Data availability
All amplicon sequencing data are publicly accessible from the National Center for Biotechnology Information BioProject database under accession number PRJNA1090890.
References
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
Chen, P. J. et al. Enhanced prime editing systems by manipulating cellular determinants of editing outcomes. Cell 184, 5635–5652 (2021).
Article
CAS
PubMed
PubMed Central
Google Scholar
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).
Article
CAS
PubMed
Google Scholar
Ayinde, D., Casartelli, N. & Schwartz, O. Restricting HIV the SAMHD1 way: through nucleotide starvation. Nat. Rev. Microbiol. 10, 675–680 (2012).
Article
CAS
PubMed
Google Scholar
Ballana, E. & Esté, J. A. SAMHD1: at the crossroads of cell proliferation, immune responses, and virus restriction. Trends Microbiol. 23, 680–692 (2015).
Article
CAS
PubMed
Google Scholar
Mauney, C. H. & Hollis, T. SAMHD1: recurring roles in cell cycle, viral restriction, cancer, and innate immunity. Autoimmunity 51, 96–110 (2018).
Article
CAS
PubMed
PubMed Central
Google Scholar
Laguette, N. et al. SAMHD1 is the dendritic- and myeloid-cell-specific HIV-1 restriction factor counteracted by Vpx. Nature 474, 654–657 (2011).
Article
CAS
PubMed
PubMed Central
Google Scholar
Hrecka, K. et al. Vpx relieves inhibition of HIV-1 infection of macrophages mediated by the SAMHD1 protein. Nature 474, 658–661 (2011).
Article
CAS
PubMed
PubMed Central
Google Scholar
Baldauf, H. M. et al. SAMHD1 restricts HIV-1 infection in resting CD4(+) T cells. Nat. Med. 18, 1682–1687 (2012).
Article
CAS
PubMed
Google Scholar
Li, D. et al. Vpx mediated degradation of SAMHD1 has only a very limited effect on lentiviral transduction rate in ex vivo cultured HSPCs. Stem Cell Res. 15, 271–280 (2015).
Article
CAS
PubMed
PubMed Central
Google Scholar
Levesque, S. et al. Marker-free co-selection for successive rounds of prime editing in human cells. Nat. Commun. 13, 5909 (2022).
Article
CAS
PubMed
PubMed Central
Google Scholar
Mikdar, M. et al. The equilibrative nucleoside transporter ENT1 is critical for nucleotide homeostasis and optimal erythropoiesis. Blood 137, 3548–3562 (2021).
Article
CAS
PubMed
PubMed Central
Google Scholar
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
Zeng, J. et al. Gene editing without ex vivo culture evades genotoxicity in human hematopoietic stem cells. Preprint at bioRxiv https://doi.org/10.1101/2023.05.27.542323 (2023).
Fiumara, M. et al. Genotoxic effects of base and prime editing in human hematopoietic stem cells. Nat. Biotechnol. https://doi.org/10.1038/s41587-023-01915-4 (2023).
Article
PubMed
Google Scholar
Ferreira da Silva, J. et al. Prime editing efficiency and fidelity are enhanced in the absence of mismatch repair. Nat. Commun. 13, 760 (2022).
Article
CAS
PubMed
PubMed Central
Google Scholar
Chen, P. J. & Liu, D. R. Prime editing for precise and highly versatile genome manipulation. Nat. Rev. Genet. 24, 161–177 (2022).
Article
CAS
PubMed
PubMed Central
Google Scholar
Nambiar, T. S., Baudrier, L., Billon, P. & Ciccia, A. CRISPR-based genome editing through the lens of DNA repair. Mol. Cell 82, 348–388 (2022).
Article
CAS
PubMed
PubMed Central
Google Scholar
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).
Article
CAS
PubMed
Google Scholar
Sharma, P. L., Nurpeisov, V. & Schinazi, R. F. Retrovirus reverse transcriptases containing a modified YXDD motif. Antivir. Chem. Chemother. 16, 169–182 (2005).
Article
CAS
PubMed
Google Scholar
Palikša, 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).
Article
PubMed
Google Scholar
Ponnienselvan, K. et al. Addressing the dNTP bottleneck restricting prime editing activity. Preprint at bioRxiv https://doi.org/10.1101/2023.10.21.563443 (2023).
Li, X. et al. Highly efficient prime editing by introducing same-sense mutations in pegRNA or stabilizing its structure. Nat. Commun. 13, 1669 (2022).
Article
CAS
PubMed
PubMed Central
Google Scholar
Su, S. S., Lahue, R. S., Au, K. G. & Modrich, P. Mispair specificity of methyl-directed DNA mismatch correction in vitro. J. Biol. Chem. 263, 6829–6835 (1988).
Article
CAS
PubMed
Google Scholar
Thomas, D. C., Roberts, J. D. & Kunkel, T. A. Heteroduplex repair in extracts of human HeLa cells. J. Biol. Chem. 266, 3744–3751 (1991).
Article
CAS
PubMed
Google Scholar
Lahue, R., Au, K. & Modrich, P. DNA mismatch correction in a defined system. Science 245, 160–164 (1998).
Article
Google Scholar
Mathews, C. K. Deoxyribonucleotide metabolism, mutagenesis and cancer. Nat. Rev. Cancer 15, 528–539 (2015).
Article
CAS
PubMed
Google Scholar
Traut, T. W. Physiological concentrations of purines and pyrimidines. Mol. Cell. Biochem. 140, 1–22 (1994).
Article
CAS
PubMed
Google Scholar
Mjelle, R. et al. Cell cycle regulation of human DNA repair and chromatin remodeling genes. DNA Repair. 30, 53–67 (2015).
Article
CAS
PubMed
Google Scholar
Longley, M. J., Pierce, A. J. & Modrich, P. D. N. A polymerase δ is required for human mismatch repair in vitro. J. Biol. Chem. 272, 10917–10921 (1997).
Article
CAS
PubMed
Google Scholar
Domínguez-González, C. et al. Deoxynucleoside therapy for thymidine kinase 2–deficient myopathy. Ann. Neurol. 86, 293–303 (2019).
Article
PubMed
PubMed Central
Google Scholar
Amtmann, D., Gammaitoni, A. R., Galer, B. S., Salem, R. & Jensen, M. P. The impact of TK2 deficiency syndrome and its treatment by nucleoside therapy on quality of life. Mitochondrion 68, 1–9 (2023).
Article
CAS
PubMed
Google Scholar
Li, C. et al. In vivo HSC prime editing rescues sickle cell disease in a mouse model. Blood 141, 2085–2099 (2023).
CAS
PubMed
PubMed Central
Google Scholar
Breda, L. et al. In vivo hematopoietic stem cell modification by mRNA delivery. Science 381, 436–443 (2023).
Article
CAS
PubMed
Google Scholar
An, M. et al. Engineered virus-like particles for transient delivery of prime editor ribonucleoprotein complexes in vivo. Nat. Biotechnol. https://doi.org/10.1038/s41587-023-02078-y (2024).
Article
PubMed
Google Scholar
Liu, B. et al. An efficient lentiviral CRISPRi approach to silence genes in primary human monocytes. Preprint at bioRxiv https://doi.org/10.1101/2020.12.23.424242 (2020).
Casirati, G. et al. Epitope editing enables targeted immunotherapies for acute myeloid leukemia. Nature 621, 404–414 (2023).
Article
CAS
PubMed
PubMed Central
Google Scholar
Brinkman, E. K., Chen, T., Amendola, M. & Van Steensel, B. Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res. 42, e168 (2014).
Article
PubMed
PubMed Central
Google Scholar
Brinkman, E. K. et al. Easy quantification of template-directed CRISPR/Cas9 editing. Nucleic Acids Res. 46, e58 (2018).
Article
PubMed
PubMed Central
Google Scholar
Xu, L., Liu, Y. & Han, R. BEAT: a Python program to quantify base editing from Sanger sequencing. Cris. J. 2, 223–229 (2019).
Article
CAS
Google Scholar
Wu, Y. et al. Highly efficient therapeutic gene editing of human hematopoietic stem cells. Nat. Med. 25, 776–783 (2019).
Article
CAS
PubMed
PubMed Central
Google Scholar
Bloh, K. et al. Deconvolution of complex DNA repair (DECODR): establishing a novel deconvolution algorithm for comprehensive analysis of CRISPR-edited Sanger sequencing data. Cris. J. 4, 120–131 (2021).
Article
CAS
Google Scholar
Conant, D. et al. Inference of CRISPR edits from Sanger trace data. Cris. J. 5, 123–130 (2022).
Article
CAS
Google Scholar
Clement, K. et al. CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat. Biotechnol. 37, 215–226 (2019).
Article
Google Scholar
Download references
Acknowledgements
This work was supported by the Doris Duke Foundation, the St. Jude Children’s Research Hospital Collaborative Research Consortium, the Harvard Stem Cell Institute and the National Institutes of Health (R01HL150669 and R01HL170629). S.L. holds a Banting Postdoctoral Fellowship from the Canadian Institutes of Health Research. A.C. was partially supported by a fellowship from the American Italian Cancer Foundation. CD34+ cells were provided by the Fred Hutch Cooperative Center of Excellence in Hematology (U54 DK106829). We thank D. Klatt and C. Brendel for their support in producing in-house Vpx VLPs, G. Casirati for his support in mRNA IVT, P. Wang and N. Kanarek for performing metabolic studies, W. Mannherz and S. Agarwal for insightful discussions, J. Zeng and N.R. Neri for technical support, and the GPP of the Broad Institute of MIT and Harvard for providing VSV-G envelope and SIV Vpx vectors.
Author information
Authors and Affiliations
Division of Hematology/Oncology, Boston Children’s Hospital, Boston, MA, USA
Sébastien Levesque, Andrea Cosentino, Archana Verma, Pietro Genovese & Daniel E. Bauer
Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA
Sébastien Levesque, Andrea Cosentino, Archana Verma, Pietro Genovese & Daniel E. Bauer
Harvard Stem Cell Institute, Cambridge, MA, USA
Sébastien Levesque, Archana Verma, Pietro Genovese & Daniel E. Bauer
Broad Institute, Cambridge, MA, USA
Sébastien Levesque, Archana Verma, Pietro Genovese & Daniel E. Bauer
Department of Pediatrics, Harvard Medical School, Boston, MA, USA
Sébastien Levesque, Andrea Cosentino, Archana Verma, Pietro Genovese & Daniel E. Bauer
Milano-Bicocca University, Milan, Italy
Andrea Cosentino
Contributions
S.L. and D.E.B. conceived the study; S.L. and D.E.B. devised the methods; S.L., A.C., A.V., P.G. and D.E.B. carried out the investigation; S.L. wrote the original draft; S.L. and D.E.B. wrote, reviewed and edited the manuscript; P.G. and D.E.B. acquired the funding; and P.G. and D.E.B. provided supervision.
Corresponding author
Ethics declarations
Competing interests
S.L. and D.E.B. have filed a provisional patent application related to this work. All other authors have no competing interests.
Peer review
Peer review information
Nature Biotechnology thanks the anonymous reviewers for their contribution to the peer review of this work.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data
Extended Data Fig. 1 Vpx VLP and dN supplementation enhance prime editing in HSPCs with the first-generation PE2 prime editor.
(a) PE quantification as determined by BEAT analysis from Sanger sequencing. 5 ×105 HSPCs were electroporated with PE3 RNAs (PE2 mRNA + epegRNA + nicking sgRNA) targeting ATP1A1 and treated with 5X Vpx VLP (GPP) or vehicle control. Genomic DNA was harvested 3 days post-nucleofection. n = 1 experiment (donor 1) replicated with HSPCs from donor 2 with similar results (See Extended Data Fig. 1b). (b) PE and indels quantification as determined by BEAT and TIDE analysis from Sanger sequencing. HSPCs were thawed and cultured in the presence or absence of 100 µM dNs for 24 hours. Following dN treatment, 5 ×105 HSPCs were electroporated with PE3 RNAs targeting ATP1A1 and cultured in the presence or absence of 100 µM each dN and the indicated concentration of Vpx VLPs (GPP) or vehicle control for 72 hours. Genomic DNA was harvested 3 days post-nucleofection. n = 1 experiment. (c) PE and indels quantification as determined by BEAT and TIDE analysis from Sanger sequencing. HSPCs were thawed and cultured in the presence or absence of the indicated concentration of each dN for 24 hours (before electroporation). Following dN treatment, 1.25 ×105 HSPCs were electroporated with PE3 RNAs targeting ATP1A1 and cultured in the presence or absence of the indicated concentration of each dN for 72 hours (after electroporation). Genomic DNA was harvested 3 days post-nucleofection. n = 1 experiment. Donor 2, circle. Donor 3, diamond.
Extended Data Fig. 2 Modulation of nucleotide metabolism has minimal impact on prime editing in cancer cell lines.
(a) PE and indels quantification as determined by BEAT and TIDE analysis from Sanger sequencing. K562 cells were electroporated with PE3max vectors targeting ATP1A1 and the indicated Vpx or SAMHD1 vector and cultured with the indicated concentration of each dN for 72 hours. An empty pUC19 vector was used as a negative control to normalize the concentration of DNA in all nucleofections. Genomic DNA was harvested 3 days post-nucleofection. n = 2 independent biological replicates performed at different times. (b) Same as in (a) with Jurkat cells.
Extended Data Fig. 3 Staggered PE3 nicks generate tandem duplications at ATP1A1 in HSPCs.
(a) CRISPResso2 amplicon sequencing allele plots after PE3 at ATP1A1. 2.5 ×105 HSPCs were electroporated with PE3max RNAs targeting ATP1A1 (Q118R_v2, +3 TTG to CCT) and cultured in the presence or absence of 5X Vpx VLPs (GPP) and 50 µM each dN. Genomic DNA was harvested 3 days post-nucleofection. Representative allele plots are from one of three independent biological replicates performed with CD34+ HSPCs from three different donors with equivalent results. Insertions are illustrated in red squares, and they represent tandem duplications of the genomic sequence found between the two nicking sites.
Extended Data Fig. 4 Modulation of nucleotide metabolism modestly improves PE at HBB.
(a) PE and indels quantification as determined by CRISPResso2 analysis from amplicon sequencing. 2.5 ×105 HSPCs were electroporated with PE3/PE3bmax RNAs targeting HBB and cultured in the presence or absence of 5X Vpx VLPs (GPP) and 50 µM each dNs. Genomic DNA was harvested 3 days post-nucleofection. Data are plotted as mean ± SEM from n = 3 independent biological replicates performed with CD34+ HSPCs from one donor homozygous for the rs713040 allele (donor 2). Each biological replicate is illustrated with the same shade of grey. (b) Same as in (a) but HSPCs were electroporated with PE2max or PE4max (PE2max mRNA + hMLH1dn mRNA) RNAs targeting HBB and cultured in the presence or absence of 50 µM each dNs. Where indicated, an equimolar ratio of Vpx mRNA was co-delivered during electroporation. The mRNA molarity was normalized between all conditions with EGFP mRNA. Data are plotted as mean ± SEM from n = 3 independent biological replicates performed with CD34+ HSPCs from three different donors. Donor 2, circle. Donor 6, empty diamond. Donor 7, triangle (down). (c) Representative CRISPResso2 amplicon sequencing allele plots from the experiments shown in (a) and (b). Representative allele plots are from one of three independent biological replicates performed with CD34+ HSPCs from three different donors with equivalent results. Reads with ≥ 0.2% frequency are shown, and the frequency of indels is indicated for each sample.
Extended Data Fig. 5 Modulation of nucleotide metabolism positively interacts with MMR-evading epegRNAs in quiescent HSPCs.
(a) Schematic representation of the RNAs used to co-deliver Vpx with PE3max components. The timeline for prime editing in quiescent CD34+ HSPCs is illustrated on the right. (b) PE and indels quantification as determined by BEAT and TIDE analysis from Sanger sequencing. 5 ×105 quiescent HSPCs were electroporated directly after thawing with Vpx mRNA and PE3max RNAs targeting HBB or B2M and cultured in the presence or absence of 50 µM each dN. Genomic DNA was harvested 3 days post-nucleofection. Data are plotted as mean ± SEM from n = 3 independent biological replicates performed with CD34+ HSPCs from three different donors. (c) Same as in (b) for prime editing at ATP1A1 with MMR-evading substitutions. Donor 2, circle. Donor 6, empty diamond. Donor 7, triangle (down).
Extended Data Fig. 6 Omitting the nicking sgRNA abrogates indels and tandem duplications at ATP1A1 in HSPCs.
(a) CRISPResso2 amplicon sequencing allele plots from Fig. 2a. 2.5 ×105 HSPCs were electroporated with PE2max RNAs targeting ATP1A1 (Q118R_v3, +3 TTGG to CCTA) and cultured in the presence or absence of 25X in-house Vpx VLPs and 50 µM each dN. Genomic DNA was harvested 3 days post-nucleofection. Representative allele plots are from one of three independent biological replicates performed with CD34+ HSPCs from three different donors with equivalent results. Reads with ≥ 0.2% frequency are shown, and the frequency of indels is indicated for each sample.
Extended Data Fig. 7 Comparison of quantification accuracy between Sanger and amplicon sequencing at ATP1A1, B2M, and HBB (related to Fig. 2 and Extended Data Fig. 4).
(a) PE and indel quantification as determined by BEAT and TIDE analysis from Sanger sequencing. (See Fig. 2a for comparison with amplicon sequencing.) The MMR evasion strategy based on the installation of additional silent mutations and the epegRNAs used to target the ATP1A1 locus are illustrated. Point mutations are illustrated in orange. As described in Figs. 2, 2.5 ×105 HSPCs were electroporated with PE2max RNAs targeting ATP1A1 and cultured in the presence or absence of 50 µM each dN and 25X in-house Vpx VLPs. Genomic DNA was harvested 3 days post-nucleofection. Data are from two out of three independent biological replicates performed with both sequencing methods. (b) Same as in (a), but HSPCs were electroporated with Vpx mRNA and PE2max RNAs targeting B2M (See Fig. 2b for comparison with amplicon sequencing). Data are plotted as mean ± SEM from n = 3 independent biological replicates performed with CD34+ HSPCs from three different donors. (c) PE and indel quantification as determined by BEAT and TIDE analysis from Sanger sequencing (See Extended Data Fig. 4a for comparison with amplicon sequencing). As described in Extended Data Fig. 4, 2.5 x 105 HSPCs were electroporated with PE3/PE3bmax RNAs targeting HBB and cultured in the presence or absence of 5X Vpx VLPs (GPP) and 50 µM each dNs. Genomic DNA was harvested 3 days post-nucleofection. Data are plotted as mean ± SEM from n = 3 independent biological replicates performed with CD34+ HSPCs from one donor homozygous for the rs713040 allele (donor 2). Each biological replicate is illustrated with the same shade of grey. (d) Correlation of Sanger and amplicon sequencing quantifications at ATP1A1 (PE2max), B2M (PE2max), and HBB (PE3max and PE3bmax) for the 96 samples shown in (a-c).
Supplementary information
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
Reprints and permissions
About this article
Cite this article
Levesque, S., Cosentino, A., Verma, A. et al. Enhancing prime editing in hematopoietic stem and progenitor cells by modulating nucleotide metabolism.
Nat Biotechnol (2024). https://doi.org/10.1038/s41587-024-02266-4
Download citation
Received: 16 October 2023
Accepted: 26 April 2024
Published: 28 May 2024
DOI: https://doi.org/10.1038/s41587-024-02266-4
>>> Read full article>>>
Copyright for syndicated content belongs to the linked Source : Nature.com – https://www.nature.com/articles/s41587-024-02266-4
