Stimuli-responsive incremental DNA machine auto-catalyzed CRISPR-Cas12a feedback amplification permits ultrasensitive molecular diagnosis of esophageal cancer-related microRNA

Development of new diagnostic methods is essential for disease diagnosis and treatment. In this work, we present a stimuli-responsive incremental DNA machine auto-catalyzed CRISPR-Cas12a (SRI-DNA machine/CRISPR-Cas12a) feedback amplification for ultrasensitive molecular detection of miRNA-21, which is an important biomarker related closely to the initiation and development of cancers, such as esophageal cancer. Strategically, the powerful SRI-DNA machine and efficient trans-cleavage activity of the CRISPR-Cas12a system are ingeniously integrated via a rationally designed probe termed as stem-elongated functional hairpin probe (SEF-HP). The SRI-DNA machine begins with the target miRNA, the trigger of the reaction, binding complementarily to the SEF-HP, followed by autonomously performed mechanical strand replication, cleavage, and displacement circuit at multiple sites. This conversion process led to the amplified generation of numerous DNA activators that are complementary with CRISPR RNA (CrRNA). Once formed the DNA activator/CrRNA heteroduplex, the trans-cleavage activity of the CRISPR-Cas12a was activated to nonspecific cleavage of single-stranded areas of a reporter probe for fluorescence emission. Under optimal conditions, the target miRNA can be detected with a wide linear range and an excellent specificity. As a proof-of-concept, this SRI-DNA machine/CRISPR-Cas12a feedback amplification system is adaptable and scalable to higher-order artificial amplification circuits for biomarkers detection, highlighting its promising potential in early diagnosis and disease treatment.