Sensitive detection of microRNA based on high-fidelity CRISPR/Cas13a trans cleavage activity coupled with template-free DNA extension-induced strongly emitting copper nanoparticles

As vital biomarkers for diagnosis of diseases, the anomalously expressed microRNAs (miRNAs) are closely linked to the occurrence and development of many diseases. Quantitative analysis of miRNAs has remarkable signifi-cance to clinical diagnosis and therapy of different cancers. In this study, the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) 13a (CRISPR/Cas13a) system was first exploited to directly and specifically recognize the miRNA at the guidance of CRISPR RNA (crRNA), which can activate the nonspecific trans-ribonuclease activity of Cas13a. Then, combining the CRISPR-Cas13a system with terminal deoxynucleotidyl transferase (TdT)-triggered template-free DNA extension to form the fluorescent copper nanoparticles (CuNCs), a simple but sensitive method (named as Cas13a-TFCN) was developed for highly specific detection of miRNA. Based on the CRISPR/Cas13a system coupled with TdT-triggered simple but effi-cient signal amplification strategy, the developed Cas13a-TFCN method showed high sensitivity for miRNA detection with the low detection limit of 0.7 fM. Furthermore, the proposed Cas13a-TFCN approach was suc-cessfully applied for accurate quantification of miRNA in complex biological samples, indicating its practical application ability. Moreover, featuring high sensitivity and simplicity without complicated probe design and fluorophore labeling, the proposed Cas13a-TFCN has a promising applications prospect in clinical diagnosis.

Automated summary

By combining the CRISPR/Cas13a system with a simple and efficient signal amplification strategy triggered by TdT, a highly specific and sensitive method (Cas13a-TFCN) was developed for miRNA detection. The Cas13a-TFCN method showed high sensitivity and was able to accurately quantify miRNA in complex biological samples, demonstrating its practical application ability.