A High-Resolution Melting Analysis with an Unlabeled Probe for CRISPR/Cas9-Induced ZBED6 Knockout Pigs Detection

Abstract

Abstract Background The zinc finger BED-type containing six knockout (ZBED6-KO) pigs were created to improve economic traits by increasing the expression of insulin-like growth factor 2. They were generated by CRISPR/CRISPR-associated protein 9 (Cas9) technology and a single-base deletion of ZBED6 was found. An efficient and rapid method was needed to detect this type of pig. Objective This study aimed to develop a high-resolution melting (HRM) method to detect ZBED6-KO pigs. Methods An unlabeled probe and two primers were designed to develop the HRM method. The limit of detection, specificity, and accuracy of the established method were tested by the constructed plasmid and DNA extracts of tissue specimens. Results The limit of detection by the established method was 102 copies/µL. The HRM method with an unlabeled probe showed good specificity and high accuracy. Conclusions The established HRM analysis with an unlabeled probe showed it to be a highly effective, rapid, and reliable method to distinguish ZBED6-KO pigs from wild-type pigs. Highlights It is the first time that HRM analysis with an unlabeled probe has been used in the detection of genome editing pigs by the CRISPR/Cas9 technology.