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Rapid generation of long, chemically modified pegRNAs for prime editing

Nature Biotechnology volume 43, pages 1156–1167 (2025) Cite this article

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Abstract

The editing efficiencies of prime editing (PE) using ribonucleoprotein (RNP) and RNA delivery are not optimal due to the challenges in solid-phase synthesis of long PE guide RNA (pegRNA) (>125 nt). Here, we develop an efficient, rapid and cost-effective method for generating chemically modified pegRNA (125–145 nt) and engineered pegRNA (epegRNA) (170–190 nt). We use an optimized splint ligation approach and achieve approximately 90% production efficiency for these RNAs, referred to as L-pegRNA and L-epegRNA. L-epegRNA demonstrates enhanced editing efficiencies across various cell lines and human primary cells with improvements of up to more than tenfold when using RNP delivery and several hundredfold with RNA delivery of PE, compared to epegRNA produced by in vitro transcription. L-epegRNA-mediated RNP delivery also outperforms plasmid-encoded PE in most comparisons. Our study provides a solution to obtaining high-quality pegRNA and epegRNA with desired chemical modifications, paving the way for the use of PE in therapeutics and various other fields.

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Fig. 1: Ligated pegRNA mediates efficient PE.
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Fig. 2: Comparison of different ligation strategies for pegRNA.
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Fig. 3: Comparison of PE efficiencies via plasmid and L-epegRNA-mediated RNP delivery.
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Fig. 4: PE efficiencies by L-epegRNA-mediated RNA delivery.
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Fig. 5: PE efficiencies using L-epegRNA in human primary T cells and hematopoietic stem cells.
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Fig. 6: Multi-fragments assembled L-epegRNA mediates efficient large insertions.
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Data availability

High-throughput sequencing data have been deposited in the National Center for Biotechnology Information Sequence Read Archive database under accession PRJNA1067838 (ref. 79). Source data are provided with this paper.

References

  1. 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 

  2. Hsu, P. D., Lander, E. S. & Zhang, F. Development and applications of CRISPR–Cas9 for genome engineering. Cell 157, 1262–1278 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Doudna, J. A. & Charpentier, E. Genome editing. The new frontier of genome engineering with CRISPR–Cas9. Science 346, 1258096 (2014).

    Article  PubMed  Google Scholar 

  4. Rees, H. A. & Liu, D. R. Base editing: precision chemistry on the genome and transcriptome of living cells. Nat. Rev. Genet. 19, 770–788 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. 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 

  6. Nelson, J. W. et al. Engineered pegRNAs improve prime editing efficiency. Nat. Biotechnol. 40, 402–410 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  7. Zhang, G. et al. Enhancement of prime editing via xrRNA motif-joined pegRNA. Nat. Commun. 13, 1856 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Liu, Y. et al. Enhancing prime editing by Csy4-mediated processing of pegRNA. Cell Research 31, 1134–1136 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Yin, H. et al. Structure-guided chemical modification of guide RNA enables potent non-viral in vivo genome editing. Nat. Biotechnol. 35, 1179–1187 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. 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 

  11. Ryan, D. E. et al. Improving CRISPR–Cas specificity with chemical modifications in single-guide RNAs. Nucleic Acids Res. 46, 792–803 (2018).

    Article  CAS  PubMed  Google Scholar 

  12. Chen, Q., Zhang, Y. & Yin, H. Recent advances in chemical modifications of guide RNA, mRNA and donor template for CRISPR-mediated genome editing. Adv. Drug Deliv. Rev. 168, 246–258 (2021).

    Article  CAS  PubMed  Google Scholar 

  13. Meisel, R. CRISPR–Cas9 gene editing for sickle cell disease and β-thalassemia. N. Engl. J. Med. 384, e91 (2021).

    PubMed  Google Scholar 

  14. Wang, J. et al. Efficient targeted insertion of large DNA fragments without DNA donors. Nat. Methods 19, 331–340 (2022).

    Article  CAS  PubMed  Google Scholar 

  15. Solomon, A. CRISPR–Cas9 in vivo gene editing for transthyretin amyloidosis. N. Engl. J. Med. 385, 1721–1722 (2021).

    Article  CAS  PubMed  Google Scholar 

  16. Finn, J. D. et al. A single administration of CRISPR/Cas9 lipid nanoparticles achieves robust and persistent in vivo genome editing. Cell Rep. 22, 2227–2235 (2018).

    Article  CAS  PubMed  Google Scholar 

  17. Chen, P. J. et al. Enhanced prime editing systems by manipulating cellular determinants of editing outcomes. Cell 184, 5635–5652.e5629 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Everette, K. A. et al. Ex vivo prime editing of patient haematopoietic stem cells rescues sickle-cell disease phenotypes after engraftment in mice. Nat. Biomed. Eng. 7, 616–628 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Liu, B. et al. Targeted genome editing with a DNA-dependent DNA polymerase and exogenous DNA-containing templates. Nat. Biotechnol. 42, 1039–1045 (2024).

    Article  CAS  PubMed  Google Scholar 

  20. Petri, K. et al. CRISPR prime editing with ribonucleoprotein complexes in zebrafish and primary human cells. Nat. Biotechnol. 40, 189–193 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  21. Ponnienselvan, K. et al. Reducing the inherent auto-inhibitory interaction within the pegRNA enhances prime editing efficiency. Nucleic Acids Res. 51, 6966–6980 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Zhang, W. et al. Enhancing CRISPR prime editing by reducing misfolded pegRNA interactions. eLife https://doi.org/10.7554/eLife.90948.2 (2024).

  23. Beaucage, S. L. & Caruthers, M. H. Deoxynucleoside phosphoramidites—a new class of key intermediates for deoxypolynucleotide synthesis. Tetrahedron Lett. 22, 1859–1862 (1981).

    Article  CAS  Google Scholar 

  24. Ryczek, M., Pluta, M., Blaszczyk, L. & Kiliszek, A. Overview of methods for large-scale RNA synthesis. Appl. Sci. 12, 1543 (2022).

    Article  CAS  Google Scholar 

  25. Wang, G. et al. mRNA produced by VSW-3 RNAP has high-level translation efficiency with low inflammatory stimulation. Cell Insight 1, 100056 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  26. Dousis, A., Ravichandran, K., Hobert, E. M., Moore, M. J. & Rabideau, A. E. An engineered T7 RNA polymerase that produces mRNA free of immunostimulatory byproducts. Nat. Biotechnol. 41, 560–568 (2023).

    Article  CAS  PubMed  Google Scholar 

  27. Karikó, K. et al. Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability. Mol. Ther. 16, 1833–1840 (2008).

    Article  PubMed  Google Scholar 

  28. Kormann, M. S. et al. Expression of therapeutic proteins after delivery of chemically modified mRNA in mice. Nat. Biotechnol. 29, 154–157 (2011).

    Article  CAS  PubMed  Google Scholar 

  29. Warren, L. et al. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell 7, 618–630 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Pardi, N., Hogan, M. J., Porter, F. W. & Weissman, D. mRNA vaccines - a new era in vaccinology. Nat. Rev. Drug Discov. 17, 261–279 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Hertler, J. et al. Synthesis of point-modified mRNA. Nucleic Acids Res. 50, e115 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Kao, C., Zheng, M. & Rüdisser, S. A simple and efficient method to reduce nontemplated nucleotide addition at the 3 terminus of RNAs transcribed by T7 RNA polymerase. RNA 5, 1268–1272 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Liu, Y. et al. Synthesis and applications of RNAs with position-selective labelling and mosaic composition. Nature 522, 368–372 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Moody, E. R., Obexer, R., Nickl, F., Spiess, R. & Lovelock, S. L. An enzyme cascade enables production of therapeutic oligonucleotides in a single operation. Science 380, 1150–1154 (2023).

    Article  CAS  PubMed  Google Scholar 

  35. Stark, M. R. & Rader, S. D. Efficient splinted ligation of synthetic RNA using RNA ligase. Methods Mol. Biol. 1126, 137–149 (2014).

    Article  CAS  PubMed  Google Scholar 

  36. Bartosik, K., Debiec, K., Czarnecka, A., Sochacka, E. & Leszczynska, G. Synthesis of nucleobase-modified RNA oligonucleotides by post-synthetic approach. Molecules 25, 3344 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Flamme, M., McKenzie, L. K., Sarac, I. & Hollenstein, M. Chemical methods for the modification of RNA. Methods 161, 64–82 (2019).

    Article  CAS  PubMed  Google Scholar 

  38. Kurschat, W. C., Müller, J., Wombacher, R. & Helm, M. Optimizing splinted ligation of highly structured small RNAs. RNA 11, 1909–1914 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Wang, S., Chen, D., Gao, L. & Liu, Y. Short oligonucleotides facilitate co-transcriptional labeling of RNA at specific positions. J. Am. Chem. Soc. 144, 5494–5502 (2022).

    Article  CAS  PubMed  Google Scholar 

  40. Van Giesen, K. J. D., Thompson, M. J., Meng, Q. & Lovelock, S. L. Biocatalytic synthesis of antiviral nucleosides, cyclic dinucleotides, and oligonucleotide therapies. JACS Au 3, 13–24 (2023).

    Article  PubMed  Google Scholar 

  41. Hengesbach, M. et al. RNA intramolecular dynamics by single-molecule FRET. Curr. Protoc. Nucleic Acid Chem. Ch. 11, Unit 11.12 (2008).

    Google Scholar 

  42. Stark, M. R., Pleiss, J. A., Deras, M., Scaringe, S. A. & Rader, S. D. An RNA ligase-mediated method for the efficient creation of large, synthetic RNAs. RNA 12, 2014–2019 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Nandakumar, J. & Shuman, S. How an RNA ligase discriminates RNA versus DNA damage. Mol. Cell 16, 211–221 (2004).

    Article  CAS  PubMed  Google Scholar 

  44. Chakravarty, A. K. & Shuman, S. The sequential 2′,3′-cyclic phosphodiesterase and 3′-phosphate/5′-OH ligation steps of the RtcB RNA splicing pathway are GTP-dependent. Nucleic Acids Res. 40, 8558–8567 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Gamper, H. et al. Enzymatic synthesis of RNA standards for mapping and quantifying RNA modifications in sequencing analysis. Methods Enzymol. 692, 127–153 (2023).

    Article  CAS  PubMed  Google Scholar 

  46. Bullard, D. R. & Bowater, R. P. Direct comparison of nick-joining activity of the nucleic acid ligases from bacteriophage T4. Biochem. J. 398, 135–144 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Viollet, S., Fuchs, R. T., Munafo, D. B., Zhuang, F. & Robb, G. B. T4 RNA ligase 2 truncated active site mutants: improved tools for RNA analysis. BMC Biotechnol. 11, 72 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Li, X. et al. Enhancing prime editing efficiency by modified pegRNA with RNA G-quadruplexes. J. Mol. Cell. Biol. 14, mjac022 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  49. Anzalone, A. V. et al. Programmable deletion, replacement, integration and inversion of large DNA sequences with twin prime editing. Nat. Biotechnol. 40, 731–740 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  50. Yarnall, M. T. N. et al. Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases. Nat. Biotechnol. 41, 500–512 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  51. Zheng, C. et al. A flexible split prime editor using truncated reverse transcriptase improves dual-AAV delivery in mouse liver. Mol. Ther. 30, 1343–1351 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Gao, Z. et al. A truncated reverse transcriptase enhances prime editing by split AAV vectors. Mol. Ther. 30, 2942–2951 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Doman, J. L. et al. Phage-assisted evolution and protein engineering yield compact, efficient prime editors. Cell 186, 3983–4002.e3926 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. An, J. et al. Enhancement of the viability of T cells electroporated with DNA via osmotic dampening of the DNA-sensing cGAS–STING pathway. Nat. Biomed. Eng. 8, 149–164 (2023).

    Article  PubMed  Google Scholar 

  55. Han, W. et al. Base editing of the HBG promoter induces potent fetal hemoglobin expression with no detectable off-target mutations in human HSCs. Cell Stem Cell 30, 1624–1639.e1628 (2023).

    Article  CAS  PubMed  Google Scholar 

  56. Qiu, H. Y., Ji, R. J. & Zhang, Y. Current advances of CRISPR-Cas technology in cell therapy. Cell Insight. 1, 100067 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  57. Yin, H. et al. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat. Biotechnol. 32, 551–553 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Yin, H. et al. Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo. Nat. Biotechnol. 34, 328–333 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Yin, H. et al. Non-viral vectors for gene-based therapy. Nat. Rev. Genet. 15, 541–555 (2014).

    Article  CAS  PubMed  Google Scholar 

  60. Sun, C. et al. Precise integration of large DNA sequences in plant genomes using PrimeRoot editors. Nat. Biotechnol. 42, 316–327 (2023).

    Article  PubMed  Google Scholar 

  61. Katrekar, D. et al. In vivo RNA editing of point mutations via RNA-guided adenosine deaminases. Nat. Methods 16, 239–242 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Khosravi, H. M. & Jantsch, M. F. Site-directed RNA editing: recent advances and open challenges. RNA Biol. 18, 41–50 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Fu, Y. et al. CRISPR-dCas9 and sgRNA scaffolds enable dual-colour live imaging of satellite sequences and repeat-enriched individual loci. Nat. Commun. 7, 11707 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Zalatan, J. G. et al. Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds. Cell 160, 339–350 (2015).

    Article  CAS  PubMed  Google Scholar 

  65. Liang, Z. et al. Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes. Nat. Commun. 8, 14261 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Nishimasu, H. et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156, 935–949 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. 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 

  68. Park, S.-J. et al. Targeted mutagenesis in mouse cells and embryos using an enhanced prime editor. Genome Biol. 22, 170 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  69. Lu, S. et al. Fast and sensitive detection of SARS-CoV-2 RNA using suboptimal protospacer adjacent motifs for Cas12a. Nat. Biomed. Eng. 6, 286–297 (2022).

    Article  CAS  PubMed  Google Scholar 

  70. Wu, J. et al. Characterization of a thermostable Cas12a ortholog. Cell Insight. 2, 100126 (2023).

    Article  PubMed  PubMed Central  Google Scholar 

  71. Zhang, H. X. et al. Cas12a-based one-pot SNP detection with high accuracy. Cell Insight. 2, 100080 (2023).

    Article  PubMed  PubMed Central  Google Scholar 

  72. Shi, Y.-J. et al. DNA topology regulates PAM-Cas9 interaction and DNA unwinding to enable near-PAMless cleavage by thermophilic Cas9. Mol. Cell 82, 4160–4175.e4166 (2022).

    Article  CAS  PubMed  Google Scholar 

  73. Xia, H. et al. Psychrophilic phage VSW-3 RNA polymerase reduces both terminal and full-length dsRNA byproducts in in vitro transcription. RNA Biol. 19, 1130–1142 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Yi, J. et al. Co-delivery of Cas9 mRNA and guide RNAs edits hepatitis B virus episomal and integration DNA in mouse and tree shrew models. Antiviral Res. 215, 105618 (2023).

    Article  CAS  PubMed  Google Scholar 

  75. Zhang, C.-P. et al. Efficient non-viral delivery of macromolecules in human primary hematopoietic stem cells and lymphocytes. J. Mol. Cell. Biol. 15, mjad018 (2023).

    Article  PubMed  PubMed Central  Google Scholar 

  76. Chen, S., Zhou, Y., Chen, Y. & Gu, J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 34, i884–i890 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  77. Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. 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 

  79. Lei, X. et al. Rapid generation of long, chemically modified pegRNAs for prime editing. NCBI Bioproject www.ncbi.nlm.nih.gov/bioproject/1067838 (2024).

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Acknowledgements

This work is kindly supported by National Science and Technology Major Project (grant no. 2023ZD0500600), National Key R&D Program of China (grant nos. 2019YFA0802801 and 2018YFA0801401 to H.Y. and 2022YFF1002801 to Ying Zhang), the Ministry of Agriculture and Rural Affairs of China, Key R&D Program of Hubei Province (grant nos. 2022BCA089 to H.Y. and 2022ACA005 to Ying Zhang), the National Natural Science Foundation of China (grant nos. 31871345 and 32071442 to H.Y. and 31972936 to Ying Zhang), the Fundamental Research Funds for the Central Universities (grant nos. 2042022dx0003 and 2042022kf1190), and the startup funding from Wuhan University (to H.Y. and Ying Zhang). We thank the members of core facility of Medical Research Institute at Wuhan University for their technical support.

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Author notes

  1. These authors contributed equally: Xinlin Lei, Anhui Huang, Didi Chen, Xuebin Wang.

Authors and Affiliations

  1. Departments of Urology and Laboratory Medicine, Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, China

    Xinlin Lei, Anhui Huang, Didi Chen, Xuebin Wang, Ruijin Ji, Jinlin Wang, Yizhou Zhang, Yuming Zhang, Shuhan Lu, Kun Zhang, Qiubing Chen, Ying Zhang & Hao Yin

  2. State Key Laboratory of Virology, TaiKang Center for Life and Medical Sciences, TaiKang Medical School, Wuhan University, Wuhan, China

    Xinlin Lei, Anhui Huang, Didi Chen, Xuebin Wang, Ruijin Ji, Jinlin Wang, Yizhou Zhang, Yuming Zhang, Shuhan Lu, Kun Zhang, Qiubing Chen, Ying Zhang & Hao Yin

  3. Department of Rheumatology and Immunology, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, China

    Ying Zhang

  4. RNA Institute, Wuhan University, Wuhan, China

    Hao Yin

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  1. Xinlin Lei
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Contributions

H.Y. conceived, designed and managed the project. X.L., A.H., D.C. and X.W. performed most of the experiments with the help of R.J., J.W., Yuming Zhang, S.L., K.Z. and Q.C. Ying Zhang edited the manuscript. Yizhou Zhang performed bioinformatic analysis. X.L. and H.Y. analyzed the data. X.L. and H.Y. wrote the paper with inputs from all authors.

Corresponding author

Correspondence to Hao Yin.

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Competing interests

H.Y., Ying Zhang, X.L. and X.W. have filed a patent application on ligation of pegRNA through Wuhan University (application number PCT/CN2024/078744). The other authors declare no competing interests.

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Nature Biotechnology thanks Chang Li and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Optimization of RNA ligation enables assembly of sgRNA.

a. (Left) Overview of design scheme for sgRNA ligation. Acceptor RNA is 20 nt spacer sequence by chemical synthesis, and donor RNA is 82 nt scaffold sequence generated by IVT. (Right) Urea-PAGE of ligated sgRNA (for VEGFA locus). The 102nt IVT RNA depicted in the figure serves as a control band, to indicate the position of bands for successful ligation. The components labeled with ‘+’ were present at equal concentrations across all bands. b. The sequence of ligated sgRNA was determined by Sanger sequencing. The ligated sgRNA was reverse transcribed, PCR amplified, and then TA cloned for Sanger sequencing. Five clones were sequenced, and the arrow indicates the ligation site. c. Urea-PAGE analysis of ligated sgRNA. A mixture of 100 pmol acceptor RNA, 100 pmol donor RNA, and 100 pmol splint DNA (40 nt) were annealed and ligated with 0.5 μl T4 RNA Ligase 2. The ligation reactions were performed at 25 °C or 37 °C, respectively. d. (Left) Overview of design scheme for sgRNA ligation using various lengths of splint DNA (20, 40, or 59 nt). (Right) Urea-PAGE analysis of ligation products. The reactions were performed at 37 °C following the conditions described above. e. Urea-PAGE analysis of sgRNA ligation products with different doses of splint DNA. f. Urea-PAGE of sgRNA ligation products with different doses of T4 RNA Ligase 2. g. The 20+82 ligated RNA: sgRNA was ligated using 20 nt synthetic acceptor RNA and 82 nt IVT-generated donor RNA; the *20+82 Ligated RNA: sgRNA was ligated using 20 nt synthetic acceptor RNA with 5′ modification and 82nt IVT donor RNA. h. In vitro cleavage of ligated sgRNA in TAE agarose gel. The molar ratio of SpCas9 protein and sgRNA was 1:1. The RNP was incubated at room temperature for 10 minutes, followed by the addition of the DNA template and incubation at 37 °C for 1 hour.

Source data

Extended Data Fig. 2 Assessment of ligation accuracy and HPLC purification of ligated pegRNA.

a. RT-PCR and deep sequencing on ligated pegRNA (*32+105) and IVT pegRNA (+5 G to T Mutation in VEGFA locus). b. HPLC purification of ligated pegRNA (+5 G to T Mutation at the VEGFA locus). c. Analysis of pegRNA purity using area under curve (AUC) of each peak. d. Detection of purity by HPLC analysis. mAU, milli-absorbance unit; time, the execution time of the program for (b) and (d). e. Urea-PAGE (6%) analysis of pegRNAs. ‘IVT pegRNA’ indicates full length pegRNA generated by IVT; ‘IVT pegRNA-HPLC’ indicates full length pegRNA generated by IVT with HPLC purification; ‘*32+ Ligated pegRNA’ indicates ligated pegRNA with 5′ end modified; ‘*32+Ligated pegRNA (HPLC)’ indicated ligated pegRNA with 5′ end modified that was HPLC purified.

Source data

Extended Data Fig. 3 PAGE analysis and comparison of non-HPLC purified pegRNA for editing.

a. Urea-PAGE (6%) analysis of ligation products before HPLC purification. b. Urea-PAGE (6%) analysis of HPLC-purified ligated pegRNA and S-pegRNA. S-pegRNA is full-length pegRNA that was solid-phase synthesized with 3 nt chemical modifications at both ends. c–e. The efficiencies of RNP-mediated prime editing in HEK293T cells were determined by deep sequencing for 3 bp insertion at HEK3 locus (c) (n = 4), +5 G to T mutation (d) (n = 5), and 3 bp deletion (e) at VEGFA locus (PE2, n = 4, 4, 5 from left to right side; PE3, n = 4). For each electroporation, 140 pmol PE protein, 186 pmol pegRNA, and 62 pmol nicking sgRNA were used. Data and error bars represent the mean and standard deviation of three or more independent biological replicates. The n values for PE2 and PE3 in figures c-e are indicated alongside each sample type. Data analysis used One-way ANOVA with Tukey's multiple comparisons test; NS, not significant; *p < 0.05; **p < 0.01; ***p < 0.001. *pegRNA*: ligated pegRNA by a 32 nt synthetic acceptor RNA with 5′ end modifications and synthetic donor RNA with 3′ end modifications. *epegRNA: ligated epegRNA by a 32 nt synthetic acceptor RNA with 5′ modifications and IVT-generated donor RNA containing evopreQ1. *epegRNA*: ligated epegRNA by a 91 nt synthetic acceptor RNA with 5′ end modifications and synthetic donor RNA with 3′ end modifications at evopreQ1 sequence.

Source data

Extended Data Fig. 4 Toxicity assessment of ligated pegRNA.

a. A dose of 180 pmol of L-pegRNA (without HPLC purification) and S-pegRNA were delivered via electroporation into 5 × 105 THP1 cells. The expression of signature genes at 4 hours after electroporation were determined by RT-qPCR. Data and error bars represent the mean and standard deviation of three independent biological replicates. Data were analyzed by two-tailed unpaired Student’s t-test; NS indicates no significance. b. Urea-PAGE (6%) analysis of L-epegRNA with or without RNase treatment. Lane 1: marker; Lane 2: 59 nt splint DNA as a reference band; Lane 3: L-epegRNA (for 1 bp insertion at HEK3 site); Lane 4: the same L-epegRNA treated with RNase.

Source data

Extended Data Fig. 5 Effects of RTT and PBS lengths on RNP editing efficiency.

a–h. Editing efficiencies by PE2 (a, e), PE3 (c, g) and respective indels (b, d, f, h) for *epegRNAs targeting HEK3 site across various RTT and PBS lengths. The pegRNAs are for 3 bp insertion (a-d) and 5 bp deletion (e-h), respectively. Each electroporation used 140 pmol PE protein, 186 pmol pegRNA, and 62 pmol nicking sgRNA. Data and error bars represent the mean and standard deviation from at least two technical replicates.

Extended Data Fig. 6 Dose optimization of RNP delivery.

a, b. Doses of pegRNA and protein were adjusted in equal proportions. The abscissa represents the protein dose. c, d. Optimization of PE protein and pegRNA ratios. For each sample, 70 pmol PE protein, and 70, 140, or 280 pmol pegRNA were used. e, f. Optimization of nickRNA dosages for PE3 system. For each sample, 70 pmol PE protein, 140 pmol pegRNA, and 10, 30, 60, or 100 pmol nickRNA were used. Dose optimizations were for editing at the VEGFA (a, c, e) and HEK3 loci (b, d, f), respectively. g–i. Optimizations for editing at the HEK3 locus in K562 cells via RNP delivery, including total dosages (g), PE protein to pegRNA ratios (h), and nicking sgRNA dosages (i). For a-i, *epegRNAs were used. Data and error bars represent the mean and standard deviation of three independent biological replicates. For each sample, 2 × 105 cells were used for electroporation.

Extended Data Fig. 7 Production of different PE proteins and their prime editing efficiencies via RNP.

a. Illustration of the protein expression vectors. b. SDS-PAGE analysis of PE proteins after NI column purification: ΔRH refers to the RT enzyme of PE lacking RNase H domain. The arrow points to the target protein, M, marker. c. Yield of PE proteins after purification. μg/L: protein yield purified from 1 L bacterial solution. Data and error bars represent the mean and standard deviation from at least two independent biological replicates (n = 6,2,2,2 from left to right side). d, e. Prime editing efficiencies mediated by different PE proteins in HEK293T cells were determined by deep sequencing for +5 G to T mutation (d) and deletion of 3 bp (e) at VEGFA locus. For each sample, 70 pmol PE protein, 140 pmol L-epegRNA, and 60 pmol nicking sgRNA were used. Data and error bars represent the mean and standard deviation of three biological replicates. Data analysis used One-way ANOVA; NS, not significant; *p < 0.05; **p < 0.01; ***p < 0.001.

Source data

Extended Data Fig. 8 The editing efficiencies of PE4max and PE5max via RNP in K562 cells.

a, b. Prime editing efficiencies of PE4max and PE5max via RNP delivery for insertion of 3 bp (a) and +1 T to A mutation (b) at the HEK3 locus in K562 cells. For each sample, purified MLH1dn of 0 pmol, 17.5 pmol, 35 pmol, 70 pmol and 140 pmol were used, with 70 pmol PEmax ΔRH protein, 140 pmol L-epegRNA, and 60 pmol nicking sgRNA. c-d. Prime editing efficiencies of PE4max and PE5max via plasmid for insertion of 3 bp (c) and +1 T to A mutation (d) at the HEK3 locus in K562 cells. For each sample, 800 ng PE expression plasmid, 200 ng pegRNA plasmid, and 83 ng nickRNA plasmid were used. Data and error bars represent the mean and standard deviation of three independent biological replicates. Data analysis used One-way ANOVA; NS, not significant; *p < 0.05; **p < 0.01; ***p < 0.001.

Extended Data Fig. 9 Comparing cost and synthesis time of pegRNAs with different production methods.

a. Urea-PAGE analysis of S-pegRNA for 17 bp insertion at the HEK3 locus. b. Urea-PAGE analysis of three-fragment ligation products for 40 bp insertion at the HEK3 locus (234 bp). M, marker. c. Comparative analysis of cost and production period between four S-pegRNAs and their corresponding L-pegRNAs. d. Cost and production period for L-pegRNAs and L-epegRNAs of varied lengths across different synthesis scales, with or without HPLC purification.

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Extended Data Fig. 10 HPLC purification of L-epegRNA and the corresponding urea-PAGE.

An example of L-epegRNA HPLC purification and collecting corresponding fractions, followed by examining each fraction via 6% urea-PAGE after purification.

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Supplementary information

Source data

Source Data Figs. 1 and 6 and Extended Data Figs. 1–4, 7, 9 and 10 (download PDF )

Uncropped gel images of Figs. 1b and 6b and Extended Data Figs. 1–4, 7, 9 and 10.

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Lei, X., Huang, A., Chen, D. et al. Rapid generation of long, chemically modified pegRNAs for prime editing. Nat Biotechnol 43, 1156–1167 (2025). https://doi.org/10.1038/s41587-024-02394-x

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