High resolution melting analysis: A rapid and sensitive method for detection of mutations induced by CRISPR/Cas9-based genome editing in oysters

Genome editing using CRISPR/Cas9 methodology holds much potential to accelerate the genetic improvement in aquaculture, and has been extensively tested and applied to various aquaculture species. However, screening and identifying successfully edited individuals based on sensitive, low-cost, and rapid methods remains challenging. Here, we developed and optimized a high-resolution melting analysis (HRM) assay to screen CRISPR/Cas9-edited larval oysters. The target amplification size range was optimized to 100–200 bp. Compared with fluorescent dyes SYBR Green and SYTO-9, saturated dye EvaGreen showed high stability and repeatability when used in PCR master mixes. The HRM assay was sensitive enough to detect a temperature shift when a mixture of 10% mutant colonies and 90% WT colonies were present. Sanger sequencing analysis confirmed that oysters with indel mutations could be successfully identified by the HRM method. Together, these data indicate that the HRM assay developed in this study is a reproducible, sensitive, and efficient method for rapidly screening and identifying gene-edited oysters.