Generation of pigs with a Belgian Blue mutation in MSTN using CRISPR/Cpf1-assisted ssODN-mediated homologous recombination
CRISPR/Cpf1 has emerged recently as an effective tool for genome editing in many organisms, but its use in pigs to generate precise genetic modifications has seldom been described. Myostatin (MSTN) is a well-characterized negative regulator of muscle development, and natural mutations in this gene c...
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KeAi Communications Co., Ltd.
2019-06-01
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author | Yun-long ZOU Zhi-yuan LI Yun-jing ZOU Hai-yang HAO Jia-xiang HU Ning LI Qiu-yan LI |
author_facet | Yun-long ZOU Zhi-yuan LI Yun-jing ZOU Hai-yang HAO Jia-xiang HU Ning LI Qiu-yan LI |
author_sort | Yun-long ZOU |
collection | DOAJ |
description | CRISPR/Cpf1 has emerged recently as an effective tool for genome editing in many organisms, but its use in pigs to generate precise genetic modifications has seldom been described. Myostatin (MSTN) is a well-characterized negative regulator of muscle development, and natural mutations in this gene cause a double-muscled phenotype in many species. However, to the best of our knowledge, no naturally occurring mutation in MSTN has been found in pigs. In addition, no living pig models with sophisticated modifications orthologous to natural mutations in MSTN have yet been reported. In this study, we exploited the CRISPR/Cpf1 system to introduce a predefined modification orthologous to the natural MSTN mutation found in Belgian Blue cattle (thus known as the Belgian Blue mutation). Our research demonstrated that the cutting efficiency of CRISPR/Cpf1 was 12.3% in mixed porcine fetal fibroblasts in drug free medium, and 41.7% in clonal colonies obtained using G418 selection. Then, the Cpf1-sgRNA vector, ssODN template, and a self-excision cassette were co-transfected into porcine fetal fibroblasts. After G418 selection, 8 clonal colonies were examined and 5 with genetic modification were found. Of these 5, 2 harbored the precise 11-bp deletion. Using 1 heterozygous clonal colony, 2 cloned Duroc piglets were successfully generated, which was heterozygous for the Belgian Blue mutation. In summary, our results demonstrate that CRISPR/Cpf1 system can be used efficiently to generate double-stranded breaks, and also to mediate homologous recombination to introduce precise genomic modifications in pigs. |
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spelling | doaj-art-7e0411ecc9f348dba3a0ffbdd33b50d72025-08-02T12:05:23ZengKeAi Communications Co., Ltd.Journal of Integrative Agriculture2095-31192019-06-0118613291336Generation of pigs with a Belgian Blue mutation in MSTN using CRISPR/Cpf1-assisted ssODN-mediated homologous recombinationYun-long ZOU0Zhi-yuan LI1Yun-jing ZOU2Hai-yang HAO3Jia-xiang HU4Ning LI5Qiu-yan LI6State Key Laboratory of Silkworm Genome Biology, Key Laboratory of Sericultural Biology and Genetic Breeding, Ministry of Agriculture and Rural Affairs, College of Biotechnology, Southwest University, Chongqing 400715, P.R. China; State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, P.R. China; ZOU Yun-long, Tel: +86-23-68251309State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, P.R. ChinaCollege of Veterinary Medicine, China Agricultural University, Beijing 100193, P.R. ChinaState Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, P.R. ChinaState Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, P.R. ChinaState Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, P.R. ChinaState Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, P.R. China; Correspondence LI Qiu-yan, Tel: +86-10-62732334CRISPR/Cpf1 has emerged recently as an effective tool for genome editing in many organisms, but its use in pigs to generate precise genetic modifications has seldom been described. Myostatin (MSTN) is a well-characterized negative regulator of muscle development, and natural mutations in this gene cause a double-muscled phenotype in many species. However, to the best of our knowledge, no naturally occurring mutation in MSTN has been found in pigs. In addition, no living pig models with sophisticated modifications orthologous to natural mutations in MSTN have yet been reported. In this study, we exploited the CRISPR/Cpf1 system to introduce a predefined modification orthologous to the natural MSTN mutation found in Belgian Blue cattle (thus known as the Belgian Blue mutation). Our research demonstrated that the cutting efficiency of CRISPR/Cpf1 was 12.3% in mixed porcine fetal fibroblasts in drug free medium, and 41.7% in clonal colonies obtained using G418 selection. Then, the Cpf1-sgRNA vector, ssODN template, and a self-excision cassette were co-transfected into porcine fetal fibroblasts. After G418 selection, 8 clonal colonies were examined and 5 with genetic modification were found. Of these 5, 2 harbored the precise 11-bp deletion. Using 1 heterozygous clonal colony, 2 cloned Duroc piglets were successfully generated, which was heterozygous for the Belgian Blue mutation. In summary, our results demonstrate that CRISPR/Cpf1 system can be used efficiently to generate double-stranded breaks, and also to mediate homologous recombination to introduce precise genomic modifications in pigs.http://www.sciencedirect.com/science/article/pii/S2095311919626948MSTNCRISPR/Cpf1Belgian Blue mutationgenetically modified pigssingle stranded oligodeoxynucleotide |
spellingShingle | Yun-long ZOU Zhi-yuan LI Yun-jing ZOU Hai-yang HAO Jia-xiang HU Ning LI Qiu-yan LI Generation of pigs with a Belgian Blue mutation in MSTN using CRISPR/Cpf1-assisted ssODN-mediated homologous recombination Journal of Integrative Agriculture MSTN CRISPR/Cpf1 Belgian Blue mutation genetically modified pigs single stranded oligodeoxynucleotide |
title | Generation of pigs with a Belgian Blue mutation in MSTN using CRISPR/Cpf1-assisted ssODN-mediated homologous recombination |
title_full | Generation of pigs with a Belgian Blue mutation in MSTN using CRISPR/Cpf1-assisted ssODN-mediated homologous recombination |
title_fullStr | Generation of pigs with a Belgian Blue mutation in MSTN using CRISPR/Cpf1-assisted ssODN-mediated homologous recombination |
title_full_unstemmed | Generation of pigs with a Belgian Blue mutation in MSTN using CRISPR/Cpf1-assisted ssODN-mediated homologous recombination |
title_short | Generation of pigs with a Belgian Blue mutation in MSTN using CRISPR/Cpf1-assisted ssODN-mediated homologous recombination |
title_sort | generation of pigs with a belgian blue mutation in mstn using crispr cpf1 assisted ssodn mediated homologous recombination |
topic | MSTN CRISPR/Cpf1 Belgian Blue mutation genetically modified pigs single stranded oligodeoxynucleotide |
url | http://www.sciencedirect.com/science/article/pii/S2095311919626948 |
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