Efficient generation of CLPG1-edited rabbits using the CRISPR/Cas9 system.

Abstract

The sheep callipyge (CLPG) phenotype, a well-known muscular hypertrophy syndrome, is caused by an A-to-G transition in the CLPG1 locus. The mechanisms of CLPG phenotype are very complicated and remain to be further studied. Lacking suitable animal models containing CLPG mutations may partially contribute to these unanswered mechanisms. In this study, we confirmed that the CLPG1 locus, especially the 12-bp CLPG1 motif, is conserved in mammalian animals including rabbit. Then, we generated seven CLPG1-edited rabbits with 100% efficiency using CRISPR/Cas9 system combined with cytoplasm injection technology. All the newborn rabbits were mosaicism with numerous kinds of mutations around the target sites. Among the nine screened potential off-target sites (POTs) for the two sgRNAs used in this study, none off-target effect was detected. This indicated that we efficiently and precisely generated CLPG1-edited rabbits, and we believe that these newly generated rabbits will do help to unravel the mechanisms of the CLPG phenotype in the future.