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Related Experiment Video

Updated: Jul 15, 2025

Field-Deployable Candidatus Liberibacter asiaticus Detection Using Recombinase Polymerase Amplification Combined with CRISPR-Cas12a

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Solid-Phase Collateral Cleavage System Based on CRISPR/Cas12 and Its Application toward Facile One-Pot Multiplex

Hiroki Shigemori^1,2, Satoshi Fujita¹, Eiichi Tamiya^1,3

¹Advanced Photonics and Biosensing Open Innovation Laboratory (PhotoBIO-OIL), National Institute of Advanced Industrial Science and Technology (AIST), Photonics Center Osaka University, 2-1 Yamada-Oka, Suita, Osaka 565-0871, Japan.

Bioconjugate Chemistry

|October 2, 2023

Summary

This study introduces a CRISPR/Cas12 system for rapid, one-pot multiplex DNA detection using solid-phase collateral cleavage. This method simplifies genotyping by enabling simultaneous identification of multiple DNA targets with high specificity.

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DNA Virus Detection System Based on RPA-CRISPR/Cas12a-SPM and Deep Learning

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Published on: May 10, 2024

Area of Science:

Molecular Biology
Biotechnology
Genetics

Background:

The CRISPR/Cas12 system offers potential for advanced nucleic acid detection.
Cas12's collateral cleavage activity upon dsDNA recognition is key for signal transduction.
Current methods for genotyping can be complex and time-consuming.

Purpose of the Study:

To develop a simplified, one-pot multiplex dsDNA detection method using CRISPR/Cas12.
To apply solid-phase collateral cleavage (SPCC) for enhanced genotyping.
To create a sensitive and specific dsDNA sensor with minimal operational steps.

Main Methods:

Developed a sensor utilizing immobilized Cas12-crRNA and ssDNA reporters on a solid phase.
Implemented a solid-phase collateral cleavage (SPCC) reaction for signal generation.
Designed a dual-target dsDNA sensor with separate spots for Cas12-crRNA and fluorophore-labeled ssDNA reporters.

Main Results:

Achieved one-pot multiplex dsDNA detection with SPCC, reducing fluorescence by 42.1-57.3% upon target binding.
Demonstrated high specificity with two-base resolution, distinguishing between different DNA sequences.
Established a detectable concentration for target dsDNA as low as 10^-9 M.

Conclusions:

The SPCC-based CRISPR/Cas12 system enables efficient and specific one-pot multiplex dsDNA detection.
This approach significantly simplifies operational steps for nucleic acid analysis.
Future applications include comprehensive genotyping using sensor arrays and automated reagent immobilization.