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Mismatch-Enhanced Specific PCR (MES-PCR): A Rapid and Cost-Effective Method for Screening CRISPR/Cas9-Induced
Peng Tian1,2, Bengang Yao1,2, Wenjing Lin1,2
1Fujian Provincial Key Laboratory of Haixia Applied Plant Systems Biology, College of Life Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China.
Biology
|July 15, 2026
Summary
We developed Mismatch-Enhanced Specific PCR (MES-PCR) for sensitive and cost-effective detection of CRISPR-Cas9 mutations. This method accelerates crop precision breeding and functional genomics research.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- CRISPR-Cas9 gene editing induces various mutations, necessitating efficient detection methods.
- Current mutation detection techniques (e.g., ACT-PCR, T7EI, HRM, high-throughput sequencing) often involve stringent conditions, high costs, or specialized equipment.
Purpose of the Study:
- To introduce Mismatch-Enhanced Specific PCR (MES-PCR) as a sensitive and cost-effective mutation detection method.
- To demonstrate the utility of MES-PCR, combined with quantitative PCR (MES-qPCR), for assessing sgRNA efficiency and screening heterozygous mutations.
Main Methods:
- Development and application of Mismatch-Enhanced Specific PCR (MES-PCR).
- Integration with quantitative PCR (MES-qPCR) for mutation quantification.
- Validation in soybean (Glycine max) and Arabidopsis thaliana.
Main Results:
- MES-PCR detects mutations under non-stringent conditions with high sensitivity.
- MES-qPCR accurately calculates sgRNA efficiency and screens for heterozygous mutations.
- The method was successfully validated in key crop and model plant species.
Conclusions:
- MES-PCR offers a practical, efficient, and economical alternative for mutation screening.
- This technique significantly advances precision breeding and functional genomics research in plants.
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