基于Cas12a靶向核酸检测的脊髓性肌萎缩症携带者筛查与诊断新技术研究

Spinal muscular atrophy (SMA) is the most common fatal genetic disease in infants, and more than 95% of patients with SMA have homozygous deletion of exon 7 of the survival motor neuron (SMN1) gene. The carrier rate is approximately 1 in 42 in China. Population screening is an effective approach for prevention and control of hereditary diseases. However, current SMA detection methods are laborious operation, high cost and long turnover time, which seriously hinder the population screening of SMA. The latest researches showed that RNA-guided DNA binding unleashes indiscriminate single-stranded DNA (ssDNA) cleavage activity by CRISPR/Cas12a, which achieved attomolar sensitivity and specificity for DNA detection. In this study, we propose to use the property of Cas12a to develop a rapid, accurate, simple and low-cost technology of SMN1 exon 7 copy number quantitative detection by designing and constructing the targeting crRNA, in combination with Recombinase Polymerase Amplification technology. Meanwhile, A visualized Paper-test system will be developed for SMA diagnosis via a targeted qualitive detection of SMN1 exon 7 using Colloidal gold-based immunochromatographic assay. This strategy of SMA mutation hotspot detection with Cas12a is expected to break through the bottleneck of SMA screening and strengthen prevention and control capability of SMA at the current stage, reducing the risk of birth defects and child mortality.

脊髓性肌萎缩症（SMA）是婴幼儿期最常见的致死性遗传病，95%以上的SMA患者由SMN1基因7号外显子纯合缺失所致，在我国人群携带率高达1/42。群体筛查是遗传病防控的有效手段，然而目前针对SMA的检测方法操作繁琐、成本高、检测周期长等严重阻碍群体筛查的开展。最新研究发现CRISPR/Cas12a能特异地识别切割靶核酸分子并产生附带切割活性，利用该特性可进行高敏感和高特异性的核酸分子检测。本研究将利用Cas12a的附带切割活性，设计构建靶向SMN1基因7号外显子的crRNA，联合RPA技术，建立快速准确、简便廉价的SMN1基因7号外显子拷贝数定量检测技术体系。通过结合胶体金免疫层析试纸条，进一步开发出可视化的SMN1基因7号外显子试纸条定性检测技术。这种基于Cas12a靶向检测SMA热点突变的策略可望突破现阶段SMA筛查的瓶颈，从而提高对SMA的防控能力、降低出生缺陷与儿童死亡率。

脊髓性肌萎缩症（SMA）是婴幼儿期最常见的致死性遗传病，95%以上的SMA患者由SMN1基因7号外显子纯合缺失所致，在我国人群携带率高达1/42。群体筛查是遗传病防控的有效手段，然而目前针对SMA的检测方法操作繁琐、成本高、检测周期长等严重阻碍群体筛查的开展。CRISPR/Cas12a能特异地识别切割靶核酸分子并产生附带切割活性，利用该特性可进行高敏感和高特异性的核酸分子检测。为此，本研究利用Cas12a的附带切割活性，通过设计构建靶向SMN1基因7号外显子的crRNA，联合RPA技术与胶体金免疫层析试纸条，开发出了可视化的SMN1基因7号外显子试纸条定性检测技术，经临床样本验证，显示Cas12a-试纸条定性检测敏感性和特异性均达100%，从样本采集到读取检测结果的全过程可在1.5h内完成。且该方法可用于MPNs检测。SMA-Cas12a具备定量检测能力，可区分低至2倍的拷贝数差异，能有效却分正常人、携带者与SMA病人，可用于SMA携带者筛查。

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