Myostatin (MSTN), a member of the transforming growth factor-β superfamily, is a negative regulator of muscle growth and development. Disruption of the MSTN gene in various mammalian species markedly promotes muscle growth. Previous studies have mainly focused on the disruption of the MSTN peptide coding region in pigs but not on the modification of the signal peptide region. In this study, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) system was used to successfully introduce two mutations (PVD20H and GP19del) in the MSTN signal peptide region of the indigenous Chinese pig breed, Liang Guang Small Spotted pig. Both mutations in signal peptide increased the muscle mass without inhibiting the production of mature MSTN peptide in the cells. Histological analysis revealed that the enhanced muscle mass in MSTN+/PVD20H pig was mainly due to an increase in the number of muscle fibers. The expression of MSTN in the longissimus dorsi muscle of MSTN+/PVD20H and MSTNKO/PVD20H pigs was significantly downregulated, whereas that of myogenic regulatory factors, including MyoD, Myogenin, and Myf-5, was significantly upregulated when compared to those in the longissimus dorsi muscle of wild-type pigs. Meanwhile, the mutations also activated the PI3K/Akt pathway. The results of this study indicated that precise editing of the MSTN signal peptide can enhance porcine muscle development without markedly affecting the expression of mature MSTN peptide, which could exert other beneficial biological functions in the edited pigs.
This is a preview of subscription content, log in to check access.
Buy single article
Instant access to the full article PDF.
Tax calculation will be finalised during checkout.
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
Tax calculation will be finalised during checkout.
Aiello D, Patel K, Lasagna E (2018) The myostatin gene: an overview of mechanisms of action and its relevance to livestock animals. Anim Genet 49:505–519. https://doi.org/10.1111/age.12696
Amthor H, Huang RJ, McKinnell I, Christ B, Kambadur R, Sharma M, Patel K (2002) The regulation and action of myostatin as a negative regulator of muscle development during avian embryogenesis. Dev Biol 251:241–257. https://doi.org/10.1006/dbio.2002.0812
Anderson SB, Goldberg AL, Whitman M (2008) Identification of a novel pool of extracellular pro-myostatin in skeletal muscle. J Biol Chem 283:7027–7035. https://doi.org/10.1074/jbc.M706678200
Cambien F, Ricard S, Troesch A, Mallet C, Generenaz L, Evans A, Arveiler D, Luc G, Ruidavets JB, Poirier O (1996) Polymorphisms of the transforming growth factor-beta 1 gene in relation to myocardial infarction and blood pressure. The Etude Cas-Temoin de l'Infarctus du Myocarde (ECTIM) study. Hypertension 28:881–887. https://doi.org/10.1161/01.hyp.28.5.881
Chan D, Ho MS, Cheah KS (2001) Aberrant signal peptide cleavage of collagen X in Schmid metaphyseal chondrodysplasia. Implications for the molecular basis of the disease. J Biol Chem 276:7992–7997. https://doi.org/10.1074/jbc.M003361200
Choo KH, Ranganathan S (2008) Flanking signal and mature peptide residues influence signal peptide cleavage. BMC Bioinform 9(Suppl 12):S15. https://doi.org/10.1186/1471-2105-9-S12-S15
Cinque L, Sparaneo A, Penta L, Mencarelli A, Rogaia D, Esposito S, Fabrizio FP, Baorda F, Verrotti A, Falorni A et al (2017) Autosomal dominant PTH gene signal sequence mutation in a family with familial isolated hypoparathyroidism. J Clin Endocrinol Metab 102:3961–3969. https://doi.org/10.1210/jc.2017-00250
Cyranoski D (2015) Super-muscly pigs created by small genetic tweak. Nature 523:13–14. https://doi.org/10.1038/523013a
Elkina Y, von Haehling S, Anker SD, Springer J (2011) The role of myostatin in muscle wasting: an overview. J Cachexia Sarcopenia Muscle 2:143–151. https://doi.org/10.1007/s13539-011-0035-5
Fikes JD, Bankaitis VA, Ryan JP, Bassford PJ Jr (1987) Mutational alterations affecting the export competence of a truncated but fully functional maltose-binding protein signal peptide. J Bacteriol 169:2345–2351. https://doi.org/10.1128/jb.169.6.2345-2351.1987
Fikes JD, Barkocy-Gallagher GA, Klapper DG, Bassford PJ Jr (1990) Maturation of Escherichia coli maltose-binding protein by signal peptidase I in vivo. Sequence requirements for efficient processing and demonstration of an alternate cleavage site. J Biol Chem 265:3417–3423
Gonzalez-Mariscal I, Montoro RA, O'Connell JF, Kim Y, Gonzalez-Freire M, Liu QR, Alfaras I, Carlson OD, Lehrmann E, Zhang Y et al (2019) Muscle cannabinoid 1 receptor regulates Il-6 and myostatin expression, governing physical performance and whole-body metabolism. FASEB J 33:5850–5863. https://doi.org/10.1096/fj.201801145R
Grobet L, Martin LJ, Poncelet D, Pirottin D, Brouwers B, Riquet J, Schoeberlein A, Dunner S, Menissier F, Massabanda J et al (1997) A deletion in the bovine myostatin gene causes the double-muscled phenotype in cattle. Nat Genet 17:71–74. https://doi.org/10.1038/ng0997-71
Hussain S, Ali JM, Jalaludin MY, Harun F (2013) Permanent neonatal diabetes due to a novel insulin signal peptide mutation. Pediatr Diabetes 14:299–303. https://doi.org/10.1111/pedi.12011
Ji Q, Zhu K, Liu Z, Song Z, Huang Y, Zhao H, Chen Y, He Z, Mo D, Cong P (2013) Improvement of porcine cloning efficiency by trichostain A through early-stage induction of embryo apoptosis. Theriogenology 79:815–823. https://doi.org/10.1016/j.theriogenology.2012.12.010
Khadempar S, Familghadakchi S, Motlagh RA, Farahani N, Dashtiahangar M, Rezaei H, Gheibi Hayat SM (2019) CRISPR-Cas9 in genome editing: Its function and medical applications. J Cell Physiol 234:5751–5761. https://doi.org/10.1002/jcp.27476
Kim H, Um E, Cho SR, Jung C, Kim H, Kim JS (2011) Surrogate reporters for enrichment of cells with nuclease-induced mutations. Nat Methods 8:941–943. https://doi.org/10.1038/nmeth.1733
Kocamis H, Gahr SA, Batelli L, Hubbs AF, Killefer J (2002) IGF-I, IGF-II, and IGF-receptor-1 transcript and IGF-II protein expression in myostatin knockout mice tissues. Muscle Nerve 26:55–63. https://doi.org/10.1002/mus.10160
Langley B, Thomas M, Bishop A, Sharma M, Gilmour S, Kambadur R (2002) Myostatin inhibits myoblast differentiation by down-regulating MyoD expression. J Biol Chem 277:49831–49840. https://doi.org/10.1074/jbc.M204291200
Lee SJ (2004) Regulation of muscle mass by myostatin. Annu Rev Cell Dev Biol 20:61–86. https://doi.org/10.1146/annurev.cellbio.20.012103.135836
Lee SJ, McPherron AC (2001) Regulation of myostatin activity and muscle growth. Proc Natl Acad Sci USA 98:9306–9311. https://doi.org/10.1073/pnas.151270098
Lillico S (2019) Agricultural applications of genome editing in farmed animals. Transgenic Res 28:57–60. https://doi.org/10.1007/s11248-019-00134-5
Lindert U, Gnoli M, Maioli M, Bedeschi MF, Sangiorgi L, Rohrbach M, Giunta C (2018) Insight into the pathology of a COL1A1 signal peptide heterozygous mutation leading to severe osteogenesis imperfecta. Calcified Tissue Int 102:373–379. https://doi.org/10.1007/s00223-017-0359-z
Liu XF, Liu HB, Wang M, Li RQ, Zeng JH, Mo DL, Cong PQ, Liu XH, Chen YS, He ZY (2019) Disruption of the ZBED6 binding site in intron 3 of IGF2 by CRISPR/Cas9 leads to enhanced muscle development in Liang Guang Small Spotted pigs. Transgenic Res 28:141–150. https://doi.org/10.1007/s11248-018-0107-9
Lokireddy S, McFarlane C, Ge X, Zhang H, Sze SK, Sharma M, Kambadur R (2011) Myostatin induces degradation of sarcomeric proteins through a Smad3 signaling mechanism during skeletal muscle wasting. Mol Endocrinol 25:1936–1949. https://doi.org/10.1210/me.2011-1124
Martoglio B, Dobberstein B (1998) Signal sequences: more than just greasy peptides. Trends Cell Biol 8:410–415. https://doi.org/10.1016/s0962-8924(98)01360-9
McCroskery S, Thomas M, Maxwell L, Sharma M, Kambadur R (2003) Myostatin negatively regulates satellite cell activation and self-renewal. J Cell Biol 162:1135–1147. https://doi.org/10.1083/jcb.200207056
McFarlane C, Langley B, Thomas M, Hennebry A, Plummer E, Nicholas G, McMahon C, Sharma M, Kambadur R (2005) Proteolytic processing of myostatin is auto-regulated during myogenesis. Dev Biol 283:58–69. https://doi.org/10.1016/j.ydbio.2005.03.039
McPherron AC, Lee SJ (2002) Suppression of body fat accumulation in myostatin-deficient mice. J Clin Invest 109:595–601. https://doi.org/10.1172/JCI13562
McPherron AC, Lawler AM, Lee SJ (1997) Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member. Nature 387:83–90. https://doi.org/10.1038/387083a0
Mitri F, Bentov Y, Behan LA, Esfandiari N, Casper RF (2014) A novel compound heterozygous mutation of the luteinizing hormone receptor-implications for fertility. J Assist Reprod Genet 31:787–794. https://doi.org/10.1007/s10815-014-0249-5
Miyake M, Hayashi S, Taketa Y, Iwasaki S, Watanabe K, Ohwada S, Aso H, Yamaguchi T (2010) Myostatin down-regulates the IGF-2 expression via ALK-Smad signaling during myogenesis in cattle. Anim Sci J 81:223–229. https://doi.org/10.1111/j.1740-0929.2009.00725.x
Morissette MR, Cook SA, Buranasombati C, Rosenberg MA, Rosenzweig A (2009) Myostatin inhibits IGF-I-induced myotube hypertrophy through Akt. Am J Physiol Cell Physiol 297:C1124–1132. https://doi.org/10.1152/ajpcell.00043.2009
Mosher DS, Quignon P, Bustamante CD, Sutter NB, Mellersh CS, Parker HG, Ostrander EA (2007) A mutation in the myostatin gene increases muscle mass and enhances racing performance in heterozygote dogs. PLoS Genet 3:e79. https://doi.org/10.1371/journal.pgen.0030079
Nothwehr SF, Gordon JI (1990) Structural features in the NH2-terminal region of a model eukaryotic signal peptide influence the site of its cleavage by signal peptidase. J Biol Chem 265:17202–17208
Qian L, Tang M, Yang J, Wang Q, Cai C, Jiang S, Li H, Jiang K, Gao P, Ma D et al (2015) Targeted mutations in myostatin by zinc-finger nucleases result in double-muscled phenotype in Meishan pigs. Sci Rep 5:14435. https://doi.org/10.1038/srep14435
Racchi M, Watzke HH, High KA, Lively MO (1993) Human coagulation factor X deficiency caused by a mutant signal peptide that blocks cleavage by signal peptidase but not targeting and translocation to the endoplasmic reticulum. J Biol Chem 268:5735–5740
Rajan S, Eames SC, Park SY, Labno C, Bell GI, Prince VE, Philipson LH (2010) In vitro processing and secretion of mutant insulin proteins that cause permanent neonatal diabetes. Am J Physiol Endocrinol Metab 298:E403–410. https://doi.org/10.1152/ajpendo.00592.2009
Rao S, Fujimura T, Matsunari H, Sakuma T, Nakano K, Watanabe M, Asano Y, Kitagawa E, Yamamoto T, Nagashima H (2016) Efficient modification of the myostatin gene in porcine somatic cells and generation of knockout piglets. Mol Reprod Dev 83:61–70. https://doi.org/10.1002/mrd.22591
Rebbapragada A, Benchabane H, Wrana JL, Celeste AJ, Attisano L (2003) Myostatin signals through a transforming growth factor beta-like signaling pathway to block adipogenesis. Mol Cell Biol 23:7230–7242. https://doi.org/10.1128/Mcb.23.20.7230-7242.2003
Rios R, Carneiro I, Arce VM, Devesa J (2002) Myostatin is an inhibitor of myogenic differentiation. Am J Physiol-Cell Physiol 282:C993–C999. https://doi.org/10.1152/ajpcell.00372.2001
Schuelke M, Wagner KR, Stolz LE, Hubner C, Riebel T, Komen W, Braun T, Tobin JF, Lee SJ (2004) Myostatin mutation associated with gross muscle hypertrophy in a child. N Engl J Med 350:2682–2688. https://doi.org/10.1056/NEJMoa040933
Snapp EL, McCaul N, Quandte M, Cabartova Z, Bontjer I, Kallgren C, Nilsson I, Land A, von Heijne G, Sanders RW et al (2017) Structure and topology around the cleavage site regulate post-translational cleavage of the HIV-1 gp160 signal peptide. Elife. https://doi.org/10.7554/eLife.26067
Tang L, Kang Y, Sun S, Zhao T, Cao W, Fan X, Guo J, Sun L, Ta D (2019) Inhibition of MSTN signal pathway may participate in LIPUS preventing bone loss in ovariectomized rats. J Bone Miner Metab. https://doi.org/10.1007/s00774-019-01029-5
Thomas M, Langley B, Berry C, Sharma M, Kirk S, Bass J, Kambadur R (2000) Myostatin, a negative regulator of muscle growth, functions by inhibiting myoblast proliferation. J Biol Chem 275:40235–40243. https://doi.org/10.1074/jbc.M004356200
Trendelenburg AU, Meyer A, Rohner D, Boyle J, Hatakeyama S, Glass DJ (2009) Myostatin reduces Akt/TORC1/p70S6K signaling, inhibiting myoblast differentiation and myotube size. Am J Physiol Cell Physiol 296:C1258–1270. https://doi.org/10.1152/ajpcell.00105.2009
Tsai SW, Wu HS, Chen IA, Chen HL, Chang GR, Fan HC, Chen CM (2019) Recombinant porcine myostatin propeptide generated by the Pichia pastoris elevates myoblast growth and ameliorates high-fat diet-induced glucose intolerance. Res Vet Sci 124:200–211. https://doi.org/10.1016/j.rvsc.2019.03.008
Tu P, Bhasin S, Hruz PW, Herbst KL, Castellani LW, Hua N, Hamilton JA, Guo W (2009) Genetic disruption of myostatin reduces the development of proatherogenic dyslipidemia and atherogenic lesions in Ldlr null mice. Diabetes 58:1739–1748. https://doi.org/10.2337/db09-0349
von Heijne G (1985) Signal sequences. The limits of variation. J Mol Biol 184:99–105. https://doi.org/10.1016/0022-2836(85)90046-4
Vouillot L, Thelie A, Pollet N (2015) Comparison of T7E1 and surveyor mismatch cleavage assays to detect mutations triggered by engineered nucleases. G3 Bethesda 5, 407–415. DOI: 10.1534/g3.114.015834.
Walker RG, Czepnik M, Goebel EJ, McCoy JC, Vujic A, Cho M, Oh J, Aykul S, Walton KL, Schang G et al (2017) Structural basis for potency differences between GDF8 and GDF11. BMC Biol 15:19. https://doi.org/10.1186/s12915-017-0350-1
Wang KK, Ouyang HS, Xie ZC, Yao CG, Guo NN, Li MJ, Jiao HP, Pang DX (2015) Efficient generation of myostatin mutations in pigs using the CRISPR/Cas9 system. Sci Rep-UK. https://doi.org/10.1038/srep16623
Wang K, Tang X, Xie Z, Zou X, Li M, Yuan H, Guo N, Ouyang H, Jiao H, Pang D (2017) CRISPR/Cas9-mediated knockout of myostatin in Chinese indigenous Erhualian pigs. Transgenic Res 26:799–805. https://doi.org/10.1007/s11248-017-0044-z
Yoon JD, Hwang SU, Kim M, Jeon Y, Hyun SH (2019) Growth differentiation factor 8 regulates SMAD2/3 signaling and improves oocyte quality during porcine oocyte maturation in vitrodagger. Biol Reprod 101:63–75. https://doi.org/10.1093/biolre/ioz066
Zimmers TA, Davies MV, Koniaris LG, Haynes P, Esquela AF, Tomkinson KN, McPherron AC, Wolfman NM, Lee SJ (2002) Induction of cachexia in mice by systemically administered myostatin. Science 296:1486–1488. https://doi.org/10.1126/science.1069525
We wish to thank past and current members of Prof. YC’s group for scientific discussions. We are particular grateful to the staffs in the Guangdong YIHAO Food Co., Ltd. for their help in animal sample collection.
This work was jointly supported by the National Transgenic Major Program of China (2016ZX08006003-006), National Key R&D Programmes of China (2018YFD0501200), The Key R&D Programmes of Guangdong Province (2018B020203003) and Guangdong Basic and Applied Basic Research Foundation (2019A1515011134).
Conflict of interest
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 procedures performed in studies involving animals were in accordance with the ethical standards of the Institutional Animal Care and Use Committee (IACUC), Sun Yat-sen University (Approval Number: IACUC DD-17-0403).
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
About this article
Cite this article
Li, R., Zeng, W., Ma, M. et al. Precise editing of myostatin signal peptide by CRISPR/Cas9 increases the muscle mass of Liang Guang Small Spotted pigs. Transgenic Res 29, 149–163 (2020). https://doi.org/10.1007/s11248-020-00188-w
- Signal peptide
- Muscle mass
- Liang Guang Small Spotted pig