AUTHOR=Lu Zhiquan , Wang Shijing , Li Ping , Yang Huasheng , Han Sanyang , Zhang Shaochong , Ma Lan 

TITLE=An ultra-sensitive suboptimal protospacer adjacent motif enhanced rolling circle amplification assay based on CRISPR/Cas12a for detection of miR-183

JOURNAL=Frontiers in Bioengineering and Biotechnology

VOLUME=Volume 12 - 2024

YEAR=2024

URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2024.1444908

DOI=10.3389/fbioe.2024.1444908

ISSN=2296-4185

ABSTRACT=MicroRNAs (miRNAs) have been recognized as promising diagnostic biomarkers for Diabetic Retinopathy (DR) due to their notable up-regulation in individuals with the condition. However, the development of highly sensitive miRNAs assays for the rapid diagnosis of DR in clinical settings remains a challenging task. In this study, we introduce an enhanced CRISPR/Cas12a assay, leveraging suboptimal PAM (sPAM)-mediated Cas12a trans-cleavage in conjunction with rolling circle amplification (RCA). We found that sPAM performed better than canonical PAM (cPAM) in the detection of Cas12a-mediated ssDNA detection at low concentrations, and by replacing cPAM with sPAM in the padlock template of RCA, ultra-high sensitivity can be achieved, with a detection  limit of 0.40 aM within 25 minutes and a linear range spanning from 1 aM to 1 pM. Notably, our assay exhibits exceptional specificity in detecting miR-183 from other miRNAs. Furthermore, we validate the applicability of our assay for the sensitive detection of miR-183 in clinical serum samples. This study introduces a groundbreaking assay with excellent performance through a simple modification, which not only addresses existing diagnostic challenges, but also opens exciting new avenues for clinical diagnosis in the realm of Diabetic Retinopathy.