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Updated: Oct 20, 2025
Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs
Published on: April 14, 2015
CRISPR/Cas14a-Based Isothermal Amplification for Profiling Plant MicroRNAs
Hao Yang1, Junbo Chen2, Sen Yang1
1College of Biomass Science and Engineering, Healthy Food Evaluation Research Center, Sichuan University, Chengdu 610065, China.
Abstract:
MicroRNAs (miRNAs) play key roles in biological processes in plants, such as stress resistance, yet can hardly be quantified by an enzyme-involved terminal polymerization process due to their 2'-O-methyl modifications at the 3'-terminal. Herein, we proposed a CRISPR/Cas14a-based rolling circle amplification (termed Cas14R) assay, allowing reverse transcription-free and demethylation-free detection of plant miRNAs with single-nucleotide resolution. The employment of target-templated rolling circle amplification circumvents the extension of the unaccessible 2'-O-methyl group at the 3'-terminal. Particularly, the activated Cas14a confers the trans-cleavage activity for identifying target single-stranded DNA sequences without the necessity of the protospacer adjacent motif, generalizing the detection of miRNA sequences and the integration of different isothermal amplification techniques. Ultimately, the Cas14R assay has been applied to profile miR156a to evaluate the ripeness process of banana, indicating its feasibility in analyzing the roles of miRNAs in biological processes of plants.
Insights
This study introduces a novel CRISPR/Cas14a-based assay for accurate plant microRNA quantification. The Cas14R method offers sensitive, single-nucleotide resolution detection without reverse transcription or demethylation.
Area of Science:
- Molecular Biology
- Biotechnology
- Plant Science
Background:
- MicroRNAs (miRNAs) are crucial regulators of plant biological processes, including stress resistance.
- Quantifying plant miRNAs is challenging due to 3'-terminal 2'-O-methyl modifications, hindering enzyme-involved assays.
- Existing methods often require complex steps like reverse transcription or demethylation.
Purpose of the Study:
- To develop a novel, sensitive, and direct detection method for plant miRNAs.
- To overcome the limitations posed by 3'-terminal modifications in miRNA quantification.
- To establish a versatile platform for miRNA profiling in plants.
Main Methods:
- Development of a CRISPR/Cas14a-based rolling circle amplification assay (Cas14R).
- Utilizing target-templated rolling circle amplification to bypass 3'-terminal modifications.
- Leveraging Cas14a's trans-cleavage activity for sequence-specific DNA target identification.
Main Results:
- The Cas14R assay enables reverse transcription-free and demethylation-free detection of plant miRNAs.
- Achieved single-nucleotide resolution for miRNA quantification.
- Demonstrated successful application in profiling miR156a during banana ripening.
Conclusions:
- The Cas14R assay provides a robust and efficient method for plant miRNA detection and profiling.
- This assay overcomes key technical hurdles in quantifying modified miRNAs.
- The Cas14R assay holds significant potential for advancing research on miRNA functions in plant biology.
