Efficient Genome Editing in Chicken DF-1 Cells Using the CRISPR/Cas9 System
In recent years, genome engineering technology has provided unprecedented opportunities for site-specific modification of biological genomes. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 is one such means that can target a specific genome locus. It has...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Oxford University Press
2016-04-01
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| Series: | G3: Genes, Genomes, Genetics |
| Subjects: | |
| Online Access: | http://g3journal.org/lookup/doi/10.1534/g3.116.027706 |
| _version_ | 1818609586748260352 |
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| author | Yichun Bai Linjie He Pengcheng Li Kun Xu Simin Shao Chonghua Ren Zhongtian Liu Zehui Wei Zhiying Zhang |
| author_facet | Yichun Bai Linjie He Pengcheng Li Kun Xu Simin Shao Chonghua Ren Zhongtian Liu Zehui Wei Zhiying Zhang |
| author_sort | Yichun Bai |
| collection | DOAJ |
| description | In recent years, genome engineering technology has provided unprecedented opportunities for site-specific modification of biological genomes. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 is one such means that can target a specific genome locus. It has been applied in human cells and many other organisms. Meanwhile, to efficiently enrich targeted cells, several surrogate systems have also been developed. However, very limited information exists on the application of CRISPR/Cas9 in chickens. In this study, we employed the CRISPR/Cas9 system to induce mutations in the peroxisome proliferator-activated receptor-γ (PPAR-γ), ATP synthase epsilon subunit (ATP5E), and ovalbumin (OVA) genes in chicken DF-1 cells. The results of T7E1 assays showed that the mutation rate at the three different loci was 0.75%, 0.5%, and 3.0%, respectively. In order to improve the mutation efficiency, we used the PuroR gene for efficient enrichment of genetically modified cells with the surrogate reporter system. The mutation rate, as assessed via the T7E1 assay, increased to 60.7%, 61.3%, and 47.3%, and subsequent sequence analysis showed that the mutation efficiency increased to 94.7%, 95%, and 95%, respectively. In addition, there were no detectable off-target mutations in three potential off-target sites using the T7E1 assay. As noted above, the CRISPR/Cas9 system is a robust tool for chicken genome editing. |
| first_indexed | 2024-12-16T15:00:54Z |
| format | Article |
| id | doaj.art-19cbb3a563d74234a1915036f528cc9c |
| institution | Directory Open Access Journal |
| issn | 2160-1836 |
| language | English |
| last_indexed | 2024-12-16T15:00:54Z |
| publishDate | 2016-04-01 |
| publisher | Oxford University Press |
| record_format | Article |
| series | G3: Genes, Genomes, Genetics |
| spelling | doaj.art-19cbb3a563d74234a1915036f528cc9c2022-12-21T22:27:18ZengOxford University PressG3: Genes, Genomes, Genetics2160-18362016-04-016491792310.1534/g3.116.02770614Efficient Genome Editing in Chicken DF-1 Cells Using the CRISPR/Cas9 SystemYichun BaiLinjie HePengcheng LiKun XuSimin ShaoChonghua RenZhongtian LiuZehui WeiZhiying ZhangIn recent years, genome engineering technology has provided unprecedented opportunities for site-specific modification of biological genomes. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 is one such means that can target a specific genome locus. It has been applied in human cells and many other organisms. Meanwhile, to efficiently enrich targeted cells, several surrogate systems have also been developed. However, very limited information exists on the application of CRISPR/Cas9 in chickens. In this study, we employed the CRISPR/Cas9 system to induce mutations in the peroxisome proliferator-activated receptor-γ (PPAR-γ), ATP synthase epsilon subunit (ATP5E), and ovalbumin (OVA) genes in chicken DF-1 cells. The results of T7E1 assays showed that the mutation rate at the three different loci was 0.75%, 0.5%, and 3.0%, respectively. In order to improve the mutation efficiency, we used the PuroR gene for efficient enrichment of genetically modified cells with the surrogate reporter system. The mutation rate, as assessed via the T7E1 assay, increased to 60.7%, 61.3%, and 47.3%, and subsequent sequence analysis showed that the mutation efficiency increased to 94.7%, 95%, and 95%, respectively. In addition, there were no detectable off-target mutations in three potential off-target sites using the T7E1 assay. As noted above, the CRISPR/Cas9 system is a robust tool for chicken genome editing.http://g3journal.org/lookup/doi/10.1534/g3.116.027706CRISPR/Cas9 systemsurrogate reporterchicken DF-1 cellmutant efficiencyoff-targetGenPredShared data resourceGenomic Selection |
| spellingShingle | Yichun Bai Linjie He Pengcheng Li Kun Xu Simin Shao Chonghua Ren Zhongtian Liu Zehui Wei Zhiying Zhang Efficient Genome Editing in Chicken DF-1 Cells Using the CRISPR/Cas9 System G3: Genes, Genomes, Genetics CRISPR/Cas9 system surrogate reporter chicken DF-1 cell mutant efficiency off-target GenPred Shared data resource Genomic Selection |
| title | Efficient Genome Editing in Chicken DF-1 Cells Using the CRISPR/Cas9 System |
| title_full | Efficient Genome Editing in Chicken DF-1 Cells Using the CRISPR/Cas9 System |
| title_fullStr | Efficient Genome Editing in Chicken DF-1 Cells Using the CRISPR/Cas9 System |
| title_full_unstemmed | Efficient Genome Editing in Chicken DF-1 Cells Using the CRISPR/Cas9 System |
| title_short | Efficient Genome Editing in Chicken DF-1 Cells Using the CRISPR/Cas9 System |
| title_sort | efficient genome editing in chicken df 1 cells using the crispr cas9 system |
| topic | CRISPR/Cas9 system surrogate reporter chicken DF-1 cell mutant efficiency off-target GenPred Shared data resource Genomic Selection |
| url | http://g3journal.org/lookup/doi/10.1534/g3.116.027706 |
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