Boosting split-crRNA CRISPR/Cas12a activity by 3′-end extension of DNA activator for direct microRNA sensing

Dec 2, 2025Analytica chimica acta

Improving CRISPR/Cas12a gene editing by extending the DNA activator's end to better detect microRNA

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Abstract

The split-crRNA CRISPR/Cas12a system with a 24-nucleotide extension showed a 6.4-fold increase in activity for detecting miRNA-375.

Direct detection of miRNA-375 was achieved without nucleic acid amplification or reverse transcription, reducing reaction time and contamination risk.
The system demonstrated a linear detection range of 5 pM to 1 nM, with a detection limit estimated at 0.6 pM.
The method successfully detected miRNA-375 in 10% diluted human serum with a recovery rate of 98%-106%.
Extending sequences at the 3'-end of the DNA activator significantly enhanced the cleavage activity of the split-crRNA CRISPR/Cas12a system.

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BACKGROUND: The unique trans-cleavage activity of CRISPR/Cas12a has been extensively utilized in the domain of biosensing. Nevertheless, the detection of miRNAs using the traditional CRISPR/Cas12a system requires nucleic acid amplification or reverse transcription to convert miRNA into DNA, which increases reaction time and the risk of contamination.

RESULTS: This study presents a split-crRNA CRISPR/Cas12a-based biosensing for direct detection of miRNA. The target miRNA-375 was utilized as the spacer region of the crRNA, facilitating its binding to the truncated scaffold RNA, thereby resulting in the formation of a complete crRNA. More importantly, we discovered that the cleavage activity of split-crRNA CRISPR/Cas12a was significantly enhanced by extending sequences at the 3'-end of the DNA activator. Compared with the conventional split-crRNA CRISPR/Cas12a system, the split-crRNA CRISPR/Cas12a with 24-nucleotide random sequence extension at the 3'-end of the DNA activator exhibited a 6.4-fold increase in activity. The enhancement mechanism of 3'-end extension of DNA activator was discussed. This proposed split-crRNA CRISPR/Cas12a system was applied to detect miRNA-375 with a linear range of 5 pM-1 nM, and the detection limit was estimated to be 0.6 pM (3σ). Furthermore, this system was used to detect miRNA-375 in 10 % diluted human serum, achieving satisfactory recovery rate (98 %-106 %).

SIGNIFICANCE: This finding indicates that it is feasible to enhance the activity of the split-crRNA CRISPR/Cas12a by extending the 3'-end of the DNA activator, thereby achieving highly sensitive direct detection of miRNA. It is a simple yet effective strategy for enhancing the sensitivity of direct miRNA detection.

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Boosting split-crRNA CRISPR/Cas12a activity by 3′-end extension of DNA activator for direct microRNA sensing

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