Rapid and Reliable Quantification of Prime Editing Targeting Within the Porcine ABCA4 Gene Using a Bioluminescence Resonance Energy Transfer-Based Sensor

0573112 - ÚŽFG 2024 RIV US eng J - Článek v odborném periodiku
Wimmer, T. - Sawinski, H. - Urban, A. M. M. - Motlík, Jan - Stieger, K.
Rapid and Reliable Quantification of Prime Editing Targeting Within the Porcine ABCA4 Gene Using a Bioluminescence Resonance Energy Transfer-Based Sensor.
Nucleic Acid Therapeutics. Roč. 33, č. 3 (2023), s. 226-232. ISSN 2159-3337. E-ISSN 2159-3345
Grant CEP: GA ČR(CZ) GC19-09628J
Institucionální podpora: RVO:67985904
Klíčová slova: abca4 * genome editing * prime editing * cellular reporter assay * bioluminescence resonance energy transfer
Obor OECD: Health-related biotechnology
Impakt faktor: 4, rok: 2022
Způsob publikování: Open access
https://www.liebertpub.com/doi/10.1089/nat.2022.0037

Stargardt disease (STGD) leads to blindness in children and young adults. So far, no curative therapy is available and gene augmentation therapies have not yet advanced to the clinics, in part, due to the limited packaging capacity of adeno-associated viruses used to transfer genes into photoreceptor cells. Prime editing offers a new perspective to treat mutations on the genomic level. A nicking variant of Cas9 fused to a reverse transcriptase complex with an elongated guideRNA force intracellular mismatch repair to correct the targeted mutation even in postmitotic cells such as photoreceptors in the eye. Using a custom-made bioluminescence resonance energy transfer (BRET)-based editing sensor in HEK293 cells, we tested 27 different prime editing guide RNAs (pegRNAs) and additional 4 nicking guide RNAs (ngRNAs) with regard to their efficiency to induce sequences changes in exon 43 of the porcine ATP binding cassette subfamily A member 4 (ABCA4) gene that eliminate a mutagenic adenine frameshift insertion, which has been associated with STGD in humans. We identified nine working pegRNAs, and in combination with ngRNAs, we achieved a correction rate of up to approximate to 92% measured with the BRET-based reporter system. Our data prove the high efficiency of prime editors to correct mutations and highlight the importance of optimal ngRNA design, thus offering a promising editing tool to correct ABCA4 mutations in the disease context.
Trvalý link: https://hdl.handle.net/11104/0343694