Construction Of Myostatin Gene Knockout Rabbit Through CRISPR/Cas9

Myostatin(MSTN) is a member of the transforming growth factor beta(TGF-β) superfamily, which acts as a negative regulator of muscle growth. It has been reported that the cattle and sheep, which have spontaneous mutations of MSTN, are called as “double muscled cattle and double muscled sheep”. These cattle and sheep have the characteristics of increased muscle mass, high feed conversion rate and high lean meat percentage. So far, the MSTN knockout(KO) mice, sheep, pigs and dogs have been successfully generated and a double-muscled phenotype was also obtained in these MSTN KO animals.It will provide an ideal animal model for the further study of the double muscled phenotypic in histomorphology and genetic.But the knockout of MSTN is not reported in rabbits so far.Rabbits are a promising animal model for biomedical research, as they show more similarities to humanbeings in terms of physiology and anatomy than mice and rats, and require low cost maintenance and short pregnancyperiod compared to pigs and monkeys. So MSTN KO rabbits constructed by CRISPR/Cas9 can be a promising model for studying muscle development in biomedicine and improving economically important traits in livestock.In this study, two g RNAs targeting the exon 1 of the rabbit MSTN gene were designed, and the efficient MSTN KO vector was constructed. Then the MSTN KO rabbits were constructed by microinjection and reimplantation of zygotes. The results showed that double muscled phenotypic was obtained in both monoallelic and biallelic rabbits. Off-target analysis showed that no off-target effect was detected in the MSTN KO rabbits. Furthermore, a similar phenotype was found in the F1 generation.The statistical analysis of body weight between MSTN+/- and WT groups demonstrated that the MSTN+/- rabbits were obviously heavier than WT rabbits after 6 weeks of age.The histological analysis showed that the increased muscle mass in MSTN KO rabbits resulted from fiber hyperplasia and hypertrophy.In all, this study successfully obtained double muscledrabbits by CRISPR/Cas9 and zygote injection. These MSTN KO rabbits can be a promising tool for studying MSTN-regulated muscle development and related disease and improving economically important traits in livestock.