Lee Yu Henson, Cao Yumeng, Lu Xiao, Hsing I-Ming
Department of Chemical and Biological Engineering, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong.
Department of Chemical and Biological Engineering, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong.
Biosens Bioelectron. 2021 Oct 1;189:113382. doi: 10.1016/j.bios.2021.113382. Epub 2021 May 29.
The sensitive and accurate detection of rare mutations has profound clinical implications; however, current methods require expensive instrumentation and are laborious and time-consuming. Thus, there is a need for a probe-based alternative that can effectively discriminate single-base mutations. Recently, several groups have shown the potential of the CRISPR/Cas12a system for sensitive and selective DNA detection but its application on single nucleotide variants (SNVs) detection is limited by the requirement of a protospacer adjacent motif (PAM) directly upstream to the SNV site and the amplification of non-specific signals due to the rapid and indiscriminate trans cleavage activity. Here, we report an ultra-selective Cas12a-based system that eliminates the need for the PAM sequence in the target with lower noise from the wild-type sequence by using its non-canonical double-stranded trans-cleavage activity. We show that our strategy can allow the detection of an EGFR gene mutation in sub-femtomolar concentrations up to 0.1% variant allele frequency using either fluorescence or electrochemical readouts.
罕见突变的灵敏且准确检测具有深远的临床意义；然而，当前方法需要昂贵的仪器设备，且费力又耗时。因此，需要一种基于探针的替代方法，能够有效区分单碱基突变。最近，几个研究小组展示了CRISPR/Cas12a系统用于灵敏且选择性DNA检测的潜力，但其在单核苷酸变异（SNV）检测中的应用受到SNV位点上游直接需要原间隔序列临近基序（PAM）以及由于快速且无差别反式切割活性导致的非特异性信号扩增的限制。在此，我们报道一种基于Cas12a的超选择性系统，该系统通过利用其非经典双链反式切割活性，消除了靶标中对PAM序列的需求，并降低了来自野生型序列的噪声。我们表明，我们的策略能够使用荧光或电化学读数，在亚飞摩尔浓度下检测EGFR基因突变，变异等位基因频率高达0.1%。
