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Ho PY, Zhang Z, Hayes ME, et al. Peptide nucleic acid-dependent artifact can lead to false-positive triplex gene editing signals. Proc Natl Acad Sci U S A. 2021;118(45)doi: 10.1073/pnas.2109175118.
Ho, P. Y., Zhang, Z., Hayes, M. E., Curd, A., Dib, C., Rayburn, M., Tam, S. N., Srivastava, T., Hriniak, B., Li, X. J., Leonard, S., Wang, L., Tarighat, S., Sim, D. S., Fiandaca, M., Coull, J. M., Ebens, A., Fordyce, M., & Czechowicz, A. (2021). Peptide nucleic acid-dependent artifact can lead to false-positive triplex gene editing signals. Proceedings of the National Academy of Sciences of the United States of America, 118(45), . https://doi.org/10.1073/pnas.2109175118
Ho, Pui Yan, et al. "Peptide nucleic acid-dependent artifact can lead to false-positive triplex gene editing signals." Proceedings of the National Academy of Sciences of the United States of America vol. 118,45 (2021). doi: https://doi.org/10.1073/pnas.2109175118
Ho PY, Zhang Z, Hayes ME, Curd A, Dib C, Rayburn M, Tam SN, Srivastava T, Hriniak B, Li XJ, Leonard S, Wang L, Tarighat S, Sim DS, Fiandaca M, Coull JM, Ebens A, Fordyce M, Czechowicz A. Peptide nucleic acid-dependent artifact can lead to false-positive triplex gene editing signals. Proc Natl Acad Sci U S A. 2021 Nov 09;118(45). doi: 10.1073/pnas.2109175118. PMID: 34732575; PMCID: PMC8609320.
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Proc Natl Acad Sci U S A. 2021 Nov 09;118(45). doi: 10.1073/pnas.2109175118.

Peptide nucleic acid-dependent artifact can lead to false-positive triplex gene editing signals.

Proceedings of the National Academy of Sciences of the United States of America

Pui Yan Ho, Zhen Zhang, Mark E Hayes, Andrew Curd, Carla Dib, Maire Rayburn, Sze Nok Tam, Tumul Srivastava, Brandon Hriniak, Xiao-Jun Li, Scott Leonard, Lan Wang, Somayeh Tarighat, Derek S Sim, Mark Fiandaca, James M Coull, Allen Ebens, Marshall Fordyce, Agnieszka Czechowicz

Affiliations

  1. Department of Pediatrics, Division of Hematology, Oncology, Stem Cell Transplantation and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305.
  2. Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305.
  3. Vera Therapeutics, Inc., South San Francisco, CA 94080.
  4. Vera Therapeutics, Inc., South San Francisco, CA 94080 [email protected] [email protected].
  5. Department of Pediatrics, Division of Hematology, Oncology, Stem Cell Transplantation and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305; [email protected] [email protected].

PMID: 34732575 PMCID: PMC8609320 DOI: 10.1073/pnas.2109175118

Abstract

Triplex gene editing relies on binding a stable peptide nucleic acid (PNA) sequence to a chromosomal target, which alters the helical structure of DNA to stimulate site-specific recombination with a single-strand DNA (ssDNA) donor template and elicits gene correction. Here, we assessed whether the codelivery of PNA and donor template encapsulated in Poly Lactic-co-Glycolic Acid (PLGA)-based nanoparticles can correct sickle cell disease and x-linked severe combined immunodeficiency. However, through this process we have identified a false-positive PCR artifact due to the intrinsic capability of PNAs to aggregate with ssDNA donor templates. Here, we show that the combination of PNA and donor templates but not either agent alone results in different degrees of aggregation that result in varying but highly reproducible levels of false-positive signal. We have identified this phenomenon in vitro and confirmed that the PNA sequences producing the highest supposed correction in vitro are not active in vivo in both disease models, which highlights the importance of interrogating and eliminating carryover of ssDNA donor templates in assessing various gene editing technologies such as PNA-mediated gene editing.

Keywords: DNA repair; gene editing; hematopoietic stem cell; peptide nucleic acid

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