Key Laboratory of Brain, Cognition and Education Sciences, Ministry of Education, Institute for Brain Research and Rehabilitation, South China Normal University, Guangzhou, 510631, China; Zhejiang Laboratory, Hangzhou, Zhejiang, 311121, China; Guangzhou Laboratory, Bio-island, Guangzhou, Guangdong, 510005, China.
Key Laboratory of Brain, Cognition and Education Sciences, Ministry of Education, Institute for Brain Research and Rehabilitation, South China Normal University, Guangzhou, 510631, China.
Biosens Bioelectron. 2022 Oct 15;214:114497. doi: 10.1016/j.bios.2022.114497. Epub 2022 Jun 30.
MicroRNAs (miRNAs) are small noncoding RNAs that posttranscriptionally regulate gene expression. The aberrant expression of miRNAs is related to many diseases. MiRNAs can serve as potential biomarkers for the prognosis and diagnosis of cancers and other human diseases. However, the short sequence and high sequence similarity of miRNAs impede detection. Herein, we propose a method to integrate polyA-tailing and CRISPR/Cas12a to amplify and detect all miRNAs with high specificity and sensitivity. PolyA-tailing enables efficient amplification of RNA and introduces a universal PAM sequence for Cas12a to unlock its PAM restriction. The CRISPR-Cas system guarantees the specific recognition of nucleic acid sequences with a single base mismatch. A limit of detection (LOD) as low as 50 fM was achieved. The practical application ability of polyA-CRISPR/Cas12a-based miRNA detection was validated by miRNA analyses in multiple cancer cell samples. With the increasing stability of RNA samples, low cost, excellent specificity, and sensitivity, this method demonstrates great potential to scale up to parallel diagnostic sets for miRNA-related disease.
微小 RNA（miRNAs）是一种小的非编码 RNA，可以在后转录水平上调节基因表达。miRNAs 的异常表达与许多疾病有关。miRNAs 可以作为癌症和其他人类疾病预后和诊断的潜在生物标志物。然而，miRNAs 的短序列和高序列相似性阻碍了检测。在此，我们提出了一种方法，将 polyA-加尾和 CRISPR/Cas12a 整合起来，以高特异性和灵敏度扩增和检测所有的 miRNAs。polyA-加尾可有效地扩增 RNA，并引入 Cas12a 的通用 PAM 序列以解锁其 PAM 限制。CRISPR-Cas 系统保证了单碱基错配的核酸序列的特异性识别。检测限（LOD）低至 50 fM。在多个癌细胞样本的 miRNA 分析中验证了基于 polyA-CRISPR/Cas12a 的 miRNA 检测的实际应用能力。随着 RNA 样本稳定性的提高、成本低、特异性和灵敏度优异，该方法具有很大的潜力扩展为 miRNA 相关疾病的平行诊断试剂盒。
