Shang Zhenlin, Liu Sitong, Liu Dongxu, Wang Yuer, Pei Xiaojing, Li Shujing, He Yifan, Tong Yigang
School of Light Industry Science and Engineering, Beijing Technology and Business University, Beijing, 100048, P. R. China.
College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, 100029, P. R. China.
J Fluoresc. 2025 May 22. doi: 10.1007/s10895-025-04360-5.
Precise identification and detection of single nucleotide variation (SNV) concomitant with excess wild-type DNA is greatly needed for invasive disease diagnosis, pathogens detection and early prediction of drug responsiveness. Many variants of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), notably the D614G and N501Y mutations, have been shown to significantly increase the infectivity of pandemics. We herein investigated CRISPR/Cas12a integrated three types fluorescent reporters and two crRNAs for SNV detection by taking D614G and N501Y variants of SARS-CoV-2 as model examples. We systematically screened all possible base substitutions from positions 0 to 19 and identified the middle position of crRNA could efficiently increase the specificity from both theoretical and experimental standpoints. With selected mutation location of crRNA, we then investigated the specificity of ssDNA, dsDNA and molecular beacon (MB) fluorescent reporters and proved the MB reporters can efficiently increase the discriminatory factors. Furthermore, we designed an additional mutation site on crRNA to increase the specificity. For user convenience, we engineered the lateral flow strips to present the results visualized with the naked eyes. Results of specific variants from Omicron proved the feasibility of clinical applications. These findings indicated that the proposed method is a powerful tool for monitoring the key mutations in pathogens and allows for modifications to incorporate newer upcoming variants.
在侵袭性疾病诊断、病原体检测以及药物反应性的早期预测中，非常需要精确识别和检测与过量野生型DNA并存的单核苷酸变异（SNV）。严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的许多变体，特别是D614G和N501Y突变，已被证明会显著增加大流行的传染性。我们在此以SARS-CoV-2的D614G和N501Y变体为模型示例，研究了CRISPR/Cas12a整合三种类型荧光报告基因和两种crRNA用于SNV检测。我们系统地筛选了从第0位到第19位所有可能的碱基替换，并从理论和实验角度确定crRNA的中间位置可以有效提高特异性。通过选择crRNA的突变位置，我们随后研究了单链DNA、双链DNA和分子信标（MB）荧光报告基因的特异性，并证明MB报告基因可以有效提高区分因子。此外，我们在crRNA上设计了一个额外的突变位点以提高特异性。为方便用户，我们设计了侧向流动试纸条，以便用肉眼直观呈现结果。来自奥密克戎的特定变体结果证明了临床应用的可行性。这些发现表明，所提出的方法是监测病原体关键突变的有力工具，并允许进行修改以纳入新出现的变体。
