Data availability
Plasmids for mRNA IVT and protein expression of the new PE variants have been deposited to Addgene for distribution. Illumina sequencing data have been submitted to the Sequence Read Archive, and datasets are available under BioProject accession number PRJNA1024467 (ref. 65). Source data are provided with this paper.
References
-
Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019).
-
Chen, P. J. et al. Enhanced prime editing systems by manipulating cellular determinants of editing outcomes. Cell 184, 5635–5652.e29 (2021).
-
Doman, J. L., Sousa, A. A., Randolph, P. B., Chen, P. J. & Liu, D. R. Designing and executing prime editing experiments in mammalian cells. Nat. Protoc. 17, 2431–2468 (2022).
-
Liu, P. et al. Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice. Nat. Commun. 12, 2121 (2021).
-
Nelson, J. W. et al. Engineered pegRNAs improve prime editing efficiency. Nat. Biotechnol. 40, 402–410 (2022).
-
Li, X. et al. Highly efficient prime editing by introducing same-sense mutations in pegRNA or stabilizing its structure. Nat. Commun. 13, 1669 (2022).
-
Ponnienselvan, K. et al. Reducing the inherent auto-inhibitory interaction within the pegRNA enhances prime editing efficiency. Nucleic Acids Res. 51, 6966–6980 (2023).
-
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 (2023).
-
Grunewald, J. et al. Engineered CRISPR prime editors with compact, untethered reverse transcriptases. Nat. Biotechnol. 41, 337–343 (2023).
-
Feng, Y. et al. Enhancing prime editing efficiency and flexibility with tethered and split pegRNAs. Protein Cell 14, 304–308 (2023).
-
Doman, J. L. et al. Phage-assisted evolution and protein engineering yield compact, efficient prime editors. Cell 186, 3983–4002.e26 (2023).
-
Liu, B. et al. Targeted genome editing with a DNA-dependent DNA polymerase and exogenous DNA-containing templates. Nat. Biotechnol. 42, 1039–1045 (2023).
-
da Silva, J. F. et al. Click editing enables programmable genome writing using DNA polymerases and HUH endonucleases. Nat. Biotechnol. https://doi.org/10.1038/s41587-024-02324-x (2024).
-
Petri, K. et al. CRISPR prime editing with ribonucleoprotein complexes in zebrafish and primary human cells. Nat. Biotechnol. 40, 189–193 (2022).
-
Davis, J. R. et al. Efficient prime editing in mouse brain, liver and heart with dual AAVs. Nat. Biotechnol. 42, 253–264 (2023).
-
Diamond, T. L. et al. Macrophage tropism of HIV-1 depends on efficient cellular dNTP utilization by reverse transcriptase. J. Biol. Chem. 279, 51545–51553 (2004).
-
Gao, W. Y., Cara, A., Gallo, R. C. & Lori, F. Low levels of deoxynucleotides in peripheral blood lymphocytes: a strategy to inhibit human immunodeficiency virus type 1 replication. Proc. Natl Acad. Sci. USA 90, 8925–8928 (1993).
-
Hakansson, P., Hofer, A. & Thelander, L. Regulation of mammalian ribonucleotide reduction and dNTP pools after DNA damage and in resting cells. J. Biol. Chem. 281, 7834–7841 (2006).
-
Traut, T. W. Physiological concentrations of purines and pyrimidines. Mol. Cell. Biochem. 140, 1–22 (1994).
-
Skasko, M. et al. Mechanistic differences in RNA-dependent DNA polymerization and fidelity between murine leukemia virus and HIV-1 reverse transcriptases. J. Biol. Chem. 280, 12190–12200 (2005).
-
Oscorbin, I. P. & Filipenko, M. L. M-MuLV reverse transcriptase: selected properties and improved mutants. Comput. Struct. Biotechnol. J. 19, 6315–6327 (2021).
-
Paliksa, S., Alzbutas, G. & Skirgaila, R. Decreased Km to dNTPs is an essential M-MuLV reverse transcriptase adoption required to perform efficient cDNA synthesis in one-step RT–PCR assay. Protein Eng. Des. Sel. 31, 79–89 (2018).
-
Kaushik, N., Chowdhury, K., Pandey, V. N. & Modak, M. J. Valine of the YVDD motif of moloney murine leukemia virus reverse transcriptase: role in the fidelity of DNA synthesis. Biochemistry 39, 5155–5165 (2000).
-
Operario, D. J., Reynolds, H. M. & Kim, B. Comparison of DNA polymerase activities between recombinant feline immunodeficiency and leukemia virus reverse transcriptases. Virology 335, 106–121 (2005).
-
Ni, P. et al. Efficient and versatile multiplex prime editing in hexaploid wheat. Genome Biol. 24, 156 (2023).
-
Halperin, S.O. Methods and compositions for directed genome editing. US patent 20230076357A1 (2023).
-
Das, D. & Georgiadis, M. M. A directed approach to improving the solubility of Moloney murine leukemia virus reverse transcriptase. Protein Sci. 10, 1936–1941 (2001).
-
Liang, S. Q. et al. Genome-wide profiling of prime editor off-target sites in vitro and in vivo using PE-tag. Nat. Methods 20, 898–907 (2023).
-
Guo, D. et al. iMyoblasts for ex vivo and in vivo investigations of human myogenesis and disease modeling. eLife 11, e70341 (2022).
-
Mathews, C. K. Deoxyribonucleotide metabolism, mutagenesis and cancer. Nat. Rev. Cancer 15, 528–539 (2015).
-
Goldstone, D. C. et al. HIV-1 restriction factor SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase. Nature 480, 379–382 (2011).
-
Laguette, N. et al. SAMHD1 is the dendritic- and myeloid-cell-specific HIV-1 restriction factor counteracted by VPX. Nature 474, 654–657 (2011).
-
Deutschmann, J. & Gramberg, T. SAMHD1 … and viral ways around it. Viruses 13, 395 (2021).
-
Bergamaschi, A. et al. The human immunodeficiency virus type 2 VPX protein usurps the CUL4A–DDB1 DCAF1 ubiquitin ligase to overcome a postentry block in macrophage infection. J. Virol. 83, 4854–4860 (2009).
-
Srivastava, S. et al. Lentiviral VPX accessory factor targets VprBP/DCAF1 substrate adaptor for cullin 4 E3 ubiquitin ligase to enable macrophage infection. PLoS Pathog. 4, e1000059 (2008).
-
Hrecka, K. et al. VPX relieves inhibition of HIV-1 infection of macrophages mediated by the SAMHD1 protein. Nature 474, 658–661 (2011).
-
Korin, Y. D. & Zack, J. A. Nonproductive human immunodeficiency virus type 1 infection in nucleoside-treated G0 lymphocytes. J. Virol. 73, 6526–6532 (1999).
-
Meyerhans, A. et al. Restriction and enhancement of human immunodeficiency virus type 1 replication by modulation of intracellular deoxynucleoside triphosphate pools. J. Virol. 68, 535–540 (1994).
-
Plesa, G. et al. Addition of deoxynucleosides enhances human immunodeficiency virus type 1 integration and 2LTR formation in resting CD4+ T cells. J. Virol. 81, 13938–13942 (2007).
-
Chabes, A. & Stillman, B. Constitutively high dNTP concentration inhibits cell cycle progression and the DNA damage checkpoint in yeast Saccharomyces cerevisiae. Proc. Natl Acad. Sci. USA 104, 1183–1188 (2007).
-
Pajalunga, D. et al. A defective dNTP pool hinders DNA replication in cell cycle-reactivated terminally differentiated muscle cells. Cell Death Differ. 24, 774–784 (2017).
-
Sawa, C. et al. High concentration of extracellular nucleotides suppresses cell growth via delayed cell cycle progression in cancer and noncancer cell lines. Heliyon 7, e08318 (2021).
-
Wakade, A. R., Przywara, D. A., Palmer, K. C., Kulkarni, J. S. & Wakade, T. D. Deoxynucleoside induces neuronal apoptosis independent of neurotrophic factors. J. Biol. Chem. 270, 17986–17992 (1995).
-
Purhonen, J., Banerjee, R., McDonald, A. E., Fellman, V. & Kallijarvi, J. A sensitive assay for dNTPs based on long synthetic oligonucleotides, EvaGreen dye and inhibitor-resistant high-fidelity DNA polymerase. Nucleic Acids Res. 48, e87 (2020).
-
Anzalone, A. V. et al. Programmable deletion, replacement, integration and inversion of large DNA sequences with twin prime editing. Nat. Biotechnol. 40, 731–740 (2022).
-
Nishimasu, H. et al. Engineered CRISPR–Cas9 nuclease with expanded targeting space. Science 361, 1259–1262 (2018).
-
Dean, M. et al. Genetic restriction of HIV-1 infection and progression to AIDS by a deletion allele of the CKR5 structural gene. Hemophilia Growth and Development Study, Multicenter AIDS Cohort Study, Multicenter Hemophilia Cohort Study, San Francisco City Cohort, ALIVE Study. Science 273, 1856–1862 (1996).
-
Liu, R. et al. Homozygous defect in HIV-1 coreceptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection. Cell 86, 367–377 (1996).
-
Samson, M. et al. Resistance to HIV-1 infection in Caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene. Nature 382, 722–725 (1996).
-
Pagliuca, F. W. et al. Generation of functional human pancreatic β cells in vitro. Cell 159, 428–439 (2014).
-
Rezania, A. et al. Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells. Nat. Biotechnol. 32, 1121–1133 (2014).
-
Philippe, C. et al. Spectrum and distribution of MECP2 mutations in 424 Rett syndrome patients: a molecular update. Eur. J. Med. Genet. 49, 9–18 (2006).
-
Kerem, B. S. et al. Identification of mutations in regions corresponding to the two putative nucleotide (ATP)-binding folds of the cystic fibrosis gene. Proc. Natl Acad. Sci. USA 87, 8447–8451 (1990).
-
Myerowitz, R. & Costigan, F. C. The major defect in Ashkenazi Jews with Tay–Sachs disease is an insertion in the gene for the α-chain of β-hexosaminidase. J. Biol. Chem. 263, 18587–18589 (1988).
-
Levesque, S., Cosentino, A., Verma, A., Genovese, P. & Bauer, D. E. Enhancing prime editing in hematopoietic stem and progenitor cells by modulating nucleotide metabolism. Nat. Biotechnol. https://doi.org/10.1038/s41587-024-02266-4 (2024).
-
Madigan, V., Zhang, F. & Dahlman. J. E. Drug delivery systems for CRISPR-based genome editors. Nat. Rev. Drug Discov. 22, 875–894 (2023).
-
Hopfner, K.-P. & Hornung, V. Molecular mechanisms and cellular functions of cGAS–STING signalling. Nat. Rev. Mol. Cell Biol. 21, 501–521 (2020).
-
Dumousseau, M., Rodriguez, N., Juty, N. & Le Novere, N. MELTING, a flexible platform to predict the melting temperatures of nucleic acids. BMC Bioinformatics 13, 101 (2012).
-
Guo, D., Daman, K., Durso, D. F., Yan, J. & Emerson, C. P. Generation of iMyoblasts from human induced pluripotent stem cells. Bio Protoc. 12, e4500 (2022).
-
Veres, A. et al. Charting cellular identity during human in vitro β-cell differentiation. Nature 569, 368–373 (2019).
-
Balboa, D. et al. Functional, metabolic and transcriptional maturation of human pancreatic islets derived from stem cells. Nat. Biotechnol. 40, 1042–1055 (2022).
-
Baiersdorfer, M. et al. A facile method for the removal of dsRNA contaminant from in vitro-transcribed mRNA. Mol. Ther. Nucleic Acids 15, 26–35 (2019).
-
Sabnis, S. et al. A novel amino lipid series for mRNA delivery: improved endosomal escape and sustained pharmacology and safety in non-human primates. Mol. Ther. 26, 1509–1519 (2018).
-
Clement, K. et al. CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat. Biotechnol. 37, 224–226 (2019).
-
Liu, P. et al. Increasing intracellular dNTP levels improves prime editing efficiency. Sequence Read Archive https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA1024467 (2024).
Acknowledgements
We thank members of the laboratories of C.P.E., D.M.H., N.D.L., J.L., C.A.S., E.J.S., J.P.W., Y. Wang, J.K.W. and S.A.W. for helpful discussions and the Rett Syndrome Research Trust for providing Rett fibroblasts. J.M.L. and C.A.S. were supported by a grant from the National Institutes of Health (R01AI150478). D.G. and C.P.E. were supported by a grant from the National Institutes of Health (U54HD0060848). S.O. and N.D.L. were supported by grants from the National Institutes of Health (R35HL140017 and R21OD030004). Z.C. and E.J.S. were supported by a grant from the National Institutes of Health (R01GM150273) and the Leducq Foundation. K.K. and J.K.W. were supported, in part, by the National Institutes of Health (UH3TR002668). T.N. and J.L. were supported by a grant from the National Institutes of Health (R37AI147868 and U54AI170856). D.M.H. and J.P.W. were supported, in part, by JDRF COE-2020-967-M-N. K.P., P.L., A.T.J. and S.A.W. were supported, in part, by the National Institutes of Health (R01HL150669, R37AI147868, R01HL170629 and UH3TR002668), Friedreich’s Ataxia Research Alliance, Doris Duke Foundation and the Rett Syndrome Research Trust.
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Competing interests
E.J.S. is a cofounder and Scientific Advisory Board member of Intellia Therapeutics and a Scientific Advisory Board member at Tessera Therapeutics. S.A.W. is a consultant for Editas Medicine. The University of Massachusetts Chan Medical School has filed patent applications related to this work. All other authors have no competing interests.
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Extended data
Extended Data Fig. 1 PEmax** outperforms other prime editor variants for precise editing by PE2 and Twin-PE systems in HEK293T cells, U2OS cells and Fibroblasts.
(a) PEmax mediated PE2 editing at HEK4 (20-bp PE-tag insertion) in HEK293T and U2OS cells with increasing amounts of PEmax and pegRNA expression plasmid delivered by transfection to define subsaturating editing conditions. Insertion of 20-bp PE-tag (blue) sequence (b) and 53-bp PE-tag+Flag-tag (blue and lowercase red) sequence (c) in HEK293T and U2OS cells with different prime editor variants (PE2 system) under unsaturated conditions (transfection with 1000 ng prime editor + 330 ng pegRNA). (d) PEmax mediated prime editing at FANCF ( + 5 G to T) in fibroblasts, EbvB cells and HBE cells with different amounts of PEmax mRNA/synthetic FANCF pegRNA delivered by electroporation to define subsaturating editing conditions. (e) Prime editing at FANCF ( + 5 G to T) in fibroblasts with various prime editor variants (PE2 system) under unsaturated conditions (electroporation with 1000 ng prime editor mRNA + 50 pmol pegRNA). (f) Evaluation various prime editor variants for the installation of the 38-bp Bxb1 attB site at HEK3 site in HEK293T cells by Twin-PE. For all datasets, values and error bars represent the mean ± SD of n = 3 independent replicates from targeted amplification deep-sequencing. Two-way ANOVA was used to compare the intended edit and unintended edit across all the groups for each graph, where PEmax was used as a control column for multiple comparisons (Supplementary table); * indicates P ≤ 0.05, ** indicates P ≤ 0.01, *** indicates P ≤ 0.001, **** indicates P ≤ 0.0001 and ns indicates not significant.
Extended Data Fig. 2 PEmax protein expression constructs and their purification.
(a) Architecture of the PEmax, PEmax* and PEmax** mammalian expression plasmid, mRNA and protein constructs. PEmax variants encoded by mRNA and protein constructs contain four nuclear localization signal (NLS) sequences7, whereas the mammalian expression plasmid encoded PEmax variants contain three NLSs2. (b) SDS-PAGE analysis of purified C-terminally His-tagged PEmax and PEmax** protein. Black arrow indicates the desired protein product (n = 1). (c) Protein yield per liter of bacterial culture for PEmax and PEmax** (n = 1).
Extended Data Fig. 3 PEmax** improves editing rates in zebrafish embryos and adult mice.
Comparison of PEmax and PEmax** RNP editing efficiency at various concentrations for introducing (a) L907F at the tek locus in zebrafish embryos (target codon indicated in green, n = 3 independent replicates). One-way ANOVA was used to compare the intended edit across all the groups for each graph; * indicates P < 0.05, **** indicates P ≤ 0.0001 (also see Supplementary table). Comparison of zebrafish embryo survival rate 24 hours post RNP injection for different treatment groups for (b) R841W and (c) L907F at the tek locus (each n = 3). One-way ANOVA was used to compare the survival across all the groups for each graph (Supplementary table); ns indicates P > 0.05, * indicates P < 0.05. (d) Comparison of in vivo PE3 editing rates for the introduction of Q155H at Pcsk9 mediated by PEmax and PEmax** mRNA LNP formulations delivered to mice through retro-orbital injection (rates determined from 3 liver punches from each of 5 mice in each cohort, n = 15). Two-way ANOVA was used to compare the intended edit and unintended edit across all the groups for each graph (Supplementary table), PEmax was used as a control column for multiple comparisons; **** indicates P ≤ 0.0001.
Extended Data Fig. 4 Comparison of 20-bp PE-tag sequence insertion at HEK4 site mediated by various prime editor variants under different conditions in HEK293T and U2OS cells.
(a) Cell viability assay for determining the optimal dN dose in growth media for use with HEK293T cells. Cell viability was monitored every 24 hours following addition of the desired dN concentration to the growth media. Symbols and error bars represent the mean ± SD, respectively, of n = 3 independent replicates. (n = 3). Arrow indicates the concentration of dNs [50 μM] used in subsequent experiments (b,c). Evaluation of editing efficiency for various prime editor variants (PE2 system) under different conditions (co-delivery of VPX, SAMHD1 and dNs [50 μM]) for insertion of a 20-bp PE-tag sequence28 at the HEK4 site in HEK293T (b) and U2OS cells (c) (unsaturated conditions: transfection with 1000 ng prime editor + 330 ng pegRNA). For b and c, bars and error bars represent the mean ± SD, respectively of n = 3 independent replicates.
Extended Data Fig. 5 Comparison of prime editing mediated by various prime editor variants at FANCF (PE2) and HEK3 (twin-PE) under different conditions in fibroblasts.
(a) Cell viability assay for determining the optimal dN dose in growth media for use with fibroblasts. Cell viability was monitored every 24 hours following addition of the desired dN concentration to the growth media. Symbols and error bars represent the mean ± SD, respectively, of n = 3 independent replicates. (n = 3). Arrow indicates the concentration of dNs [30 μM] used in subsequent experiments (b) Evaluation of editing efficiency for various prime editor variants (PE2 system) under different conditions (co-delivery of VPX, SAMHD1 and dNs [30 μM]) for FANCF ( + 5 G to T) base conversion in fibroblasts (electroporation with 1000 ng prime editor mRNA + 50pmol pegRNA). (c) Evaluation of editing efficiency for various prime editor variants (PE2 system) under different conditions (co-delivery of VPX, SAMHD1 and dNs [30 μM]) for Twin-PE in fibroblasts for the installation of the 38-bp Bxb1 attB site at HEK3 site. For b and c, bars and error bars reflect the mean ± SD, respectively, of n = 3 independent replicates from targeted amplification deep-sequencing.
Extended Data Fig. 6 Comparison of 53-bp PE-tag+Flag-tag sequence insertion at HEK4 site mediated by various prime editor variants under different conditions in HEK293T and U2OS cells.
(a,b) Evaluation of editing efficiency for various prime editor variants (PE2 system) under different conditions (co-delivery of VPX, SAMHD1 and dNs [50 μM]) for insertion of a 53-bp tag sequence at the HEK4 site in HEK293T (a) and U2OS cells (b) with various prime editor variants mediated PE2 approach under different conditions (unsaturated conditions: transfection with 1000 ng prime editor + 330 ng pegRNA). For a and b, bars and error bars reflect the mean ± SD, respectively, of n = 3 independent replicates.
Extended Data Fig. 7 Delivery of VPX increases the intracellular dNTP pool by down regulating SAMHD1 expression in fibroblasts and human primary T cells.
(a,b) Measurement of intracellular dNTP pool in (a) fibroblasts and (b) human primary T cells using long synthetic oligonucleotides and EvaGreen dye based assay44. For perturbation of intracellular dNTP levels VPX, VPX mutant (Q76A) or SAMHD1 mRNA are electroporated into patient derived fibroblasts (a) or primary T cells (b). Cells are harvested 48 hours post treatment and the intracellular dNTP concentration was determined. For a and b, bars and error bars reflect the mean ± SD, respectively, of n = 2 biological replicates with 3 technical replicates each. SAMHD1 protein levels in fibroblasts (n = 1) (c) and primary T cells (n = 1) (d) electroporated with VPX, VPX mutant or SAMHD1 mRNA was detected by immunoblotting with GAPDH used as a loading control.
Extended Data Fig. 8 PEmax** and co-delivery of VPX protein improves prime editing rates in resting T cells.
(a) PEmax-mRNA mediated PE3 editing at FANCF ( + 5 G to T) in resting human primary T cells with co-delivery of synthetic pegRNA/sgRNA and increasing amounts of VPX protein (n = 3 different donors). Two-way ANOVA was used to compare the intended edit and unintended edits across all the groups for each graph, and the no VPX group was used as a control column for multiple comparisons (Supplementary table); ns indicates P > 0.05, * indicates P ≤ 0.05, ** indicates P ≤ 0.01 and **** indicates P ≤ 0.0001. (b) PEmax, PEmax*, and PEmax** mRNA mediated PE3 editing at FANCF ( + 5 G to T) in resting human primary T cells with co-delivery of synthetic pegRNA/sgRNA and 0.5 µg VPX protein (n = 3 different donors). Two-way ANOVA was used to compare the intended edit and unintended edits across all the groups for each graph, and the no VPX group was used as a control column for multiple comparisons (Supplementary table); ns indicates P > 0.05, * indicates P ≤ 0.05, ** indicates P ≤ 0.01. (c) PEmax and PEmax* mRNA-mediated PE3 editing at FANCF ( + 5 G to T) in resting human primary T cells with co-delivery of synthetic pegRNA/sgRNA and VPX or VPX Q76A mRNA (n = 3 different donors). One-way ANOVA was used to compare the intended edits across all the groups for multiple comparisons (Supplementary table); ns indicates P > 0.05, * indicates P ≤ 0.05, ** indicates P ≤ 0.01 and **** indicates P ≤ 0.0001.
Extended Data Fig. 9 SC-islets are glucose responsive and express appropriate markers.
(a) Glucose‐stimulated insulin secretion (GSIS) was performed to assess in vitro function of SC-islets at time of nucleofection. The GSIS index, calculated by dividing the amount of insulin secreted following incubation in high glucose (20 mM) by that following incubation with basal glucose (2.8 mM), was 3.7. Bars and error bars reflect the mean ± SD. Each point represents an independent sample (n = 4). *** indicates P ≤ 0.001 and **** indicates P < 0.0001, one-way ANOVA (Supplementary table). (b) Representative flow cytometry staining of dispersed SC-islet clusters for differentiation and proliferation markers at time of nucleofection, with representative scatter plots (left) and data from replicate samples (right, n = 2). Bars and error bars reflect the mean ± SD.
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Liu, P., Ponnienselvan, K., Nyalile, T. et al. Increasing intracellular dNTP levels improves prime editing efficiency.
Nat Biotechnol (2024). https://doi.org/10.1038/s41587-024-02405-x
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DOI: https://doi.org/10.1038/s41587-024-02405-x
