Rabbits are long-eared ground-dwelling mammals belonging to the family Leporidae order Lagomorpha and domesticated about 1,400 years ago (1) due to their delicious high-quality meat. They are geographically distributed across the desert, wetland, and tropical forests (2). Rabbits are being reared at the domestic level as a livestock species for meat, wool, or fur production (3), and nowadays rabbit production has become a minor agricultural enterprise in Western European countries such as Italy, Spain, and France (1, 3, 4). The worldwide per capita rabbit meat consumption is 0.242 kg, while in some European countries like Italy the per capita consumption is about 4.39 kg (5, 6). Furthermore, a stable rate of rabbit meat consumption has also been reported from 2000 to 2013 in many European countries (5).

The body growth is primarily regulated through complex interactive pathways that cause cell proliferation and cell enlargement (7). However, several factors including hormonal, genetic, environmental, and nutritional can remarkably predict the growth patterns (8). Myostatin (MSTN) is a member of the transforming growth factor (TGF-β) superfamily which is considered to be a negative regulator through inhibiting muscle development and regeneration (9). MSTN is an extracellular hormone that occurs in the skeletal muscle in an inactive state and restores its activity by binding to the precursor peptide, follistatin 3, and TGF-β binding protein, and transmitting the signals through the receptors (9, 10). The binding affinity of MSTN and ActRIIB could activate a chain of signal transmission that inhibits the myocyte differentiation and proliferation (10–12).

The MSTN gene inactivation might have an effect on muscle development and regeneration since MSTN gene deletion in mice resulted in a double muscle phenomenon (hyperplasia or hypertrophy) and a significant increase in muscle mass (10–12). Natural mutations of MSTN, which have an obvious double muscle phenotypic effect were found in cattle (13, 14), dogs (15), sheep (16, 17), pig (18), and humans (19, 20). CRISPR/Cas9 system is an efficient genome editing tool and has widely been used for functional gene study. Knock out of MSTN with CRISPR/Cas9 system has been processed in sheep (21, 22), goats (23, 24), and pigs (23, 25). Qian et al. have reported the MSTN-KO gene in Meishan pig fetal fibroblasts by engineered zinc-finger proteins and prepared the MSTN-KO pig by somatic cell nuclear transfer technology. In comparison to wild-type Meishan pigs, the lean meat rate of Meishan pigs with MSTN knockout increased by 11.62% (26). In addition, MSTN-KO goats' meat production was 32% higher than the wild-type (27).

Rabbits offer quick breeding sources and meat with excellent nutritive and dietetic properties which contain high-quality protein and low fat and cholesterol contents, that is fine-grained white meat which can substitute chicken, and is also ideal for obese and cardiovascular patients (28). In the present study, we have designed a highly efficient novel regulatory site and additionally verified that the exon 3 region can regulate the MSTN gene. Furthermore, in our study, the rabbits obtained after the MSTN gene editing are able to pass the gene-edited traits to the next-generation normally, and homozygous gene-edited rabbits obtained in the F2 generation have normal production performance and stable double muscle buttock characteristics.

Humans are entirely reliant on livestock for their daily food supply, which comes in the form of eggs, meat, and milk (30–33). Thus, genetic alteration offers an opportunity for significant production and gains in a short time (34, 35). Since the last decade, many gene-edited organisms have been approved and are being used by the public, such as the gene-edited goats' milk and chicken eggs are being used for drug extraction. Further, the salmon was the first gene-edited species to be approved to be consumed as food, and recently the GalSafe pigs have also been certified to be used for food and medical purpose (36). Rabbit farming is now very well developed all around the world and systematically reared on a large scale, and the global rabbit meat production is reaching 1.8 million metric tons per year (37). As high-quality meat, the demand for rabbit meat is worth looking forward to in the future, but rabbits' muscular development is still a challenge. Therefore, MSTN gene-edited rabbits have the possibility of being approved and used by the public under the advancement of our multi-generation continuous observational research. In the present study, we used CRISPR-Cas9 to delete a long gene fragment of the MSTN gene, which ensured the inactivation of target gene function, and the results of our study presented a successful production of MSTN-KO rabbit with heritable ability, and the loss of the MSTN fragment could lead to muscle growth. In both the F0 and F1 generations, there were significant differences in body weight before it reached the plateau, and the average body weight of the edited rabbits before the slaughter age was also higher than that of the WT rabbits.

Although the increase in muscle mass makes MSTN-KO livestock production more attractive, in these MSTN mutant animals calving difficulties are often caused by various reasons, such as large offspring syndrome (LOS) has been reported in MSTN-deficient animals (15, 38). Compared with other livestock, MSTN gene-edited rabbits rarely report dystocia due to large fetuses, even though in our study the MSTN-KO rabbits exhibited a typical double-muscle phenotype with increased body weight but, at birth, no significant differences have been observed in body weight and size as compared with the control WT. Further, the MSTN-KO rabbits appeared healthy and normal without reproduction difficulties, demonstrating that CRISPR/Cas9 system-generated MSTN-KO rabbits are best suited for studying muscle development and associated diseases, and the results of our study are in line with those of Lv et al. (39).

MSTN gene can code for TGF-beta (transforming growth factor-beta) superfamily ligand that can bind with different TGF-beta receptors and ultimately recruit or activate the SMAD family transcription factors, which regulate the gene expression (40). MSTN is a key regulator of muscle cell proliferation and differentiation throughout muscle development, and MSTN expression signals were detected from the early myogenic stage of embryonic sarcomere formation to the adult skeletal muscle development stage (14, 41, 42). Earlier, the birth weight of MSTN mutant animals has also been reported in sheep, cattle, and goats (43, 44). Wang et al. have also described the MSTN gene-modification and their effects in goats with larger muscle fiber size resulting in enhanced bodyweight (43). Therefore, the MSTN gene knockout might affect muscular development at the embryonic stage (43, 44). Furthermore, it has been stated that MSTN can only express in skeletal muscle during fetal development, thereby controlling the differentiation and proliferation of myoblasts (43, 44). In our study, MSTN-KO rabbits exhibited a typical double-muscle phenotypic trait, but there was no significant difference in body size at birth (P > 0.05) and body weight in the first 60 days compared with the WT group. This might be because of the IGF-1 which is an important positive regulator of muscle cell proliferation and differentiation in the skeletal muscle (45). The IGF1 signaling pathway is critically involved in long-term health regulation, which ultimately plays an essential role to control various homeostatic mechanisms related to growth or development (46). Despite the inhibition of MSTN signal expression, IGF signaling still might upregulate the expression of myostatin in skeletal muscle tissue models, which indicated the presence of an autoregulatory inhibitory loop in a muscular system (47, 48).

Moreover, the muscle fiber hypertrophy, hyperplasia, or a combination thereof triggered by the lack of the MSTN gene subsequently results in increased mammalian muscle production (49–52). The quantity of muscle fibers is mainly determined before birth because the diameter of muscle fibers might be increased after birth (53, 54). The mechanism of muscle hypertrophy and hyperplasia may not occur simultaneously, and it appears that the mechanism by which MSTN mutations enhance the muscle mass varies among species (55, 56). In MSTN mutant mice and goats, the muscle mass gain was caused by muscle fiber hyperplasia and diameter hypertrophy (39, 52, 57), while only muscle fiber hyperplasia was observed in MSTN mutant cattle and pigs (13, 50). The results of the current study presented that the gluteus maximus of the MSTN-KO rabbits was also bigger than the WT rabbits, and histological analysis showed that in MSTN-KO rabbits the density and diameter of the myofiber were significantly large. Therefore, we hypothesized that the enhanced muscular phenotype in the MSTN-KO rabbits is attributed to muscles fiber hyperplasia and/or hypertrophy. Furthermore, previously it has been reported that the muscle mass of MSTN-KO mice was 2–3 times more than that of the WT mice, which was the result of the combined action of muscle fiber hyperplasia and diameter hypertrophy (13, 52). On the other hand, the cattle with double-muscle trait showed that muscle mass increased by 20–25%, and the increased muscle weight appears to be the result of muscle fiber diameter proliferation, rather than an increase in muscle number (13, 50). In this study, compared with WT rabbits, the average body weight of F0 and F1 generation edited rabbits increased by 15 and 15.99% at the age of 180 and 140 days, respectively. The fragment deletion targeting a specific site in MSTN-KO rabbits could suggest that the sgRNA-based CRISPR/Cas9 system might be a useful tool for gene knockout in the mammalian genome (58). Earlier in rabbits, Lv et al. (39) designed two knockout sites at the first exon to edit the MSTN gene (39), while we designed a total of 8 sites in two exons (exon 1 and exon 3) each with four sites. After selection, we determined that the knockout efficiency of the third exon is better than the first exon, thus, we obtained edited rabbits with different knockout sites from already reported MSTN knockout studies. Moreover, at present, most of the experimental research regarding animals' gene-editing is still in the F0 generation (24, 59, 60), including the reported gene editing in rabbits (39). While, no study has yet been performed on the F1 or their subsequent generations, we not only developed MSTN gene-edited rabbits in the F0 generation but also continued the follow-up research in successive F1 and F2 generations with stable heritable mutant populations. Also, the difference between the body type of mutant and the wild type can be observed within 1 month after birth. Gene-editing technology can accelerate the breeding process and improve the meat production of livestock, but the congenital defects of gene edited animals and the uncertainty of trait stability have a great impact on the commercialization of edited animals (51, 61). In our study medium- and long-term breeding can greatly reduce concerns about the phenotypic and genetic stability of MSTN gene-edited animals.

Moreover, various studies reported different types of skeletal-related problems in gene-edited animals such as MSTN gene-edited rabbits had bone deformities, pelvic tilt, and tooth dislocation, while MSTN gene knock-out Meishan pigs had an extrathoracic vertebra (26, 37). Abnormal pelvic structure may cause difficulties in calving in female animals. But, in our study, the MSTN gene-edited rabbits obtained by knocking out the third exon did not show these symptoms in appearance, which are more suitable for the breeding of strains. Additionally, in the previous study, the MSTN-KO animals exhibited the muscular feature, but the MSTN-KO caused severe health issues, such as the fetus having “large tongue syndrome,” which results in milk suckling problems and death (52). In our study, the MSTN-KO rabbits not only exhibited double mushy buttocks but were also able to inherit this trait to subsequent generations without any problem. The majority of MSTN-KO rabbits showed a rapid increase in muscle mass after the age of 60 days, and the MSTN-KO rabbits were healthy and could inherit the knocked-out gene fragments, demonstrating that the MSTN-KO rabbits produced by the CRISPR/Cas9 system are suitable for studying muscle development and related diseases.

The rabbit meat is an enriched source of minerals with low fat and calories and is easy to digest, making it valuable for the consumers, especially for persons suffering from cardiovascular diseases or obesity. Taken together, we developed MSTN-KO rabbits with a typical phenotypic trait of double muscle buttocks that are likely to obtain edited rabbits lines which would help to improve the rabbit's meat production efficiency and promote the development of the rabbit industry. Furthermore, these MSTN-KO rabbits could be a promising tool for studying the development of muscles in other livestock species and improving their important economic trait.

The datasets presented in this study can be found in Genbank repository accession number MZ427879-MZ427887 at: https://www.ncbi.nlm.nih.gov/nuccore/.

The animal study was reviewed and approved by Experimental Animal Care and Use Committee of Guangxi University (Permit Code: 2022-GXU-006). Written informed consent was obtained from the owners for the participation of their animals in this study.

SR, KC, and QL: conceptualization and resources. YZ, YLZ, and LW: data curation. YLZ, YZ, and ZL: methodology. HR, ZL, and DS: software. QL and KC: supervision. YZ and YLZ: writing—original draft preparation. YLZ, YZ, HR, ZL, DS, SR, QL, and KC: writing—review and editing. All authors have read and agreed to the published version of the manuscript.

The present study was granted and supported by the National Natural Science Fund (Grant Nos. 31760648 and 31860638) and Guangxi Natural Science Foundation (Grant No. AB16380042).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.