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CRISPR/Cas9-Mediated Specific Integration of Fat-1 and IGF-1 at the pRosa26 Locus

Many researchers have focused on knock-in pigs for site-specific integration, but little attention has been given to genetically modified pigs with the targeted integration of multiple recombinant genes. To establish a multigene targeted knock-in editing system, we used the internal ribosome entry s...

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Autores principales: You, Wenni, Li, Mengjing, Qi, Yilin, Wang, Yanbing, Chen, Yiwu, Liu, Ying, Li, Li, Ouyang, Hongsheng, Pang, Daxin
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8305104/
https://www.ncbi.nlm.nih.gov/pubmed/34356043
http://dx.doi.org/10.3390/genes12071027
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author You, Wenni
Li, Mengjing
Qi, Yilin
Wang, Yanbing
Chen, Yiwu
Liu, Ying
Li, Li
Ouyang, Hongsheng
Pang, Daxin
author_facet You, Wenni
Li, Mengjing
Qi, Yilin
Wang, Yanbing
Chen, Yiwu
Liu, Ying
Li, Li
Ouyang, Hongsheng
Pang, Daxin
author_sort You, Wenni
collection PubMed
description Many researchers have focused on knock-in pigs for site-specific integration, but little attention has been given to genetically modified pigs with the targeted integration of multiple recombinant genes. To establish a multigene targeted knock-in editing system, we used the internal ribosome entry site (IRES) and self-cleaving 2A peptide technology to construct a plasmid coexpressing the fatty acid desaturase (Fat-1) and porcine insulin-like growth factor-1 (IGF-1) genes at equal levels. In this study, pigs were genetically modified with multiple genes that were precisely inserted into the pRosa26 locus by using the clustered regularly spaced short palindrome repeat sequence (CRISPR)/CRISPR-related 9 (Cas9) system and somatic cell nuclear transfer technology (SCNT) in combination. Single copies of the Fat-1 and IGF-1 genes were expressed satisfactorily in various tissues of F0-generation pigs. Importantly, gas chromatography analysis revealed a significantly increased n-3 polyunsaturated fatty acid (PUFA) level in these genetically modified pigs, which led to a significant decrease of the n-6 PUFA/n-3 PUFA ratio from 6.982 to 3.122 (*** p < 0.001). In conclusion, the establishment of an editing system for targeted double-gene knock-in in this study provides a reference for the precise integration of multiple foreign genes and lays a foundation for the development of new transgenic pig breeds with multiple excellent phenotypes.
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spelling pubmed-83051042021-07-25 CRISPR/Cas9-Mediated Specific Integration of Fat-1 and IGF-1 at the pRosa26 Locus You, Wenni Li, Mengjing Qi, Yilin Wang, Yanbing Chen, Yiwu Liu, Ying Li, Li Ouyang, Hongsheng Pang, Daxin Genes (Basel) Article Many researchers have focused on knock-in pigs for site-specific integration, but little attention has been given to genetically modified pigs with the targeted integration of multiple recombinant genes. To establish a multigene targeted knock-in editing system, we used the internal ribosome entry site (IRES) and self-cleaving 2A peptide technology to construct a plasmid coexpressing the fatty acid desaturase (Fat-1) and porcine insulin-like growth factor-1 (IGF-1) genes at equal levels. In this study, pigs were genetically modified with multiple genes that were precisely inserted into the pRosa26 locus by using the clustered regularly spaced short palindrome repeat sequence (CRISPR)/CRISPR-related 9 (Cas9) system and somatic cell nuclear transfer technology (SCNT) in combination. Single copies of the Fat-1 and IGF-1 genes were expressed satisfactorily in various tissues of F0-generation pigs. Importantly, gas chromatography analysis revealed a significantly increased n-3 polyunsaturated fatty acid (PUFA) level in these genetically modified pigs, which led to a significant decrease of the n-6 PUFA/n-3 PUFA ratio from 6.982 to 3.122 (*** p < 0.001). In conclusion, the establishment of an editing system for targeted double-gene knock-in in this study provides a reference for the precise integration of multiple foreign genes and lays a foundation for the development of new transgenic pig breeds with multiple excellent phenotypes. MDPI 2021-07-01 /pmc/articles/PMC8305104/ /pubmed/34356043 http://dx.doi.org/10.3390/genes12071027 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
You, Wenni
Li, Mengjing
Qi, Yilin
Wang, Yanbing
Chen, Yiwu
Liu, Ying
Li, Li
Ouyang, Hongsheng
Pang, Daxin
CRISPR/Cas9-Mediated Specific Integration of Fat-1 and IGF-1 at the pRosa26 Locus
title CRISPR/Cas9-Mediated Specific Integration of Fat-1 and IGF-1 at the pRosa26 Locus
title_full CRISPR/Cas9-Mediated Specific Integration of Fat-1 and IGF-1 at the pRosa26 Locus
title_fullStr CRISPR/Cas9-Mediated Specific Integration of Fat-1 and IGF-1 at the pRosa26 Locus
title_full_unstemmed CRISPR/Cas9-Mediated Specific Integration of Fat-1 and IGF-1 at the pRosa26 Locus
title_short CRISPR/Cas9-Mediated Specific Integration of Fat-1 and IGF-1 at the pRosa26 Locus
title_sort crispr/cas9-mediated specific integration of fat-1 and igf-1 at the prosa26 locus
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8305104/
https://www.ncbi.nlm.nih.gov/pubmed/34356043
http://dx.doi.org/10.3390/genes12071027
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