Detection of mutations induced by CRISPR/Cas9 in cabbage protoplasts using qPCR and dPCR

Stajič, Ester (author), Javornik, Žiga (author), Štajner, Nataša (author)

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.

• Language: English
• Keywords: CRISPR/Cas9, detection methods, dPCR, editing efficiency, protoplasts, qPCR
• Work type: Article
• Typology: 1.01 - Original Scientific Article
• Organization: BF - Biotechnical Faculty
• Publication status: Published
• Publication version: Version of Record
• Year: 2026
• Number of pages: Str. 260-268
• Numbering: Vol. 188, iss. 1
• PID: 20.500.12556/RUL-176947
• UDC: 631
• ISSN on article: 0003-4746
• DOI: 10.1111/aab.70048
• COBISS.SI-ID: 248417027
• Publication date in RUL: 16.12.2025

Record is a part of a journal

• Title: Annals of applied biology
• Shortened title: Ann. appl. biol.
• Publisher: Wiley, Association of Applied Biologists
• ISSN: 0003-4746
• COBISS.SI-ID: 13699589

Licences

• License: CC BY-NC-ND 4.0, Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International
• Link: http://creativecommons.org/licenses/by-nc-nd/4.0/
• Description: The most restrictive Creative Commons license. This only allows people to download and share the work for no commercial gain and for no other purposes.

Secondary language

• Language: Slovenian
• Keywords: žlahtnjenje rastlin, mutacije, CRISPR/Cas9, rastlinska genetika

Projects

• Funder: ARRS - Slovenian Research Agency
• Project number: Z4-3215
• Name: Optimizacija protokola za CRISPR/Cas9 in tarčno preurejanje nukleotidov pri zelju

• Funder: ARRS - Slovenian Research Agency
• Project number: P4-0077
• Name: Kmetijske rastline - genetika in sodobne tehnologije