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2019-12-20

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cc-by-nc-nd (c) Félix, Álex J. et al., 2019
Please use this identifier to cite or link to this item: https://hdl.handle.net/2445/173989

Correction of the aprt Gene Using Repair-Polypurine Reverse Hoogsteen Hairpins in Mammalian Cells

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https://doi.org/10.1016/j.omtn.2019.12.015

Abstract

In this study, we describe the correction of single-point mutations in mammalian cells by repair-polypurine reverse Hoogsteen hairpins (repair-PPRHs). These molecules consist of (1) a PPRH hairpin core that binds to a polypyrimidine target sequence in the double-stranded DNA (dsDNA), producing a triplex structure, and (2) an extension sequence homologous to the DNA sequence to be repaired but containing the wild-type nucleotide instead of the mutation and acting as a donor DNA to correct the mutation. We repaired different point mutations in the adenosyl phosphoribosyl transferase (aprt) gene contained in different aprt-deficient Chinese hamster ovary (CHO) cell lines. Because we had previously corrected mutations in the dihydrofolate reductase (dhfr) gene, in this study, we demonstrate the generality of action of the repair-PPRHs. Repaired cells were analyzed by DNA sequencing, mRNA expression, and enzymatic activity to confirm the correction of the mutation. Moreover, whole-genome sequencing analyses did not detect any off-target effect in the repaired genome. We also performed gel-shift assays to show the binding of the repair-PPRH to the target sequence and the formation of a displacement-loop (D-loop) structure that can trigger a homologous recombination event. Overall, we demonstrate that repair-PPRHs achieve the permanent correction of point mutations in the dsDNA at the endogenous level in mammalian cells without off-target activity.

Subject

Cèl·lules, Mamífers, Mutació (Biologia), Reparació de l'ADN

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Cells, Mammals, Mutation (Biology), DNA repair

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Articles publicats en revistes (Bioquímica i Fisiologia)

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FÉLIX, Álex J., CIUDAD I GÓMEZ, Carlos Julián and NOÉ MATA, Verónica. Correction of the aprt Gene Using Repair-Polypurine Reverse Hoogsteen Hairpins in Mammalian Cells. Molecular Therapy-Nucleic Acids. 2019. Vol. 19, num. 683-695. ISSN 2162-2531. [consulted: 10 of August of 2026]. Available at: https://hdl.handle.net/2445/173989

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