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Updated: Jun 11, 2025
CRISPR Gene Editing Tool for MicroRNA Cluster Network Analysis
Published on: April 25, 2022
An ultra-sensitive suboptimal protospacer adjacent motif enhanced rolling circle amplification assay based on
Zhiquan Lu1, Shijing Wang2,3, Ping Li2
1Precision Medicine and Healthcare Research Center, Tsinghua-Berkeley Shenzhen Institute (TBSI), Tsinghua Shenzhen International Graduate School, University Town of Shenzhen, Shenzhen, China.
Introduction:
MicroRNAs (miRNAs) have been recognized as promising diagnostic biomarkers for Diabetic Retinopathy (DR) due to their notable upregulation 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.
Methods:
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). sPAM was found to perform better than canonical PAM (cPAM) in the detection of Cas12a-mediated ssDNA detection at low concentrations and was used instead of canonical PAM (cPAM) to mediate the detection. The parameters of reactions have also been optimized.
Results And Discussion:
In comparison with cPAM, sPAM has higher sensitivity in the detection of ssDNA at concentrations lower than 10 pM by Cas12a. By replacing cPAM with sPAM in the padlock template of RCA, ultra-high sensitivity for miR-183 detection is achieved, with a detection limit of 0.40 aM. within 25 min and a linear range spanning from 1 aM. to 1 pM. Our assay also exhibits exceptional specificity in detecting miR-183 from other miRNAs. Furthermore, the applicability of our assay for the sensitive detection of miR-183 in clinical serum samples is also validated. 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 DR.
Insights
This study presents a novel CRISPR/Cas12a assay for sensitive Diabetic Retinopathy (DR) diagnosis. The enhanced assay achieves ultra-high sensitivity and specificity for microRNA detection in clinical samples.
Area of Science:
- Biochemistry
- Molecular Biology
- Biotechnology
Background:
- MicroRNAs (miRNAs) are upregulated in Diabetic Retinopathy (DR), making them potential diagnostic biomarkers.
- Current miRNA assays lack the sensitivity and speed required for rapid clinical diagnosis of DR.
Purpose of the Study:
- To develop a highly sensitive and specific assay for Diabetic Retinopathy (DR) diagnosis.
- To improve upon existing CRISPR/Cas12a assays for miRNA detection.
Main Methods:
- An enhanced CRISPR/Cas12a assay was developed using suboptimal Protospacer Adjacent Motif (sPAM)-mediated Cas12a trans-cleavage.
- Rolling Circle Amplification (RCA) was integrated with the CRISPR/Cas12a system.
- Reaction parameters were optimized for enhanced sensitivity and specificity.
Main Results:
- The sPAM-enhanced CRISPR/Cas12a assay demonstrated superior sensitivity compared to canonical PAM (cPAM) for single-stranded DNA (ssDNA) detection at low concentrations.
- The assay achieved an ultra-high sensitivity for miR-183 detection, with a limit of detection of 0.40 attomolar (aM) within 25 minutes.
- Exceptional specificity was observed in detecting miR-183 from other miRNAs, and the assay's applicability was validated in clinical serum samples.
Conclusions:
- The modified CRISPR/Cas12a assay offers a groundbreaking solution for sensitive and rapid DR diagnosis.
- This enhanced assay addresses current diagnostic challenges and opens new avenues for clinical applications in DR.
- The study highlights the potential of sPAM-mediated Cas12a trans-cleavage combined with RCA for sensitive biomarker detection.
