Generation of Cashmere Goats Carrying an EDAR Gene Mutant Using CRISPR-Cas9-Mediated Genome Editing

: In recent years, while the use of the clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 9 (Cas9) (CRISPR-Cas9) system for targeted genome editing has become a research hotspot, it has, to date, not proved adequate for genome editing in large mammals, such as goats. In this study, two opposite single-guide RNAs (sgRNAs) were designed for complete EDAR gene targeting in Cashmere goats, and co-transfected with a plasmid encoding Cas9 into goat fibroblasts. Among the 89 cell lines obtained through the cultivation of clonal cell lines, 62 were positive for EDAR gene targeting. Nine types of mutations were identified by sequencing analysis, and the mutation efficiency was 69.7%. Using one of these cell lines, EDAR gene-targeted Cashmere goat embryos were prepared by somatic cell cloning. Developed embryos were transferred to 79 Cashmere goat recipients, and, after a gestation period of five months six male EDAR gene-targeted Cashmere goats were born. Although only two of these goats survived, they had abnormal primary hair follicles and no hair on the top of their heads, which are the distinctive features of the EDAR gene-targeted Cashmere goats. Thus, this study provides a valuable animal model for future studies on EDAR gene-related phenotypes and hair follicle growth and development and shows that the CRISPR-Cas9 system can be used to edit genes in large mammals.