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Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells
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Position-independent single-nucleotide polymorphism discrimination by CRISPR/Cas12a via rational activator strand
Qing-Nan Li1, Hao-Ran Huang1, Ruo-Yan Li2
1State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, Research Centre for Analytical Sciences, College of Chemistry, Nankai University, Tianjin, 300071, PR China.
Biosensors & Bioelectronics
|August 29, 2025
Summary
This study introduces an improved CRISPR/Cas12a biosensing system for accurate single-nucleotide polymorphism (SNP) detection. The novel activator strand design enables sensitive identification of low-abundance mutations for advanced molecular diagnostics.
Area of Science:
- Molecular Biology
- Biotechnology
- Genomics
Background:
- Single-nucleotide polymorphisms (SNPs) are vital biomarkers for disease diagnosis and genetic research.
- Sensitive and specific detection of SNPs remains a significant challenge in current methodologies.
Purpose of the Study:
- To develop an enhanced CRISPR/Cas12a biosensing system for improved SNP discrimination.
- To optimize activator strand design for high-sensitivity, single-nucleotide resolution SNP detection.
Main Methods:
- Systematic optimization of crRNA-complementary region length and 3"-terminal random extension sequence.
- Development of an engineered CRISPR/Cas12a platform utilizing the "RESET" effect.
- One-pot detection assay for low-abundance mutations without pre-amplification.
Main Results:
- Achieved single-nucleotide resolution SNP discrimination irrespective of mutation position.
- Demonstrated sensitive detection of mutations as low as 0.1% without target pre-amplification.
- Showcased broad applicability across diverse genomic contexts due to sequence flexibility and length tolerance.
Conclusions:
- The engineered CRISPR/Cas12a platform offers a versatile and streamlined approach for SNP detection.
- This strategy significantly enhances SNP discrimination capabilities, surpassing conventional methods.
- The platform holds promise for applications in molecular diagnostics, pathogen surveillance, and precision medicine.
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