Abstract
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 <5% of mutant DNA containing one bp indel can be detected using this method. The vector developed in this study can be used with a Gateway technology-compatible derivative of pCUT vectors, with which off-target mutations could not be detected even by a whole genome sequencing.
Highlights
CRISPR-Cas9 technology made targeted mutagenesis readily possible for many organisms
Indel Detection Using High-resolution melting (HRM) Analysis small indels effectively cause loss-of-function through a frame shift if they are in a protein coding region, they cannot be detected using a DNA-agarose gel due to the small size shift
We describe the method used in our laboratory to detect CRISPR/Cas9 based gene editing in Arabidopsis thaliana
Summary
CRISPR-Cas9 technology made targeted mutagenesis readily possible for many organisms. High-resolution melting (HRM) analysis detects the decreased melting temperature in heteroduplex DNA fragments compared to the homoduplex ones. The resulting entry vector carrying the U6promoter-sgRNA repeats can be cloned into pMTN3164, a binary vector carrying the CAS9 coding sequence tagged with the N7 nuclear localization signal under the ubiquitin promoter of Arabidopsis (Supplemental Figure 2).
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