Abstract
Myostatin (MSTN), a protein encoded by growth differentiation factor 8 (GDF8), is primarily expressed in skeletal muscle and negatively regulates the development and regeneration of muscle. Accordingly, myostatin-deficient animals exhibit a double-muscling phenotype. The CRISPR/Cas9 system has proven to be an efficient genome-editing tool and has been applied to gene modification in cells from many model organisms such as Drosophila melanogaster, zebrafish, mouse, rat, sheep, and human. Here, we edited the GDF8 gene in fibroblasts and embryos of Debao pig and swamp buffalo using the CRISPR/Cas9 system. The CRISPR/Cas9-mediated mutation efficiency in fibroblasts was as high as 87.5% in pig and 78.9% in buffalo. We then obtained single-cell clones with mutations at the specific sites of the GDF8 gene by screening with G418 in fibroblasts of pig and buffalo. In addition, the frequencies of Cas9/gRNA-mediated mutations were at 36 and 25% in the intracytoplasmic sperm injection embryos of pig and in vitro fertilization embryos of buffalo, respectively. Our work demonstrates that the Cas9/gRNA system is a highly efficient and fast tool for genome editing in cultured cells and embryos of Debao pig and swamp buffalo. These results can be helpful for the establishment of a new animal strain that can generate more meat.
Similar content being viewed by others
References
Bollag RJ, Waldman AS, Liskay RM (1989) Homologous recombination in mammalian cells. Annu Rev Genet 23(1):199–225. http://www.ncbi.nlm.nih.gov/pubmed/2694931. https://doi.org/10.1146/annurev.ge.23.120189.001215
Courtney DG, Moore JE, Atkinson SD, Maurizi E, Allen EH, Pedrioli DM, McLean WH, Nesbit MA, Moore CB (2016) CRISPR/Cas9 DNA cleavage at SNP-derived PAM enables both in vitro and in vivo KRT12 mutation-specific targeting. Gene Ther 23(1):108–112. http://www.ncbi.nlm.nih.gov/pubmed/26289666. https://doi.org/10.1038/gt.2015.82
Crispo M, Mulet AP, Tesson L, Barrera N, Cuadro F, dos Santos-Neto PC, Nguyen TH, Creneguy A, Brusselle L, Anegon I, Menchaca A (2015) Efficient generation of myostatin knock-out sheep using CRISPR/Cas9 technology and microinjection into zygotes. PLoS One 10(8):e0136690. http://www.ncbi.nlm.nih.gov/pubmed/26305800. https://doi.org/10.1371/journal.pone.0136690
Guo R, Wan Y, Xu D, Cui L, Deng M, Zhang G, Jia R, Zhou W, Wang Z, Deng K, Huang M, Wang F, Zhang Y (2016) Generation and evaluation of myostatin knock-out rabbits and goats using CRISPR/Cas9 system. Sci Rep 6(29855) http://www.ncbi.nlm.nih.gov/pubmed/27417210
Huang XJ, Zhang HX, Wang H, Xiong K, Qin L, Liu H (2014) Disruption of the myostatin gene in porcine primary fibroblasts and embryos using zinc-finger nucleases. Molecules and cells 37(4):302–306. http://www.ncbi.nlm.nih.gov/pubmed/24802055. https://doi.org/10.14348/molcells.2014.2209
Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD, Peterson RT, Yeh JR, Joung JK (2013) Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol 31(3):227–229. http://www.ncbi.nlm.nih.gov/pubmed/23360964. https://doi.org/10.1038/nbt.2501
Jinek M, East A, Cheng A, Lin S, Ma E, Doudna J (2013) RNA-programmed genome editing in human cells. elife 2:e00471 http://www.ncbi.nlm.nih.gov/pubmed/23386978
Kambadur R, Sharma M, Smith TPL, Bass JJ (1997) Mutations inmyostatin(GDF8) in double-muscled Belgian blue and Piedmontese cattle. Genome Res 7(9):910–915. https://doi.org/10.1101/gr.7.9.910
Liu C, Li W, Zhang X, Zhang N, He S, Huang J, Ge Y, Liu M (2014) Knockdown of endogenous myostatin promotes sheep myoblast proliferation. In vitro cellular & developmental biology Animal 50(2):94–102. http://www.ncbi.nlm.nih.gov/pubmed/24052475. https://doi.org/10.1007/s11626-013-9689-y
Luo J, Song Z, Yu S, Cui D, Wang B, Ding F, Li S, Dai Y, Li N (2014) Efficient generation of myostatin (MSTN) biallelic mutations in cattle using zinc finger nucleases. PLoS One 9(4):e95225. http://www.ncbi.nlm.nih.gov/pubmed/24743319. https://doi.org/10.1371/journal.pone.0095225
Lv Q, Yuan L, Deng J, Chen M, Wang Y, Zeng J, Li Z, Lai L (2016) Efficient generation of myostatin gene mutated rabbit by CRISPR/Cas9. Sci Rep 6(25029) http://www.ncbi.nlm.nih.gov/pubmed/27113799
Mashiko D, Fujihara Y, Satouh Y, Miyata H, Isotani A, Ikawa M (2013) Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA. Sci Rep 3(3355. http://www.ncbi.nlm.nih.gov/pubmed/24284873). https://doi.org/10.1038/srep03355
Mashimo T, Kaneko T, Sakuma T, Kobayashi J, Kunihiro Y, Voigt B, Yamamoto T, Serikawa T (2013) Efficient gene targeting by TAL effector nucleases coinjected with exonucleases in zygotes. Sci Rep 3(1253. http://www.ncbi.nlm.nih.gov/pubmed/23409244). https://doi.org/10.1038/srep01253
McPherron AC, Lawler AM, Lee SJ (1997) Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member. Nature 387(6628):83–90. http://www.ncbi.nlm.nih.gov/pubmed/9139826. https://doi.org/10.1038/387083a0
Morbitzer R, Elsaesser J, Hausner J, Lahaye T (2011) Assembly of custom TALE-type DNA binding domains by modular cloning. Nucleic Acids Res 39(13):5790–5799. http://www.ncbi.nlm.nih.gov/pubmed/21421566. https://doi.org/10.1093/nar/gkr151
Ni W, Qiao J, Hu S, Zhao X, Regouski M, Yang M, Polejaeva IA, Chen C (2014) Efficient gene knockout in goats using CRISPR/Cas9 system. PLoS One 9(9):e106718. http://www.ncbi.nlm.nih.gov/pubmed/25188313. https://doi.org/10.1371/journal.pone.0106718
Ochiai H, Harashima H, Kamiya H (2006) Intranuclear disposition of exogenous DNA in vivo: silencing, methylation and fragmentation. FEBS Lett 580:918–922 http://www.ncbi.nlm.nih.gov/pubmed/16427048
Patel AK, Tripathi AK, Patel UA, Shah RK, Joshi CG (2014) Myostatin knockdown and its effect on myogenic gene expression program in stably transfected goat myoblasts. In vitro cellular & developmental biology Animal 50(7):587–596. http://www.ncbi.nlm.nih.gov/pubmed/24682647. https://doi.org/10.1007/s11626-014-9743-4
Proudfoot C, Carlson DF, Huddart R, Long CR, Pryor JH, King TJ, Lillico SG, Mileham AJ, McLaren DG, Whitelaw CB, Fahrenkrug SC (2015) Genome edited sheep and cattle. Transgenic Res 24(1):147–153. http://www.ncbi.nlm.nih.gov/pubmed/25204701. https://doi.org/10.1007/s11248-014-9832-x
Santiago Y, Chan E, Liu PQ, Orlando S, Zhang L, Urnov FD, Holmes MC, Guschin D, Waite A, Miller JC, Rebar EJ, Gregory PD, Klug A, Collingwood TN (2008) Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases. Proc Natl Acad Sci U S A 105(15):5809–5814. http://www.ncbi.nlm.nih.gov/pubmed/18359850 . https://doi.org/10.1073/pnas.0800940105
Suzuki H, Saito Y, Kagawa N, Yang X (2003) In vitro fertilization and polyspermy in the pig: factors affecting fertilization rates and cytoskeletal reorganization of the oocyte. Microsc Res Tech 61(4):327–334. http://www.ncbi.nlm.nih.gov/pubmed/12811737. https://doi.org/10.1002/jemt.10345
Ui-Tei K, Maruyama S, Nakano Y (2017) Enhancement of single guide RNA transcription for efficient CRISPR/Cas-based genomic engineering. Genome 60:537–545 http://www.ncbi.nlm.nih.gov/pubmed/28177825
Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R (2013) One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153(4):910–918. http://www.ncbi.nlm.nih.gov/pubmed/23643243. https://doi.org/10.1016/j.cell.2013.04.025
Wang K, Ouyang H, Xie Z, Yao C, Guo N, Li M, Jiao H, Pang D (2015) Efficient generation of myostatin mutations in pigs using the CRISPR/Cas9 system. Sci Rep 5(16623) http://www.ncbi.nlm.nih.gov/pubmed/26564781
Yu B, Lu R, Yuan Y, Zhang T, Song S, Qi Z, Shao B, Zhu M, Mi F, Cheng Y (2016) Efficient TALEN-mediated myostatin gene editing in goats. BMC Dev Biol 16(26. http://www.ncbi.nlm.nih.gov/pubmed/27461387):26. https://doi.org/10.1186/s12861-016-0126-9
Yu S, Luo J, Song Z, Ding F, Dai Y, Li N (2011) Highly efficient modification of beta-lactoglobulin (BLG) gene via zinc-finger nucleases in cattle. Cell Res 21(11):1638–1640. http://www.ncbi.nlm.nih.gov/pubmed/21912434. https://doi.org/10.1038/cr.2011.153
Funding
This work was supported by the China 863 High Technique Project (2013AA102504), the National Natural Science Foundation (31760648), and the Guangxi Natural Science Foundation (Grant No. 31260552).
Author information
Authors and Affiliations
Corresponding authors
Additional information
Editor: Tetsuji Okamoto
Electronic supplementary material
ESM 1
(DOCX 4879 kb)
ESM 2
Flow cytometry data showing CRISPR/Cas9-mediated efficiency of non-homologous end joining using the RGS-CR reporter. (A) gRNAs targeting the GDF8 gene of Debao pig. (B) gRNAs targeting the GDF8 gene of buffalo. (JPG 54.5 kb)
ESM 3
Comparative analysis of the amino acid mutations induced by CRISPR/Cas9 around the target sites of the GDF8 gene in Debao pig fibroblasts. (A) Comparison of the amino acid sequence around target site #2 in the fibroblasts versus wild-type (WT). (B) Comparison of the amino acid sequence around target site #3 in the fibroblasts versus WT. (C) Comparison of the amino acid sequence around target site #2 in the fibroblast cell clones versus WT. Note: * indicates a stop codon. (JPG 139 kb)
ESM 4
Comparative analysis of the amino acid mutations induced by CRISPR/Cas9 around the target sites of the GDF8 gene in swamp buffalo fibroblasts. (A) Comparison of the amino acid sequence around target site #3 in the fibroblasts versus wild-type (WT). (B) Comparison of the amino acid sequence around target site #3 in the fibroblast cell clones versus WT. Note: * indicates a stop codon. (JPG 117 kb)
ESM 5
Comparative analysis of the amino acid mutations induced by CRISPR/Cas9 around target sites of the GDF8 gene in embryos of pig and buffalo. (A) Comparison of the amino acid sequence around target site #3 in wild-type (WT) versus ICSI embryos of pig. (B) Comparison of the amino acid sequence around target site #3 in WT versus IVF embryos of buffalo. Note: * indicates a stop codon. (JPG 96.7 kb)
ESM 6
The ICSI embryos of pig and IVF embryos of buffalo. (A) ICSI embryos of pig. (B) IVF embryos of buffalo. (JPG 59.0 kb)
ESM 7
(DOCX 15.9 kb)
ESM 8
(DOCX 15.6 kb)
ESM 9
(DOCX 15.8 kb)
Rights and permissions
About this article
Cite this article
Su, X., Cui, K., Du, S. et al. Efficient genome editing in cultured cells and embryos of Debao pig and swamp buffalo using the CRISPR/Cas9 system. In Vitro Cell.Dev.Biol.-Animal 54, 375–383 (2018). https://doi.org/10.1007/s11626-018-0236-8
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11626-018-0236-8