Amplification-free miRNA detection with CRISPR/Cas12a system based on fragment complementary activation strategy

Area of Science:

• Molecular Biology
• Biotechnology
• Diagnostics

Background:

• CRISPR/Cas12a systems are known for DNA targeting and trans-cleavage activity.
• Previous understanding limited CRISPR/Cas12a applications to DNA detection.
• The need for sensitive and direct RNA detection methods is growing.

Purpose of the Study:

• To investigate the intrinsic trans-cleavage activity of CRISPR/Cas12a (LbCas12a) with non-DNA targets.
• To develop a novel strategy for direct miRNA detection using CRISPR/Cas12a.
• To establish a CRISPR/Cas12a-based diagnostic platform for clinical applications.

Main Methods:

• Systematic investigation of LbCas12a trans-cleavage activity with fragmented single-stranded DNA (ssDNA) activators.
• Development of the Fragment Complementary Activation Strategy (FCAS) by replacing ssDNA activators with target miRNA sequences.
• Design of reverse fluorescence-enhanced lateral flow test strips (rFLTS) for miRNA detection.
• Application of FCAS for miRNA in situ imaging in tumor cells.

Main Results:

• CRISPR/Cas12a can be activated by fragmented ssDNA activators.
• Target miRNA sequences can directly activate CRISPR/Cas12a without pre-amplification.
• FCAS enabled direct detection of miRNA-10b with a limit of detection (LOD) of 5.53 fM.
• miRNA-10b was quantified in clinical serum samples from glioma patients.
• FCAS-based CRISPR/Cas12a was successfully used for miRNA in situ imaging in tumor cells.

Conclusions:

• The Fragment Complementary Activation Strategy (FCAS) expands CRISPR/Cas12a targeting capabilities to RNA, specifically miRNAs.
• FCAS provides a sensitive and direct method for miRNA detection and quantification in clinical samples.
• This approach facilitates CRISPR/Cas12a applications in molecular diagnostics and in situ imaging, particularly for cancer research.