A highly-efficient isothermal nano-detection platform coupling CRISPR/Cas technology for detection of circRNA

Abstract

Triple-negative breast cancer (TNBC), an aggressive molecular subtype of breast cancer with poor prognosis, is characterized by a high rate of metastasis and proliferation, which makes early detection particularly challenging. Early diagnosis of TNBC through biomarkers and prompt development of treatment methods can lower its mortality rate. This work has designed a nano-detection platform for TNBC biomarker circRNA based on the CRISPR/Cas system and isothermal amplification strategy. Specifically, this detection system uses functional nucleic acid molecules for recognition of circCD44, as well as dual signal amplification using Klenow(3′–5′exo-) and Cas9n. Furthermore, it combines Cas12a and immunomagnetic beads for an extra signal boost and output. After confirming its feasibility and optimizing the conditions, the detection system achieved a 1.73-fold enhancement in sensitivity, offering a linear detection range of 1 pM to 100 nM, with the limit of detection lowered to 95.1 fM. It also showed good specificity through testing against 5 biomarkers. Therefore, this detection system provides a novel strategy for the early diagnosis of TNBC and other diseases.