20.500.12556/RUL-176947 Detection of mutations induced by CRISPR/Cas9 in cabbage protoplasts using qPCR and dPCR CRISPR/Cas9 technology is a widely used plant breeding innovation that employs site-directed nucleases to target and modify DNA with great accuracy. It has received significant attention in recent years due to its broad range of applications, including biological research of gene function and breeding new plant varieties. After genome editing reagents are delivered into plant cells, a fast, simple, accurate, and cost-effective method is necessary to confirm the successful occurrence of genetic changes in the samples. In this study, we explored the use of two PCR-based methods: real-time PCR (qPCR) and digital PCR (dPCR) for detecting mutations induced by CRISPR/Cas9 in transformed protoplast samples. Designed primers and probes were tested in a duplex qPCR assay, and dPCR was employed for higher sensitivity. Using the qPCR method, we confirmed mutations in 80% of the samples, while with dPCR we could confirm mutations in all tested samples. Both methods were compared with next-generation sequencing (NGS) results, and dPCR showed more comparable mutation frequencies than qPCR. Overall, both methods effectively distinguished edited from wild-type protoplasts and provide time-saving, cost-efficient alternatives to traditional molecular tools for detecting gene-edited mutations. Furthermore, they could be used to quantify induced mutations, with dPCR offering higher sensitivity but at a higher cost than qPCR. CRISPR/Cas9 detection methods dPCR editing efficiency protoplasts qPCR žlahtnjenje rastlin mutacije CRISPR/Cas9 rastlinska genetika true false true Angleški jezik Slovenski jezik Članek v reviji 2025-12-16 11:46:25 2025-12-16 11:46:32 2025-12-26 03:53:25 0000-00-00 00:00:00 2026 0 0 Str. 260-268 iss. 1 Vol. 188 2026 0000-00-00 Zaloznikova Objavljeno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 631 0003-4746 10.1111/aab.70048 248417027 Annals_of_Applied_Biology_-_2025_-_Stajic_-_Detecti.pdf Annals_of_Applied_Biology_-_2025_-_Stajic_-_Detecti.pdf 1 A015F1804ACBEBA3600DA673D7225FC6 e2508a19319061c886773dedc57953ca24bce752f7a0257b72cd57c83c99c74b 15000a77-da79-11f0-9328-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=223798 https://onlinelibrary.wiley.com/doi/10.1111/aab.70048 1 0cbb36ca-da6c-11f0-9328-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=223782 Biotehniška fakulteta 0 0 0