Detection of rare variant alleles using the AsCas12a double-stranded DNA trans-cleavage activity.

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. Copyright © 2021 Elsevier B.V. All rights reserved.

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Henson Lee Yu, Yumeng Cao, Xiao Lu, I-Ming Hsing. Detection of rare variant alleles using the AsCas12a double-stranded DNA trans-cleavage activity. Biosensors & bioelectronics. 2021 Oct 01;189:113382

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PMID: 34087725

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