Generation of beta-lactoglobulin knock-out goats using CRISPR/Cas9.
Goat's milk, considered a substitute for cow's milk, has a high nutritional value. However, goat's milk contains various allergens, predominantly β-lactoglobulin (BLG). In this study, we employed the CRISPR/Cas9 system to target the BLG locus in goat fibroblasts for sgRNA optimization...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2017-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC5634636?pdf=render |
| _version_ | 1818127517705306112 |
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| author | Wenjun Zhou Yongjie Wan Rihong Guo Mingtian Deng Kaiping Deng Zhen Wang Yanli Zhang Feng Wang |
| author_facet | Wenjun Zhou Yongjie Wan Rihong Guo Mingtian Deng Kaiping Deng Zhen Wang Yanli Zhang Feng Wang |
| author_sort | Wenjun Zhou |
| collection | DOAJ |
| description | Goat's milk, considered a substitute for cow's milk, has a high nutritional value. However, goat's milk contains various allergens, predominantly β-lactoglobulin (BLG). In this study, we employed the CRISPR/Cas9 system to target the BLG locus in goat fibroblasts for sgRNA optimization and generate BLG knock-out goats through co-injection of Cas9 mRNA and small guide RNAs (sgRNAs) into goat embryos at the one-cell stage. We firstly tested sgRNA editing efficiencies in goat fibroblast cells, and approximately 8.00%-9.09% of the cells were modified in single sgRNA-guided targeting experiment. Among the kids, the genome-targeting efficiencies of single sgRNA were 12.5% (10 ng/μL sg1) and 0% (10 ng/μL sg2) and efficiencies of dual sgRNAs were 25.0% (25 ng/μL sg2+sg3 group) and 28.6% (50 ng/μL sg2+sg3 group). Relative expression of BLG in BLG knock-out goat mammary glands significantly (p < 0.01) decreased as well as other milk protein coding genes, such as CSN1S1, CSN1S2, CSN2, CSN3 and LALBA (p < 0.05). As expected, BLG protein had been abolished in the milk of the BLG knock-out goat. In addition, most of the targeted kids were chimeric (3/4), and their various body tissues were edited simultaneously. Our study thus provides a basis for optimizing the quality of goat milk, which can be applied to biomedical and agricultural research. |
| first_indexed | 2024-12-11T07:18:37Z |
| format | Article |
| id | doaj.art-14452ea161564fe3a4d4e100fb1834dc |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-11T07:18:37Z |
| publishDate | 2017-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-14452ea161564fe3a4d4e100fb1834dc2022-12-22T01:16:09ZengPublic Library of Science (PLoS)PLoS ONE1932-62032017-01-011210e018605610.1371/journal.pone.0186056Generation of beta-lactoglobulin knock-out goats using CRISPR/Cas9.Wenjun ZhouYongjie WanRihong GuoMingtian DengKaiping DengZhen WangYanli ZhangFeng WangGoat's milk, considered a substitute for cow's milk, has a high nutritional value. However, goat's milk contains various allergens, predominantly β-lactoglobulin (BLG). In this study, we employed the CRISPR/Cas9 system to target the BLG locus in goat fibroblasts for sgRNA optimization and generate BLG knock-out goats through co-injection of Cas9 mRNA and small guide RNAs (sgRNAs) into goat embryos at the one-cell stage. We firstly tested sgRNA editing efficiencies in goat fibroblast cells, and approximately 8.00%-9.09% of the cells were modified in single sgRNA-guided targeting experiment. Among the kids, the genome-targeting efficiencies of single sgRNA were 12.5% (10 ng/μL sg1) and 0% (10 ng/μL sg2) and efficiencies of dual sgRNAs were 25.0% (25 ng/μL sg2+sg3 group) and 28.6% (50 ng/μL sg2+sg3 group). Relative expression of BLG in BLG knock-out goat mammary glands significantly (p < 0.01) decreased as well as other milk protein coding genes, such as CSN1S1, CSN1S2, CSN2, CSN3 and LALBA (p < 0.05). As expected, BLG protein had been abolished in the milk of the BLG knock-out goat. In addition, most of the targeted kids were chimeric (3/4), and their various body tissues were edited simultaneously. Our study thus provides a basis for optimizing the quality of goat milk, which can be applied to biomedical and agricultural research.http://europepmc.org/articles/PMC5634636?pdf=render |
| spellingShingle | Wenjun Zhou Yongjie Wan Rihong Guo Mingtian Deng Kaiping Deng Zhen Wang Yanli Zhang Feng Wang Generation of beta-lactoglobulin knock-out goats using CRISPR/Cas9. PLoS ONE |
| title | Generation of beta-lactoglobulin knock-out goats using CRISPR/Cas9. |
| title_full | Generation of beta-lactoglobulin knock-out goats using CRISPR/Cas9. |
| title_fullStr | Generation of beta-lactoglobulin knock-out goats using CRISPR/Cas9. |
| title_full_unstemmed | Generation of beta-lactoglobulin knock-out goats using CRISPR/Cas9. |
| title_short | Generation of beta-lactoglobulin knock-out goats using CRISPR/Cas9. |
| title_sort | generation of beta lactoglobulin knock out goats using crispr cas9 |
| url | http://europepmc.org/articles/PMC5634636?pdf=render |
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