Genotoxic effects of base and prime editing in human hematopoietic stem cells

Abstract

Base and prime editors (BEs and PEs) may provide more precise genetic engineering than nuclease-based approaches because they bypass the dependence on DNA double-strand breaks. However, little is known about their cellular responses and genotoxicity. Here, we compared state-of-the-art BEs and PEs and Cas9 in human hematopoietic stem and progenitor cells with respect to editing efficiency, cytotoxicity, transcriptomic changes and on-target and genome-wide genotoxicity. BEs and PEs induced detrimental transcriptional responses that reduced editing efficiency and hematopoietic repopulation in xenotransplants and also generated DNA double-strand breaks and genotoxic byproducts, including deletions and translocations, at a lower frequency than Cas9. These effects were strongest for cytidine BEs due to suboptimal inhibition of base excision repair and were mitigated by tailoring delivery timing and editor expression through optimized mRNA design. However, BEs altered the mutational landscape of hematopoietic stem and progenitor cells across the genome by increasing the load and relative proportions of nucleotide variants. These findings raise concerns about the genotoxicity of BEs and PEs and warrant further investigation in view of their clinical application.

References 64

1. 1.

   Naldini L. Genetic engineering of hematopoiesis: current stage of clinical translation and future perspectives. EMBO Mol. Med. 2019;11:e9958.

   Not in our library10.15252/emmm.201809958

2. 2.
   The promise and challenge of therapeutic genome editing · 2020

   No notice
3. 3.

   Ferrari S, et al. Genetic engineering meets hematopoietic stem cell biology for next-generation gene therapy. Cell Stem Cell. 2023;30:549-570.

   Not in our library10.1016/j.stem.2023.04.014

4. 4.

   Porteus MH. A new class of medicines through DNA editing. N. Engl. J. Med. 2019;380:947-959.

   Not in our library10.1056/nejmra1800729

5. 5.

   Schiroli G, et al. Precise gene editing preserves hematopoietic stem cell function following transient p53-mediated DNA damage response. Cell Stem Cell. 2019;24:551-565.

   Not in our library10.1016/j.stem.2019.02.019

6. 6.

   Ferrari S, et al. Efficient gene editing of human long-term hematopoietic stem cells validated by clonal tracking. Nat. Biotechnol. 2020;38:1298-1308.

   Not in our library10.1038/s41587-020-0551-y

7. 7.

   Adikusuma F, et al. Large deletions induced by Cas9 cleavage. Nature. 2018;560:E8-E9.

   Not in our library10.1038/s41586-018-0380-z

8. 8.

   Leibowitz ML, et al. Chromothripsis as an on-target consequence of CRISPR–Cas9 genome editing. Nat. Genet. 2021;53:895-905.

   Not in our library10.1038/s41588-021-00838-7

9. 9.

   Park SH, et al. Comprehensive analysis and accurate quantification of unintended large gene modifications induced by CRISPR–Cas9 gene editing. Sci. Adv. 2022;8:eabo7676.

   Not in our library10.1126/sciadv.abo7676

10. 10.

    Turchiano G, et al. Quantitative evaluation of chromosomal rearrangements in gene-edited human stem cells by CAST-seq. Cell Stem Cell. 2021;28:1136-1147.

    Not in our library10.1016/j.stem.2021.02.002

11. 11.

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

    Not in our library10.1038/nbt.4192

12. 12.

    Takata M, et al. Homologous recombination and non-homologous end-joining pathways of DNA double-strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells. EMBO J. 1998;17:5497-5508.

    Not in our library10.1093/emboj/17.18.5497

13. 13.

    Anzalone AV, Koblan LW, Liu DR. Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors. Nat. Biotechnol. 2020;38:824-844.

    Not in our library10.1038/s41587-020-0561-9

14. 14.

    Ferrari S, et al. Choice of template delivery mitigates the genotoxic risk and adverse impact of editing in human hematopoietic stem cells. Cell Stem Cell. 2022;29:1428-1444.

    Not in our library10.1016/j.stem.2022.09.001

15. 15.

    Rees HA, Liu DR. Base editing: precision chemistry on the genome and transcriptome of living cells. Nat. Rev. Genet. 2018;19:770-788.

    Not in our library10.1038/s41576-018-0059-1

16. 16.

    Komor AC, Kim YB, Packer MS, Zuris JA, Liu DR. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature. 2016;533:420-424.

    Not in our library10.1038/nature17946

17. 17.

    Antoniou P, Miccio A, Brusson M. Base and prime editing technologies for blood disorders. Front. Genome Ed. 2021;3:618406.

    Not in our library10.3389/fgeed.2021.618406

18. 18.

    Wang L, et al. Reactivation of γ-globin expression through Cas9 or base editor to treat β-hemoglobinopathies. Cell Res. 2020;30:276-278.

    Not in our library10.1038/s41422-019-0267-z

19. 19.

    Zeng J, et al. Therapeutic base editing of human hematopoietic stem cells. Nat. Med. 2020;26:535-541.

    Not in our library10.1038/s41591-020-0790-y

20. 20.

    Gaudelli NM, et al. Directed evolution of adenine base editors with increased activity and therapeutic application. Nat. Biotechnol. 2020;38:892-900.

    Not in our library10.1038/s41587-020-0491-6

21. 21.

    Newby GA, et al. Base editing of haematopoietic stem cells rescues sickle cell disease in mice. Nature. 2021;595:295-302.

    Not in our library10.1038/s41586-021-03609-w

22. 22.

    Anzalone AV, et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature. 2019;576:149-157.

    Not in our library10.1038/s41586-019-1711-4

23. 23.

    Petri K, et al. CRISPR prime editing with ribonucleoprotein complexes in zebrafish and primary human cells. Nat. Biotechnol. 2022;40:189-193.

    Not in our library10.1038/s41587-021-00901-y

24. 24.
    Enhanced prime editing systems by manipulating cellular determinants of editing outcomes · 2021

    No notice
25. 25.
    Ex vivo prime editing of patient haematopoietic stem cells rescues sickle-cell disease phenotypes after engraftment in mice · 2023

    No notice
26. 26.

    Nelson JW, et al. Engineered pegRNAs improve prime editing efficiency. Nat. Biotechnol. 2021;40:402-410.

    Not in our library10.1038/s41587-021-01039-7

27. 27.

    Koblan LW, et al. Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction. Nat. Biotechnol. 2018;36:843-846.

    Not in our library10.1038/nbt.4172

28. 28.

    Kurt IC, et al. CRISPR C-to-G base editors for inducing targeted DNA transversions in human cells. Nat. Biotechnol. 2021;39:41-46.

    Not in our library10.1038/s41587-020-0609-x

29. 29.

    Bai T, et al. Expansion of primitive human hematopoietic stem cells by culture in a zwitterionic hydrogel. Nat. Med. 2019;25:1566-1575.

    Not in our library10.1038/s41591-019-0601-5

30. 30.

    Scala S, et al. Dynamics of genetically engineered hematopoietic stem and progenitor cells after autologous transplantation in humans. Nat. Med. 2018;24:1683-1690.

    Not in our library10.1038/s41591-018-0195-3

31. 31.
    Prime editing efficiency and fidelity are enhanced in the absence of mismatch repair · 2022

    No notice
32. 32.
    Evolution of an adenine base editor into a small, efficient cytosine base editor with low off-target activity · 2022

    No notice
33. 33.
    Improved cytosine base editors generated from TadA variants · 2023

    No notice
34. 34.

    Al-Bahlani S, et al. P73 regulates cisplatin-induced apoptosis in ovarian cancer cells via a calcium/calpain-dependent mechanism. Oncogene. 2011;30:4219-4230.

    Not in our library10.1038/onc.2011.134

35. 35.

    Long SL, Morales JC, Hwang A, Wagner MW, Boothman DA. DNA mismatch repair-dependent activation of c-Abl/p73α/GADD45α-mediated apoptosis. J. Biol. Chem. 2008;283:21394-21403.

    Not in our library10.1074/jbc.m709954200

36. 36.

    Kuzminov A. Single-strand interruptions in replicating chromosomes cause double-strand breaks. Proc. Natl Acad. Sci. USA. 2001;98:8241-8246.

    Not in our library10.1073/pnas.131009198

37. 37.

    Beerman I, Seita J, Inlay MA, Weissman IL, Rossi DJ. Quiescent hematopoietic stem cells accumulate DNA damage during aging that is repaired upon entry into cell cycle. Cell Stem Cell. 2014;15:37-50.

    Not in our library10.1016/j.stem.2014.04.016

38. 38.

    Canarutto D, et al. Peripheral blood stem and progenitor cell collection in pediatric candidates for ex vivo gene therapy: a 10-year series. Mol. Ther. Methods Clin. Dev. 2021;22:76-83.

    Not in our library10.1016/j.omtm.2021.05.013

39. 39.
    Inheritable Silencing of Endogenous Genes by Hit-and-Run Targeted Epigenetic Editing · 2016

    No notice
40. 40.

    Nuñez JK, et al. Genome-wide programmable transcriptional memory by CRISPR-based epigenome editing. Cell. 2021;184:2503-2519.

    Not in our library10.1016/j.cell.2021.03.025

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• Durable and efficient gene silencing in vivo by hit-and-run epigenome editing · 2024
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