Citation: Xinrui Fei, Chao Lei, Zhaowei Tian, Xinyu Zhang, Wei Ren, Chenghui Liu. Artificial mismatch-assisted Cas12a crRNA splicing mechanism enabling interference-free discrimination of homologous microRNAs with single-nucleotide resolution[J]. Chinese Chemical Letters, ;2026, 37(9): 112450. doi: 10.1016/j.cclet.2026.112450 shu

Artificial mismatch-assisted Cas12a crRNA splicing mechanism enabling interference-free discrimination of homologous microRNAs with single-nucleotide resolution

Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry & Chemical Engineering, Shaanxi Normal University, Xi'an 710119, China

wei.ren@snnu.edu.cn

liuch@snnu.edu.cn

Received Date: 21 November 2025
Revised Date: 21 January 2026
Accepted Date: 22 January 2026
Available Online: 24 January 2026

Figures(4)

How to precisely discriminate homologous microRNA sequences from each other has always been a big challenge. Herein, we proposed an artificial mismatch-assisted Cas12a crRNA splicing mechanism that enables completely interference-free discrimination of homologous microRNAs with single-nucleotide resolution. Specifically, a truncated crRNA (tcrRNA) can recover its ability to activate Cas12a trans-cleavage activity when spliced with a miRNA. Since the miRNA splicing region itself is relatively short, rationally tuning the tcrRNA splicing length can render the proposed system with high sensitivity for base-variation discrimination. More importantly, we innovatively introduced an extra artificial mismatch in the miRNA splicing region, which enables the system to distinguish single-nucleotide variants in an interference-free manner. In this way, only the perfectly matched target miRNA can trigger the complete assembly of the spliced crRNA to active Cas12a, while non-target RNAs even with a single-base mismatch cannot. This mechanism ensures that the target miRNA can be accurately hooked even in the presence of a large excess of non-target homologous RNAs (100-fold). The discrimination capability of the proposed method has been proven by achieving the interference-free discrimination of the members within the let-7 family, which can hardly be achieved by conventional hybridization-based amplification methods. This work fully taps into the potential of the Cas12a system in interference-free homologous miRNA discrimination, offering a new tool for precise RNA analysis in molecular diagnostics.
- Homologous microRNA,
- CRISPR/Cas12a,
- CrRNA splicing,
- Single-base discrimination,
- Interference-free
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