Use of CRISPR/Cas9 technology efficiently targetted goat myostatin through zygotes microinjection resulting in double-muscled phenotype in goats

Abstract

Myostatin gene (MSTN) can inhibit the proliferation of myoblast, which in turn promotes muscle growth and inhibits adipocyte differentiation in livestock. MSTN mutation may lead to muscle hypertrophy or double-muscled (DM) phenotype. MSTN mutation animal, such as sheep, dog, and rabbit have been generated through CRISPR/Cas9 technology. However, goats with promising MSTN mutation have not been generated. We designed two sgRNAs loci targetting exon3 of MSTN gene to destroy the MSTN cysteines knots. We got seven goats from seven recipients, in which six were MSTN knocked-out (KO) goats, with a mutation rate of 85.7%. Destroyed cysteine knots caused MSTN structure inactivation. The average body weight gain (BWG) per day of MSTN KO goats was significantly higher than that of wild-type (WT) goats. MSTN KO goats showed abnormal sugar, fat, and protein metabolism compared with wild-type controls (MSTN+/+). Inheritance of mutations was observed in offspring of MSTN KO goats by PCR analysis.

Muscle Hyperplasia in Japanese Quail by Single Amino Acid Deletion in MSTN Propeptide

“…MSTN is mainly expressed in skeletal muscle and negatively regulates the growth of muscle [2]. This inhibitory effect on muscle growth has been further confirmed by MSTN mutation resulting in increased muscle mass in humans and animals including cattle, dogs, pigs, goats, sheep, rabbits, and fishes [3][4][5][6][7][8][9][10][11][12][13].…”

“…Function of MSTN in negative regulation of muscle growth is conserved across species and natural or genetically engineered disruption of the MSTN gene gives increased muscle mass in mice, cattle, dogs, pigs, goats, sheep, rabbits, fishes, and humans [2][3][4][5][6][7][8][9][10][11][12][13]. However, the mechanism of enhancing muscle mass by MSTN mutation is somewhat different across species.…”

“…To inhibit the anti-myogenic function of MSTN, the entire MSTN gene [10] or mature domain encoding region [7,8] can be disrupted by genetic mutations using CRISPR/Cas9, a powerful genome editing tool. On the other hand, disruption of mature MSTN binding to ACVR2B and overexpression of a dominant-negative form of ACVR2B resulted in increased muscle mass in mice [15,18].…”

Livestock breeding for the 21st century: the promise of the editing revolution

“…As an alternative to cloning, edited animals have been produced by directly microinjecting mRNAs encoding the genome editor reagents into the cytoplasm of an early zygote [9] . Litter sizes and viability are good with this approach, and while early demonstrations had relatively low editing frequency, evolution of the tools and refinement in delivery has resulted in substantive improvements [10,11] . A combination of SCNT followed by zygote microinjection with Cas9 ribonucleoprotein (Cas9 protein pre-complexed with sgRNA) has also been shown to be a very efficient strategy for generation of genome edited pigs [12] .…”

“…Breeds that have been selected for increased muscle mass, such as Belgian Blue cattle, have mutations within the coding sequence of the myostatin gene [18] , while others, such as Texel sheep, have reduced myostatin expression due to altered non-coding sequence [19] . Given the striking phenotype that can be achieved by perturbing myostatin expression, coupled with its perceived value for agriculture, it is not surprising that it has now been edited in most major livestock species including cattle [20] , sheep [21] , pig [22] and goat [11] . It is important to note that such animals require a more energy-dense diet than comparators and as such are not suitable for all agricultural systems.…”