期刊
BIOSENSORS & BIOELECTRONICS
卷 189, 期 -, 页码 -出版社
ELSEVIER ADVANCED TECHNOLOGY
DOI: 10.1016/j.bios.2021.113382
关键词
AsCas12a trans-cleavage activity; CRISPR-Based diagnostics; Single nucleotide variant (SNV) discrimination; Low variant allele frequency (VAF) detection; Immobilization-free electrochemical detection
类别
资金
- Research Grants Council of the Hong Kong Special Administrative Region [GRF 16306519]
- Hong Kong PhD Fellowship Scheme
We present an ultra-selective Cas12a-based system that does not require a PAM sequence in the target and reduces noise from the wild-type sequence using its non-canonical double-stranded trans-cleavage activity. Our strategy enables the detection of an EGFR gene mutation in sub-femtomolar concentrations up to 0.1% variant allele frequency using either fluorescence or electrochemical readouts.
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 doublestranded 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.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据