Peptide nucleic acid-dependent artifact can lead to false-positive triplex gene editing signals
| Autor: | Allen Ebens, Pui Yan Ho, Maire Rayburn, Mark E Hayes, Scott W. Leonard, James M. Coull, Tumul Srivastava, Somayeh S. Tarighat, Zhen Zhang, Xiao-Jun Li, Agnieszka Czechowicz, Carla Dib, Sze Nok Tam, Andrew Curd, Lan Wang, Mark J. Fiandaca, Marshall Fordyce, Brandon Hriniak, Derek S. Sim |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
Gene Editing
Peptide Nucleic Acids Multidisciplinary Peptide nucleic acid Chemistry DNA repair Molecular Probe Techniques Anemia Sickle Cell Mice SCID Biological Sciences In vitro chemistry.chemical_compound Template Biochemistry Genome editing Polylactic Acid-Polyglycolic Acid Copolymer In vivo Animals False Positive Reactions Gene DNA Interleukin Receptor Common gamma Subunit |
| Zdroj: | Proc Natl Acad Sci U S A |
| ISSN: | 1091-6490 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|