Department of Ophthalmology, Justus-Liebig-University Giessen, Giessen, Germany.
Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, Libechov, Czech Republic.
Nucleic Acid Ther. 2023 Jun;33(3):226-232. doi: 10.1089/nat.2022.0037. Epub 2023 Mar 1.
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 () 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 mutations in the disease context.
斯塔加特病(STGD)可导致儿童和青年失明。迄今为止,尚无有效的治疗方法,并且由于用于将基因转入光感受器细胞的腺相关病毒的包装能力有限,基因增强疗法尚未进入临床应用。Prime editing 为在基因组水平上治疗突变提供了新的视角。与延长的 guideRNA 融合的 Cas9 的 nicking 变体迫使细胞内错配修复纠正靶向突变,即使在有丝分裂后细胞(如眼内的光感受器)中也是如此。我们使用定制的基于生物发光共振能量转移(BRET)的编辑传感器在 HEK293 细胞中测试了 27 种不同的 Prime editing guide RNA(pegRNA)和另外 4 种 nicking guide RNA(ngRNA),以评估它们在诱导猪 ATP 结合盒亚家族 A 成员 4(ABCA4)基因第 43 外显子序列变化的效率,该突变可消除与人类 STGD 相关的诱变腺嘌呤移码插入。我们确定了九个有效的 pegRNA,并且与 ngRNA 结合使用,我们使用基于 BRET 的报告系统实现了高达约 92%的校正率。我们的数据证明了 Prime editors 校正突变的高效性,并强调了优化 ngRNA 设计的重要性,从而为在疾病背景下校正突变提供了一种很有前途的编辑工具。
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