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Engineered pegRNAs improve prime editing efficiency

Nature Biotechnology volume 40, pages 402–410 (2022)Cite this article

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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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References

  1. Komor, A. C., Badran, A. H. & Liu, D. R. CRISPR-based technologies for the manipulation of eukaryotic genomes. Cell 168, 20–36 (2017).

    Article  CAS  PubMed  Google Scholar 

  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).

    Article  CAS  PubMed  Google Scholar 

  3. Cullot, G. et al. CRISPR–Cas9 genome editing induces megabase-scale chromosomal truncations. Nat. Commun. 10, 1136 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  4. Kosicki, M., Tomberg, K. & Bradley, A. Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements. Nat. Biotechnol. 36, 765–771 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Boroviak, K., Fu, B., Yang, F., Doe, B. & Bradley, A. Revealing hidden complexities of genomic rearrangements generated with Cas9. Sci. Rep. 7, 12867 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  6. Enache, O. M. et al. Cas9 activates the p53 pathway and selects for p53-inactivating mutations. Nat. Genet. 52, 662–668 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Haapaniemi, E., Botla, S., Persson, J., Schmierer, B. & Taipale, J. CRISPR–Cas9 genome editing induces a p53-mediated DNA damage response. Nat. Med. 24, 927–930 (2018).

    Article  CAS  PubMed  Google Scholar 

  8. Ihry, R. J. et al. p53 inhibits CRISPR–Cas9 engineering in human pluripotent stem cells. Nat. Med. 24, 939–946 (2018).

    Article  CAS  PubMed  Google Scholar 

  9. Leibowitz, M. L. et al. Chromothripsis as an on-target consequence of CRISPR–Cas9 genome editing. Nat. Genet. 53, 895–905 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Burgio, G. & Teboul, L. Anticipating and identifying collateral damage in genome editing. Trends Genet. 36, 905–914 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Cox, D. B., Platt, R. J. & Zhang, F. Therapeutic genome editing: prospects and challenges. Nat. Med. 21, 121–131 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A. & Liu, D. R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Gaudelli, N. M. et al. Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 551, 464–471 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. 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 

  15. Houseley, J. & Tollervey, D. The many pathways of RNA degradation. Cell 136, 763–776 (2009).

    Article  CAS  PubMed  Google Scholar 

  16. Hendel, A. et al. Chemically modified guide RNAs enhance CRISPR–Cas genome editing in human primary cells. Nat. Biotechnol. 33, 985–989 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Geisberg, J. V., Moqtaderi, Z., Fan, X., Ozsolak, F. & Struhl, K. Global analysis of mRNA isoform half-lives reveals stabilizing and destabilizing elements in yeast. Cell 156, 812–824 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Wu, X. & Bartel, D. P. Widespread influence of 3′-end structures on mammalian mRNA processing and stability. Cell 169, 905–917 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Brown, J. A. et al. Structural insights into the stabilization of MALAT1 noncoding RNA by a bipartite triple helix. Nat. Struct. Mol. Biol. 21, 633–640 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Roth, A. et al. A riboswitch selective for the queuosine precursor preQ1 contains an unusually small aptamer domain. Nat. Struct. Mol. Biol. 14, 308–317 (2007).

    Article  CAS  PubMed  Google Scholar 

  21. Anzalone, A. V., Lin, A. J., Zairis, S., Rabadan, R. & Cornish, V. W. Reprogramming eukaryotic translation with ligand-responsive synthetic RNA switches. Nat. Methods 13, 453–458 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Houck-Loomis, B. et al. An equilibrium-dependent retroviral mRNA switch regulates translational recoding. Nature 480, 561–564 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Lorenz, R. et al. ViennaRNA package 2.0. Algorithms Mol. Biol. 6, 26 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Kim, D. Y., Moon, S. B., Ko, J. H., Kim, Y. S. & Kim, D. Unbiased investigation of specificities of prime editing systems in human cells. Nucleic Acids Res. 48, 10576–10589 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Gao, P. et al. Prime editing in mice reveals the essentiality of a single base in driving tissue specific gene expression. Genome Biol. 22, 83 (2020).

    Article  CAS  Google Scholar 

  27. Lin, Q. et al. High-efficiency prime editing with optimized, paired pegRNAs in plants. Nat. Biotechnol. 39, 923–927 (2021).

    Article  CAS  PubMed  Google Scholar 

  28. 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 

  29. Schmid, M. & Jensen, T. H. The exosome: a multipurpose RNA-decay machine. Trends Biochem. Sci. 33, 501–510 (2008).

    Article  CAS  PubMed  Google Scholar 

  30. Ibrahim, H., Wilusz, J. & Wilusz, C. J. RNA recognition by 3′-to-5′ exonucleases: the substrate perspective. Biochim. Biophys. Acta 1779, 256–265 (2008).

    Article  CAS  PubMed  Google Scholar 

  31. Green, L., Kim, C. H., Bustamante, C. & Tinoco, I. Jr. Characterization of the mechanical unfolding of RNA pseudoknots. J. Mol. Biol. 375, 511–528 (2008).

    Article  CAS  PubMed  Google Scholar 

  32. Chavez, A. et al. Highly efficient Cas9-mediated transcriptional programming. Nat. Methods 12, 326–328 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Hu, J. H. et al. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature 556, 57–63 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Nahar, S. et al. A G-quadruplex motif at the 3′ end of sgRNAs improves CRISPR–Cas9 based genome editing efficiency. Chem. Commun. 54, 2377–2380 (2018).

    Article  CAS  Google Scholar 

  35. Steckelberg, A. L. et al. A folded viral noncoding RNA blocks host cell exoribonucleases through a conformationally dynamic RNA structure. Proc. Natl Acad. Sci. USA 115, 6404–6409 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Cate, J. H. et al. Crystal structure of a group I ribozyme domain: principles of RNA packing. Science 273, 1678–1685 (1996).

    Article  CAS  PubMed  Google Scholar 

  37. Fedor, M. J. & Westhof, E. Ribozymes: the first 20 years. Mol. Cell 10, 703–704 (2002).

    Article  CAS  PubMed  Google Scholar 

  38. Chen, B. et al. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell 155, 1479–1491 (2014).

    Article  CAS  Google Scholar 

  39. Jost, M. et al. Titrating gene expression using libraries of systematically attenuated CRISPR guide RNAs. Nat. Biotechnol. 38, 355–364 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Basila, M., Kelley, M. L. & Smith, A. V. B. Minimal 2′-O-methyl phosphorothioate linkage modification pattern of synthetic guide RNAs for increased stability and efficient CRISPR–Cas9 gene editing avoiding cellular toxicity. PLoS ONE 12, e0188593 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  41. Mead, S. et al. A novel protective prion protein variant that colocalizes with Kuru exposure. N. Engl. J. Med. 361, 2056–2065 (2009).

    Article  CAS  PubMed  Google Scholar 

  42. Asante, E. A. et al. A naturally occurring variant of the human prion protein completely prevents prion disease. Nature 522, 478–481 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Kim, H. K. et al. Predicting the efficiency of prime editing guide RNAs in human cells. Nat. Biotechnol. 39, 198–206 (2021).

    Article  CAS  PubMed  Google Scholar 

  44. Jonsson, T. et al. A mutation in APP protects against Alzheimer’s disease and age-related cognitive decline. Nature 488, 96–99 (2012).

    Article  CAS  PubMed  Google Scholar 

  45. Abifadel, M. et al. Mutations in PCSK9 cause autosomal dominant hypercholesterolemia. Nat. Genet. 34, 154–156 (2003).

    Article  CAS  PubMed  Google Scholar 

  46. Bustami, J. et al. Cholesteryl ester transfer protein (CETP) I405V polymorphism and cardiovascular disease in eastern European Caucasians—a cross-sectional study. BMC Geriatr. 16, 144 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  47. Flannick, J. et al. Loss-of-function mutations in SLC30A8 protect against type 2 diabetes. Nat. Genet. 46, 357–363 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Sakuntabhai, A. et al. A variant in the CD209 promoter is associated with severity of dengue disease. Nat. Genet. 37, 507–513 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Olson, H. E. et al. Cyclin-dependent kinase-like 5 deficiency disorder: clinical review. Pediatr. Neurol. 97, 18–25 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  50. Al-Saaidi, R. et al. The LMNA mutation p.Arg321Ter associated with dilated cardiomyopathy leads to reduced expression and a skewed ratio of lamin A and lamin C proteins. Exp. Cell. Res. 319, 3010–3019 (2013).

    Article  CAS  PubMed  Google Scholar 

  51. Ip, J. P. K., Mellios, N. & Sur, M. Rett syndrome: insights into genetic, molecular and circuit mechanisms. Nat. Rev. Neurosci. 19, 368–382 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Christodoulou, J., Grimm, A., Maher, T. & Bennetts, B. RettBASE: the IRSA MECP2 variation database—a new mutation database in evolution. Hum. Mutat. 21, 466–472 (2003).

    Article  CAS  PubMed  Google Scholar 

  53. Gaudelli, N. M. et al. Directed evolution of adenine base editors with increased activity and therapeutic application. Nat. Biotechnol. 38, 892–900 (2020).

    Article  CAS  PubMed  Google Scholar 

  54. 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 

  55. Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta CT) method. Methods 25, 402–408 (2001).

    Article  CAS  PubMed  Google Scholar 

  56. Fang, W. & Bartel, D. P. The menu of features that define primary microRNAs and enable de novo design of microRNA genes. Mol. Cell 60, 131–145 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Romani, A. M. P. Cellular magnesium homeostasis. Arch. Biochem. Biophys. 512, 1–23 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Bertsimas, D. & Tsitsiklis, J. Simulated Annealing. Stat. Sci. 8, 10–15 (1993).

    Article  Google Scholar 

  59. Win, M. N. & Smolke, C. D. A modular and extensible RNA-based gene-regulatory platform for engineering cellular function. Proc. Natl Acad. Sci. USA 104, 14283–14288 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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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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Author notes

  1. These authors contributed equally: James W. Nelson, Peyton B. Randolph.

Authors and Affiliations

  1. Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, MA, USA

    James W. Nelson, Peyton B. Randolph, Simon P. Shen, Kelcee A. Everette, Peter J. Chen, Andrew V. Anzalone, Meirui An, Gregory A. Newby, Jonathan C. Chen, Alvin Hsu & David R. Liu

  2. Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA

    James W. Nelson, Peyton B. Randolph, Simon P. Shen, Kelcee A. Everette, Peter J. Chen, Andrew V. Anzalone, Meirui An, Gregory A. Newby, Jonathan C. Chen, Alvin Hsu & David R. Liu

  3. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA

    James W. Nelson, Peyton B. Randolph, Simon P. Shen, Kelcee A. Everette, Peter J. Chen, Andrew V. Anzalone, Meirui An, Gregory A. Newby, Jonathan C. Chen, Alvin Hsu & David R. Liu

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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.

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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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