Mismatch-Enhanced Specific PCR (MES-PCR): A Rapid and Cost-Effective Method for Screening CRISPR/Cas9-Induced Mutations

2026-07-07

Peng Tian

Bengang Yao

Wenjing Lin

Maoting Yuan

Shuran Li

Yuzhu Qin

Shuang Chen

Tao Lai

Zhenbiao Yang

Wenwei Lin *

Xiang Zhou *

DOI：10.3390/biology15131089

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CRISPR-associated protein 9 (Cas9)-mediated editing generates numerous mutations. Existing detection methods, such as ACT-PCR, T7EI endonuclease cleavage, HRM analysis, and high-throughput sequencing, often require stringent conditions, expensive reagents, or specialized instruments. Here, we introduce Mismatch-Enhanced Specific PCR (MES-PCR), a method that offers sensitivity for detecting mutations under non-stringent experimental conditions. Combined with quantitative PCR (MES-qPCR), it facilitates the calculation of sgRNA efficiency and enables screening for heterozygous mutations. We validated this method in soybean (Glycine max (L.) Merr.) and Arabidopsis thaliana, confirming its practicality. This approach significantly enhances the efficiency and reduces the cost of mutation screening, presenting a powerful tool to accelerate precision breeding and functional genomics research in crops.

Keywords:

ACT-PCR

MES-PCR

mutation

CRISPR/Cas9

genotyping

Mismatch-Enhanced Specific PCR (MES-PCR): A Rapid and Cost-Effective Method for Screening CRISPR/Cas9-Induced Mutations

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