CRISPR/Cas12a platform activated by protospacer adjacent motifengineered DNA circuit for specific target sensing

Abstract

CRISPR/Cas systems have found extensive applications in nucleic acid diagnostics. However, the generalizability of this approach, particularly for the sensing of non-nucleic acid targets, remains a challenge. This study presents the development of a CRISPR/Cas12a platform activated by a protospacer adjacent motif (PAM)-engineered DNA circuit. Initially, the influence of the presence or absence of the PAM on the DNA circuit and the subsequent CRISPR/Cas12a system was investigated, demonstrating that a PAM-engineered DNA circuit functions as an effective activator of Cas12a, whereas a DNA circuit lacking the PAM does not induce activation. Subsequently, through the strategic design of recognition elements, sensitive and selective detection of specific targets is achieved, with limits of detection (LODs) of 0.023 fM for circulating tumor DNA (ctDNA), 0.00004 U/mL for uracil-DNA glycosylase (UDG), and 0.12 fM for acetamiprid (ACE). This approach exemplifies a two-stage signal amplification mechanism, achieving improved sensitivity relative to either the CRISPR/Cas12a system or the DNA circuits alone. Moreover, quantitative assays for these targets were successfully conducted in real samples, suggesting the practical applicability of the proposed method. This research establishes a versatile sensing platform for various targets, which holds significant promise for advancements in molecular diagnostics, food safety assessment, and environmental monitoring.