Hunan Provincial Key Laboratory of Environmental Catalysis and Waste Recycling, College of Materials and Chemical Engineering, Hunan Institute of Engineering, Xiangtan 411104, China.
Anal Methods. 2024 Oct 17;16(40):6810-6818. doi: 10.1039/d4ay01131g.
The aberrant expression of piRNAs in germ cells is a potential cause of male infertility. Establishing diagnostic methods with highly specific biomarkers for male infertility is important for accurate diagnosis and treatment of male infertility. In this study, we proposed a novel method combining rolling circle transcription (RCT) and Cas13a techniques, which utilized the high amplification efficiency of RCT and the two different RNase activities possessed by Cas13a, establishing a highly sensitive and specific assay for male infertility-associated piRNA. First, a circular DNA template was synthesized by hybridizing linear ssDNA with the T7 promoter. The nick in the circular DNA was closed by T4 DNA ligase. In the presence of T7 RNA polymerase, the closed circular DNA produced tandemly repeated pre-crRNA. The RNase activity of Cas13a was used to process pre-crRNAs to form mature crRNA. Guided by crRNA, Cas13a specifically recognized piRNA and activated collateral activity. Activated Cas13a disaggregated thousands of fluorescent probes for each target RNA detected, resulting in powerful signal amplification. As a proof of concept, piR-hsa-14 was used as the validation target. The limit of detection was as low as 3.32 fM with a good linearity in the range of 100 fM to 50 pM. Recovery of piR-hsa-14 ranged from 91.33% to 112.63% in spiked recovery experiments using human serum samples. The results revealed that this method has the advantages of high sensitivity, sufficient accuracy and good reproducibility. We believe that this method could have a promising future as a potential tool for clinical diagnosis of male infertility.
piRNA 在生殖细胞中的异常表达是男性不育的潜在原因。建立具有高度特异性生物标志物的男性不育诊断方法对于男性不育的准确诊断和治疗非常重要。在这项研究中，我们提出了一种结合滚环转录（RCT）和 Cas13a 技术的新方法，该方法利用 RCT 的高扩增效率和 Cas13a 所具有的两种不同的 RNase 活性，建立了一种用于男性不育相关 piRNA 的高灵敏度和特异性检测方法。首先，通过将线性 ssDNA 与 T7 启动子杂交合成环形 DNA 模板。T4 DNA 连接酶封闭环形 DNA 中的缺口。在 T7 RNA 聚合酶的存在下，闭合的环形 DNA 产生串联重复的前 crRNA。Cas13a 的 RNase 活性用于处理前 crRNA 以形成成熟的 crRNA。crRNA 指导 Cas13a 特异性识别 piRNA 并激活旁侧活性。激活的 Cas13a 使检测到的每个靶 RNA 的数千个荧光探针解聚，从而实现强大的信号放大。作为概念验证，使用 piR-hsa-14 作为验证靶标。检测限低至 3.32 fM，在 100 fM 至 50 pM 的范围内具有良好的线性度。在使用人血清样本进行的加标回收实验中，piR-hsa-14 的回收率在 91.33%至 112.63%之间。结果表明，该方法具有灵敏度高、准确性高、重现性好等优点。我们相信，该方法有望成为男性不育临床诊断的潜在工具。
