Gene point mutation information translation and detection: Leveraging single base extension and CRISPR/Cas12a,Biosensors and Bioelectronics

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Gene point mutation information translation and detection: Leveraging single base extension and CRISPR/Cas12a
Biosensors and Bioelectronics ( IF 10.7 ) Pub Date : 2023-12-20 , DOI: 10.1016/j.bios.2023.115936
Zhujun Liu ₁ , Jie Xu ₁ , Shan Huang ₁ , Wei Dai ₂ , Wei Zhang ₃ , Longjie Li ₂ , Xianjin Xiao ₃ , Tongbo Wu ₁

Affiliation

Gene point mutations play a significant role in the development of cancer. Therefore, developing a sensitive, specific, and universally applicable method for detecting gene point mutation is crucial for clinical diagnosis, prognosis, and cancer treatment. Recently, gene point mutation detection methods based on CRISPR/Cas12a detection have emerged. However, existing methods generally lack universality and specificity. In this study, we have developed a CRISPR/Cas12a-based method that combines improved allele-specific polymerase chain reaction and single base extension to translate the point mutation information in the target dsDNA into length information in ssDNA activators to overcome the limitations associated with PAM sequences in the CRISPR/Cas12a system. Our method achieved a detection limit of 0.002% for clinically significant T790M mutation. The CRISPR/Cas12a system we constructed demonstrates high sensitivity, specificity, and universality in detecting gene point mutations, making it a promising tool for clinical cancer screening.

中文翻译：

基因点突变信息翻译和检测：利用单碱基延伸和CRISPR/Cas12a

基因点突变在癌症的发展中发挥着重要作用。因此，开发一种灵敏、特异、普适的基因点突变检测方法对于临床诊断、预后判断和癌症治疗至关重要。近年来，基于CRISPR/Cas12a检测的基因点突变检测方法出现。然而，现有方法普遍缺乏通用性和特异性。在本研究中，我们开发了一种基于 CRISPR/Cas12a 的方法，结合改进的等位基因特异性聚合酶链反应和单碱基延伸，将目标 dsDNA 中的点突变信息转化为 ssDNA 激活剂中的长度信息，以克服与 PAM 相关的限制CRISPR/Cas12a 系统中的序列。我们的方法对临床显着的 T790M 突变实现了 0.002% 的检测限。我们构建的CRISPR/Cas12a系统在检测基因点突变方面表现出高灵敏度、特异性和普适性，使其成为临床癌症筛查的有前途的工具。

更新日期：2023-12-20

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