{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T14:17:36Z","timestamp":1785421056894,"version":"3.56.0"},"reference-count":22,"publisher":"MDPI AG","issue":"13","license":[{"start":{"date-parts":[[2026,7,7]],"date-time":"2026-07-07T00:00:00Z","timestamp":1783382400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100012166","name":"National Key Research and Development Program of China","doi-asserted-by":"publisher","award":["2025YFA0921600"],"award-info":[{"award-number":["2025YFA0921600"]}],"id":[{"id":"10.13039\/501100012166","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["32470768"],"award-info":[{"award-number":["32470768"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["32270335"],"award-info":[{"award-number":["32270335"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"award":["32470768"],"award-info":[{"award-number":["32470768"]}],"id":[{"id":"https:\/\/ror.org\/01h0zpd94","id-type":"ROR","asserted-by":"publisher"}]},{"award":["32270335"],"award-info":[{"award-number":["32270335"]}],"id":[{"id":"https:\/\/ror.org\/01h0zpd94","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biology"],"abstract":"<jats:p>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.<\/jats:p>","DOI":"10.3390\/biology15131089","type":"journal-article","created":{"date-parts":[[2026,7,7]],"date-time":"2026-07-07T09:53:46Z","timestamp":1783418026000},"page":"1089","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Mismatch-Enhanced Specific PCR (MES-PCR): A Rapid and Cost-Effective Method for Screening CRISPR\/Cas9-Induced Mutations"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0009-0005-7313-2885","authenticated-orcid":false,"given":"Peng","family":"Tian","sequence":"first","affiliation":[{"name":"Fujian Provincial Key Laboratory of Haixia Applied Plant Systems Biology, College of Life Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China"},{"name":"Horticultural Plant Biology and Metabolomics Center, Haixia Institute of Science and Technology, Fujian Agriculture and Forestry University, Fuzhou 350002, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-3278-8369","authenticated-orcid":false,"given":"Bengang","family":"Yao","sequence":"additional","affiliation":[{"name":"Fujian Provincial Key Laboratory of Haixia Applied Plant Systems Biology, College of Life Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China"},{"name":"Horticultural Plant Biology and Metabolomics Center, Haixia Institute of Science and Technology, Fujian Agriculture and Forestry University, Fuzhou 350002, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wenjing","family":"Lin","sequence":"additional","affiliation":[{"name":"Fujian Provincial Key Laboratory of Haixia Applied Plant Systems Biology, College of Life Science, Fujian Agriculture and Forestry 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