CRISPR/Cas9 technology is a widely used method for precise genome editing in plants. The CRISPR/Cas system was originally discovered in bacteria and archaea, where the guide RNA recognizes viral DNA and breaks it down into smaller fragments using an endonuclease. For genome editing in plants, a complementary gRNA sequence must be synthesized that binds to the target gene. Targeted genome editing using CRISPR/Cas9 technology is used to investigate gene function, study gene regulation and develop plants with improved traits. This method is also more time and cost efficient than conventional genetic modification techniques. After targeted genome editing, a fast, simple, accurate and cost-effective method is required to confirm the successful occurrence of genetic changes in the samples. In this master thesis, we aimed to develop dPCR and qPCR methods for the detection of mutations after targeted editing of the cenh3 gene using CRISPR/Cas9 technology. Using the qPCR method, we successfully confirmed the presence of induced mutations in 75 % of the samples tested, while using the dPCR method we confirm mutations in 89 % of the samples. We compared the mutation percentages obtained with the qPCR and dPCR methods with the results of the NGS method and found that the dPCR method yields more comparable mutation percentages than the qPCR method. Based on our results, both methods can be used to successfully confirm mutations after targeted genome editing using the CRISPR/Cas9 system, reducing the financial and time burden of analysis.
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