Center for Medical Genetics & Hunan Key Laboratory of Medical Genetics, School of Life Sciences, Central South University, Changsha 410078, China.
Biosensors (Basel). 2022 Apr 23;12(5):268. doi: 10.3390/bios12050268.
Spinal muscular atrophy (SMA) is the main genetic cause of infant death. In >95% of the patients with SMA, the disease is caused by a single hotspot pathogenic mutation: homozygous deletion of exon 7 of the survival motor neuron 1 gene (SMN1). Recently, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein (Cas)-based assays have been developed as a promising new option for nucleic acid detection. Here, we developed a Cas14a1-based assay combined with asymmetric PCR to establish a method for detection of the homozygous deletion of SMN1 exon 7 in SMA patients. The minimum detectable concentration of genomic DNA reached 5.26 aM with our method, and the assessment of its detection performance in 33 clinical samples revealed that the results were completely consistent with those of multiple ligation-dependent probe amplification and quantitative PCR. Thus, our novel nucleic acid diagnostics combining CRISPR/Cas14a1 and asymmetric PCR not only provides specific and sensitive testing of the deletion of SMN1 exon 7, but also holds promise for an accurate detection platform of genetic diseases and pathogens in multiple sample types.
脊髓性肌萎缩症（SMA）是导致婴儿死亡的主要遗传原因。在 >95%的 SMA 患者中，该疾病是由单一热点致病突变引起的：生存运动神经元 1 基因（SMN1）第 7 外显子的纯合缺失。最近，基于成簇规律间隔短回文重复序列（CRISPR）/CRISPR 相关蛋白（Cas）的检测已被开发为核酸检测的一种很有前途的新选择。在这里，我们开发了一种基于 Cas14a1 的检测方法，并结合不对称 PCR 来建立一种用于检测 SMA 患者 SMN1 外显子 7 纯合缺失的方法。我们的方法对基因组 DNA 的最低检测浓度达到 5.26 aM，对 33 个临床样本的检测性能评估表明，结果与多重连接依赖探针扩增和定量 PCR 的结果完全一致。因此，我们将 CRISPR/Cas14a1 和不对称 PCR 相结合的新型核酸诊断方法不仅提供了对 SMN1 外显子 7 缺失的特异性和敏感性检测，而且为多种样本类型的遗传疾病和病原体的准确检测平台提供了可能。
