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

Abstract

Simple Summary The CRISPR/Cas9 system has been widely used for genome editing, enabling the efficient generation of mutations in target genes. However, current methods for identifying these mutations, such as ACT-PCR, T7 endonuclease I (T7EI) cleavage, high-resolution melting (HRM) analysis, and high-throughput sequencing, suffer from limitations including stringent reaction conditions, high-cost reagents or instruments, operational complexity, or insufficient sensitivity for heterozygous and single-nucleotide variants. To address this, we developed a novel PCR-based method termed Mismatch-Enhanced Specific PCR (MES-PCR) for the rapid identification of CRISPR/Cas9-induced mutations. This method employs primers with artificially introduced mismatched bases, enabling high-efficiency discrimination between wild-type and mutant alleles without precise annealing temperature control. It is suitable for detecting both known and unknown mutations, including those distant from the PAM site. When coupled with quantitative PCR, the method can calculate the editing efficiency of sgRNAs and screen heterozygous mutants. We validated MES-PCR in soybean and Arabidopsis thaliana gene-edited materials, demonstrating that it has high accuracy in preliminary screening while being significantly simpler and more cost effective. Thus MES-PCR provides an accessible and high-throughput-compatible strategy for screening genome-edited mutants and for the preliminary evaluation of sgRNA efficiency in plant functional genomics and breeding applications.