Department of Ophthalmology, Shanghai General Hospital (Shanghai First People's Hospital), Shanghai Jiao Tong University, School of Medicine, Shanghai, China; National Clinical Research Center for Eye Diseases, Shanghai, China; Shanghai Key Laboratory of Fundus Diseases, Shanghai, China; Shanghai Engineering Center for Visual Science and Photomedicine, Shanghai, China.
Department of Hematology, Zhongnan Hospital of Wuhan University, Wuhan, China.
J Mol Diagn. 2023 Aug;25(8):540-554. doi: 10.1016/j.jmoldx.2023.04.006.
Leber hereditary optic neuropathy (LHON) is the most common maternally inherited mitochondrial disease, with >90% of cases harboring one of three point variants (m.3460G>A, m.11778G>A, and m.14484T>C). Rapid and sensitive diagnosis of LHON variants is urgently needed for early diagnosis and timely treatment after onset, which is currently limited. Herein, we adapted the Cas12a-based DNA detection platform for LHON mitochondrial variant diagnosis. Single-strand guide CRISPR RNAs and enzymatic recombinase amplification primers were first screened, the CRISPR/Cas12a system was then optimized with restriction enzymes, and finally compared with Sanger sequencing and next-generation sequencing (NGS) in multicenter clinical samples. This approach can be completed within 30 minutes using only one drop of blood and could reach a sensitivity of 1% of heteroplasmy. Among the 182 multicenter clinical samples, the CRISPR/Cas12a detection system showed high consistency with Sanger sequencing and NGS in both specificity and sensitivity. Notably, a sample harboring a de novo 3.78% m.11778G>A variant detected by NGS, but not by Sanger sequencing, was successfully confirmed using the CRISPR/Cas12a assay, which proved the effectiveness of our method. Overall, our CRISPR/Cas12a detection system provides an alternative for rapid, convenient, and sensitive detection of LHON variants, exhibiting great potential for clinical practice.
Leber 遗传性视神经病变（LHON）是最常见的母系遗传性线粒体疾病，超过 90%的病例携带三种点突变之一（m.3460G>A、m.11778G>A 和 m.14484T>C）。快速、敏感地诊断 LHON 变体对于发病后的早期诊断和及时治疗至关重要，但目前这方面的诊断手段有限。在此，我们将 Cas12a 为基础的 DNA 检测平台用于 LHON 线粒体变体诊断。首先筛选单链引导 CRISPR RNA 和酶重组扩增引物，然后用限制酶优化 CRISPR/Cas12a 系统，最后在多中心临床样本中与 Sanger 测序和下一代测序（NGS）进行比较。该方法仅需一滴血，可在 30 分钟内完成，杂合性灵敏度可达 1%。在 182 个多中心临床样本中，CRISPR/Cas12a 检测系统在特异性和敏感性方面与 Sanger 测序和 NGS 均具有高度一致性。值得注意的是，NGS 检测到但 Sanger 测序未检测到的一个新发生的 3.78% m.11778G>A 变体的样本，使用 CRISPR/Cas12a 检测成功得到了确认，证明了我们方法的有效性。总的来说，我们的 CRISPR/Cas12a 检测系统为 LHON 变体的快速、方便、敏感检测提供了一种替代方法，具有很大的临床应用潜力。
