CRISPR-SDA: an integrated isothermal amplification and CRISPR-Cas12a biosensing platform for sensitive detection of microRNA-21

Sep 1, 2025Analytical methods : advancing methods and applications

Sensitive detection of microRNA-21 using combined DNA amplification and CRISPR-Cas12a sensing

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Abstract

A detection limit of 10.1 fM was achieved using a novel CRISPR-SDA biosensing platform for microRNA-21.

The platform integrates strand displacement amplification with CRISPR-Cas12a for enhanced detection of low-abundance miRNA-21.
Segmented linear ranges for detection were established at 0.05-25 pM and 25-500 pM.
High selectivity against other microRNAs was demonstrated, alongside resistance to interference from complex biological samples.
Spike recovery experiments in fetal bovine serum indicated reliable recoveries ranging from 92.0 ± 7.8% to 105.6 ± 3.3%.

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MicroRNA-21 (miRNA-21), a critical oncogenic biomarker, poses detection challenges due to low abundance and limitations of conventional methods. Herein, we developed a novel CRISPR-SDA biosensing platform by integrating strand displacement amplification (SDA) with CRISPR-Cas12a, leveraging SDA's efficient isothermal amplification of miRNA-21 and Cas12a's precise target recognition and-cleavage activity for signal amplification. Optimized conditions achieved high sensitivity with a detection limit of 10.1 fM and segmented linear ranges of 0.05-25 pM and 25-500 pM. It showed excellent selectivity against other miRNAs and anti-interference in complex matrices (, salmon sperm DNA). Spike recovery experiments in fetal bovine serum yielded recoveries of 92.0 ± 7.8% to 105.6 ± 3.3%, confirming its reliability in complex biological samples. This CRISPR-SDA platform overcomes the drawbacks of conventional methods, enabling rapid, sensitive, and equipment-friendly detection. It holds great potential for early cancer diagnosis and point-of-care testing and provides a versatile framework for detecting other disease-associated nucleic acids. trans e.g.

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CRISPR-SDA: an integrated isothermal amplification and CRISPR-Cas12a biosensing platform for sensitive detection of microRNA-21

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