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...

Full description

Saved in:
Bibliographic Details
Main Authors: Yun-long ZOU, Zhi-yuan LI, Yun-jing ZOU, Hai-yang HAO, Jia-xiang HU, Ning LI, Qiu-yan LI
Format: Article
Language:English
Published: KeAi Communications Co., Ltd. 2019-06-01
Series:Journal of Integrative Agriculture
Subjects:
Online Access:http://www.sciencedirect.com/science/article/pii/S2095311919626948
Tags: Add Tag
No Tags, Be the first to tag this record!
_version_ 1839600643337093120
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.
format Article
id doaj-art-7e0411ecc9f348dba3a0ffbdd33b50d7
institution Matheson Library
issn 2095-3119
language English
publishDate 2019-06-01
publisher KeAi Communications Co., Ltd.
record_format Article
series Journal of Integrative Agriculture
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
work_keys_str_mv AT yunlongzou generationofpigswithabelgianbluemutationinmstnusingcrisprcpf1assistedssodnmediatedhomologousrecombination
AT zhiyuanli generationofpigswithabelgianbluemutationinmstnusingcrisprcpf1assistedssodnmediatedhomologousrecombination
AT yunjingzou generationofpigswithabelgianbluemutationinmstnusingcrisprcpf1assistedssodnmediatedhomologousrecombination
AT haiyanghao generationofpigswithabelgianbluemutationinmstnusingcrisprcpf1assistedssodnmediatedhomologousrecombination
AT jiaxianghu generationofpigswithabelgianbluemutationinmstnusingcrisprcpf1assistedssodnmediatedhomologousrecombination
AT ningli generationofpigswithabelgianbluemutationinmstnusingcrisprcpf1assistedssodnmediatedhomologousrecombination
AT qiuyanli generationofpigswithabelgianbluemutationinmstnusingcrisprcpf1assistedssodnmediatedhomologousrecombination