دورية أكاديمية
Rapid and Reliable Quantification of Prime Editing Targeting Within the Porcine ABCA4 Gene Using a BRET-Based Sensor.
| العنوان: | Rapid and Reliable Quantification of Prime Editing Targeting Within the Porcine ABCA4 Gene Using a BRET-Based Sensor. |
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| المؤلفون: | Wimmer T; Department of Ophthalmology, Justus-Liebig-University Giessen, Giessen, Germany., Sawinski H; Department of Ophthalmology, Justus-Liebig-University Giessen, Giessen, Germany., Urban AM; Department of Ophthalmology, Justus-Liebig-University Giessen, Giessen, Germany., Motlik J; Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, Libechov, Czech Republic., Stieger K; Department of Ophthalmology, Justus-Liebig-University Giessen, Giessen, Germany. |
| المصدر: | Nucleic acid therapeutics [Nucleic Acid Ther] 2023 Jun; Vol. 33 (3), pp. 226-232. Date of Electronic Publication: 2023 Mar 01. |
| نوع المنشور: | Journal Article; Research Support, Non-U.S. Gov't |
| اللغة: | English |
| بيانات الدورية: | Publisher: Mary Ann Liebert, Inc Country of Publication: United States NLM ID: 101562758 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 2159-3345 (Electronic) Linking ISSN: 21593337 NLM ISO Abbreviation: Nucleic Acid Ther Subsets: MEDLINE |
| أسماء مطبوعة: | Original Publication: New Rochelle, N.Y. : Mary Ann Liebert, Inc. |
| مواضيع طبية MeSH: | ATP-Binding Cassette Transporters*/genetics, Child ; Young Adult ; Humans ; Animals ; Swine ; HEK293 Cells ; Stargardt Disease/genetics ; Mutation ; Energy Transfer |
| مستخلص: | 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 ≈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. |
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| فهرسة مساهمة: | Keywords: ABCA4; bioluminescence resonance energy transfer; cellular reporter assay; genome editing; prime editing |
| المشرفين على المادة: | 0 (ATP-Binding Cassette Transporters) 0 (ABCA4 protein, human) |
| تواريخ الأحداث: | Date Created: 20230301 Date Completed: 20230608 Latest Revision: 20230621 |
| رمز التحديث: | 20240628 |
| مُعرف محوري في PubMed: | PMC10278032 |
| DOI: | 10.1089/nat.2022.0037 |
| PMID: | 36857739 |
| قاعدة البيانات: | MEDLINE |
| تدمد: | 2159-3345 |
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| DOI: | 10.1089/nat.2022.0037 |