ACS sensorsFeb 13, 2026

Self-Assembly of a Cas13a Circuit that Boosts Biosensing

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Feb 14, 2026

Abstract

The PRA-Cas13a system achieves an attomolar (aM) level detection limit for various targets.

Site-specific splitting within the crRNA seed region enables effective activation of Cas13a trans-cleavage.
The system utilizes engineered pre-crRNA as a molecular switch that self-processes upon target binding.
Integration of a dual-UUU site DNA switch and a signal amplification module constructs a cycle for enhanced detection.
Validation shows successful detection of miRNA, mRNA, and viral DNA within 10 minutes using a lateral flow test strip.
Sensitivity analysis of single-base mutations indicates superior performance compared to conventional CRISPR-based methods.
The system accurately detects survivin mRNA in various cell lines and HPV16 in clinical samples, aligning with qPCR results.

Simplified

Despite the success of non-classical crRNA designs in Cas12a-based biosensing, application to Cas13a systems faces fundamental challenges. Our research discovers that site-specific splitting within the crRNA seed region enables effective activation of Cas13a trans-cleavage. Here, we developed an autocatalytic Cas13a circuit that transitions from self-processing to responsive assembly for enhanced biosensing (PRA-Cas13a). The system employs engineered pre-crRNA as a molecular switch, which undergoes self-processing upon target binding to assemble an active Cas13a complex and activate its trans-cleavage activity. By integrating a dual-UUU site DNA switch template and a T7 RNA polymerase-mediated signal amplification module, a "processing-assembly-amplification" cycle is constructed to enhance the detection signal. Through validation using various targets including miRNA, mRNA, and viral DNA, the PRA-Cas13a system not only achieves an attomolar (aM) level detection limit but also enables visual field detection within 10 min using a lateral flow test strip. Analysis of single-base mutations demonstrated that its sensitivity is significantly superior to conventional CRISPR-based methods. Moreover, the system successfully enabled accurate detection of survivin mRNA in different cell lines and HPV16 in clinical cervical swab samples, showing strong concordance with qPCR gold standard methods. The PRA-Cas13a strategy leverages intrinsic self-processing assembly and autocatalytic signal amplification, addresses the critical issue of off-target cleavage inherent in conventional Cas13a systems while expanding the range of applicable targets, and demonstrates high specificity and point-of-care testing potential in cancer and viral diagnostics.

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Self-Assembly of a Cas13a Circuit that Boosts Biosensing

Abstract

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