Efficient Generation of Myostatin Gene Mutated Rabbit by CRISPR/Cas9

Abstract

CRISPR/Cas9 has been widely used in generating site-specific genetically modified animal models. Myostatin (MSTN) is a negative regulator of muscle mass, related to muscle growth and differentiation. The knockout of MSTN with the desired phenotype of double muscle has been successfully generated in mice, goats, pigs and cattle, but not in rabbits. In this study, the MSTN knockout (KO) rabbits were generated by co-injection of Cas9 mRNA and sgRNA into zygotes. The typical phenotype of double muscle with hyperplasia or hypertrophy of muscle fiber was observed in MSTN KO rabbits. Furthermore, a similar phenotype was found in the F1 generation, suggesting that the mutation of MSTN could be stably inherited in the MSTN KO rabbits. In summary, we have successfully generated MSTN KO rabbits using CRISPR/Cas9 system with high efficiency, which is a reliable and effective animal model for the study of muscle development and related diseases.

Figure 1. CRISPR/Cas9-mediated gene knockout of MSTN in zygote.

(A) Schematic diagram of sgRNA targeting the rabbit MSTN gene loci. Two sgRNA sequences, sgRNA1 and sgRNA2, are marked in red and the protospacer adjacent moti (PAM) sequences are presented in green. (B) Mutation detection in blastocyst by T-cloning and Sanger sequencing. The WT sequence is shown at the top of the targeting sequence. E: embryos; WT: wild type; deletions “−”; insertion “+”. (C) Mutation detection in blastocyst by T7E1 cleavage assay. M, DL2000; 1–12 represent different blastocysts used in this study.

Figure 2. Generation of MSTN KO rabbit via zygote injection.

(A) T-cloning and Sanger sequencing of 20 pups. F₀1-1–F₀1–9 represent the F0 pups from the first time of microinjection. F₀2-1–F₀2–11 represent the F0 pups from the second microinjection. The sequences of sgRNA are shown in red and the PAM sequences are presented in green. The length of deletions is noted to the right of each sequence (- deletion). WT: wild type; deletions “−”; insertion “+”. (B) T7E1 cleavage assay for the mutation detection of F₀1-1–F₀1–9. M, DL2000; 1–9 represent the pups used in this study. (C) T7E1 cleavage assay for the mutation detection of F₀2-1–F₀2–11. M, DL2000; 1–11 represent the pups used in this study. (D) Photos of MSTN^−/− (F₀2–3), MSTN^+/− (F₀2–6) and WT (F₀2–11) rabbits (4-month old) in F0. Note that an obvious double muscular phenotype was found in MSTN^−/− and MSTN^+/− rabbits, which compared to the WT.

Figure 3. Heritability of the MSTN KO rabbit.

(A) T-cloning and Sanger sequencing analyses of MSTN KO rabbits in F1. F₁1–F₁9 represent the F1 MSTN KO rabbits. The sequences of sgRNA are shown in red and the PAM sequences are presented in green. WT: wild type; deletions “−”. (B) The mutation detection of F1 rabbits by T7E1 cleavage assay. M, DL2000; F₁1–F₁9 represent the offspring pups used in this study. (C) Determination of MSTN protein in skeletal muscle by Western blot. Equal amounts of protein were used and the β-actin was used as reference control.

Figure 4. Increased body weight and muscle mass in MSTN KO rabbits.

(A) The average body weight of MSTN^+/− and WT rabbits from F1 (n = 4). (B) Photos of MSTN^+/− (F₁ 6) and WT (F₁ 1) rabbits (2-month old) in F1. (C) The average weight of heart (H), tongue (T), gluteus maximus (G) and vastus lateralis (V) from MSTN^+/− and WT rabbits (n = 4). Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, Student’s t test. (D) Representative of heart (H), tongue (T), gluteus maximus (G) and vastus lateralis (V) from MSTN^+/− and WT rabbits.

Figure 5. A hyperplasia and/or hypertrophy of muscle fibers was observed in MSTN KO rabbits.

(A) H&E staining of the muscle fibers from tongue and gluteus maximus. (a) Cross and longitudinal section in tongue; (b) Cross section in gluteus maximus. (B) The statistical analysis of the fiber size and number in tongue and gluteus maximus from MSTN^+/− and WT rabbits (2-month old). Average fiber size and number in tongue (a,b); and in gluteus maximus (c,d). Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, Student’s t test.