Gateway-Compatible CRISPR-Cas9 Vectors and a Rapid Detection by High-Resolution Melting Curve Analysis

CRISPR-Cas9 system rapidly became an indispensable tool in plant biology to perform targeted mutagenesis. A CRISPR-Cas9-mediated double strand break followed by non-homologous end joining (NHEJ) repair most frequently results in a single base pair deletion or insertions (indels), which is hard to detect using methods based on enzymes that detect heteroduplex DNA. In addition, somatic tissues of the T1 generation inevitably contain a mosaic population, in which the portion of cells carrying the mutation can be too small to be detected by the enzyme-based methods. Here we report an optimized experimental protocol for detecting Arabidopsis mutants carrying a CRISPR-Cas9 mediated mutation, using high-resolution melting (HRM) curve analysis. Single-base pair insertion or deletion (indel) can be easily detected using this method. We have also examined the detection limit for the template containing a one bp indel compared to the WT genome. Our results show that