McGreary and K. Xiang for helpful discussions on northern blot procedures; and M. Shen for helpful discussions on pegLIT coding.

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Authors and Affiliations

Authors

Contributions

J.W.N. and P.B.R. contributed equally and designed the research, performed experiments and analyzed data. A.V.A. and S.P.S. designed the research. K.A.E., G.A.N., P.J.C., P.C., M.A., J.C.C. and A.H. performed experiments. D.R.L. designed and supervised the research. J.W.N., P.B.R. and D.R.L. wrote the paper.

Corresponding author

Correspondence to David R. Liu.

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Competing interests

The authors are co-inventors on patents filed by the Broad Institute on prime editing. D.R.L. is a consultant and co-founder of Prime Medicine, Beam Therapeutics and Pairwise Plants, which are companies that use genome editing. A.V.A. is currently an employee of Prime Medicine.

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Supplementary Information

Supplementary Discussion, Figs. 1–16 and Notes 1–3

Reporting Summary

Supplementary Tables 1–6

PegRNAs, RNA motifs, sequencing primers, genomic loci, RTqPCR primers and reference SNPs installed.

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Nelson, J.W., Randolph, P.B., Shen, S.P. et al. Engineered pegRNAs improve prime editing efficiency. Nat Biotechnol 40, 402–410 (2022). https://doi.org/10.1038/s41587-021-01039-7

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