High-Resolution Melting (HRM) analysis for screening edited lines: a case study in Vitis spp.

In recent years, CRISPR-Cas9 technology has become a powerful and indispensable tool for targeted mutagenesis in plants, including applications such as gene knockout, prime-editing, multiplex gene editing, and regulation of gene transcription. As the number of potential genome editing approaches expands at a very fast pace, rapid, efficient, and cost-effective analytical strategies are needed to screen large numbers of mutants, including the detection of off-target events. In this study, we reported a detection method based on High-Resolution Melting (HRM) analysis to discriminate between wild-type (wt) and edited lines of different varieties of Vitis vinifera and grapevine rootstocks. Those edited lines were obtained through Agrobacterium tumefaciens mediated transformation of embryogenic calli using the CRISPR/SpCas9 system and targeting VviEPFL9-1 and VviEPFL9-2, two paralogous genes involved in stomata cell fate induction. The method clearly distinguished between the wt allele and the mutated one and was partially effective in distinguishing different types of mutation. Moreover, HRM data elaboration based on a Principal Component Analysis (PCA) allowed one to group populations of lines which originated from the same transformation event. Our study demonstrates the reliability of HRM as a fast and cost-effective diagnostic tool for the screening of edited lines and the evaluation of off-target events