High Resolution Melting Temperature Analysis to IdentifyCRISPR/Cas9 Mutants from Arabidopsis

• Published in: Bio-protocol Vol. 8; no. 14; p. e2944
• Main Authors: Denbow, Cynthia, Ehivet, Sonia Carole, Okumoto, Sakiko
• Format: Journal Article
• Language: English
• Published: United States Bio-Protocol 20-07-2018
• Subjects: Biology; Methods; Targeted mutagenesis; CRISPR/Cas9; Arabidopsis; High-resolution melting analysis; Indel detection
• DOI: 10.21769/BioProtoc.2944
• EISSN: 2331-8325

CRISPR/Cas9 made targeted mutagenesis and genome editing possible for many plant species. One of the ways that the endonuclease is used for plant genetics is the creation of loss-of-function mutants, which typically result from erroneous DNA repair through non-homologous end joining (NHEJ) pathway. The majority of erroneous repair events results in single-bp insertion or deletion. While single-bp insertions or deletions (indels) effectively destroy the function of protein-coding genes through frameshift, detection is difficult due to the small size shift. High-resolution melting temperature analysis allows quick detection, and it does not require any additional pipetting steps after the PCR amplification of the region of interest. In this protocol, we will describe the steps required for the analysis of potential homozygous mutants.