Targeted knock-in into the OVA locus of chicken cells using CRISPR/Cas9 system with homology-independent targeted integration

Ming Shi, Yoshinori Kawabe, Akira Ito, Masamichi Kamihira

Research output: Contribution to journalArticle

Abstract

It is anticipated that transgenic avian species will be used as living bioreactors for the production of biopharmaceutical proteins. Precise tissue-specific expression of exogenous genes is a major challenge for the development of avian bioreactors. No robust vector is currently available for highly efficient and specific expression. In recent years, genome-editing techniques such as the CRISPR/Cas9 system have emerged as efficient and user-friendly genetic modification tools. Here, to apply the CRISPR/Cas9 system for the development of transgenic chickens, guide RNA sequences (gRNAs) of the CRISPR/Cas9 system for the ovalbumin (OVA) locus were evaluated for the oviduct-specific expression of exogenous genes. An EGFP gene expression cassette was introduced into the OVA locus of chicken DF-1 and embryonic fibroblasts using the CRISPR/Cas9 system mediated by homology-independent targeted integration. For the knock-in cells, EGFP expression was successfully induced by activation of the endogenous OVA promoter using the dCas9-VPR transactivation system. The combination of gRNAs designed around the OVA TATA box was important to induce endogenous OVA gene expression with high efficiency. These methods provide a useful tool for studies on the creation of transgenic chicken bioreactors and the activation of tissue-specific promoters.

Original languageEnglish
JournalJournal of Bioscience and Bioengineering
DOIs
Publication statusAccepted/In press - Jan 1 2019

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Clustered Regularly Interspaced Short Palindromic Repeats
Ovalbumin
Bioreactors
Guide RNA
Chickens
Genes
Gene expression
Gene Expression
Chemical activation
Tissue
Fibroblasts
RNA
TATA Box
Oviducts
Proteins
Transcriptional Activation

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

Cite this

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title = "Targeted knock-in into the OVA locus of chicken cells using CRISPR/Cas9 system with homology-independent targeted integration",
abstract = "It is anticipated that transgenic avian species will be used as living bioreactors for the production of biopharmaceutical proteins. Precise tissue-specific expression of exogenous genes is a major challenge for the development of avian bioreactors. No robust vector is currently available for highly efficient and specific expression. In recent years, genome-editing techniques such as the CRISPR/Cas9 system have emerged as efficient and user-friendly genetic modification tools. Here, to apply the CRISPR/Cas9 system for the development of transgenic chickens, guide RNA sequences (gRNAs) of the CRISPR/Cas9 system for the ovalbumin (OVA) locus were evaluated for the oviduct-specific expression of exogenous genes. An EGFP gene expression cassette was introduced into the OVA locus of chicken DF-1 and embryonic fibroblasts using the CRISPR/Cas9 system mediated by homology-independent targeted integration. For the knock-in cells, EGFP expression was successfully induced by activation of the endogenous OVA promoter using the dCas9-VPR transactivation system. The combination of gRNAs designed around the OVA TATA box was important to induce endogenous OVA gene expression with high efficiency. These methods provide a useful tool for studies on the creation of transgenic chicken bioreactors and the activation of tissue-specific promoters.",
author = "Ming Shi and Yoshinori Kawabe and Akira Ito and Masamichi Kamihira",
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AU - Shi, Ming

AU - Kawabe, Yoshinori

AU - Ito, Akira

AU - Kamihira, Masamichi

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AB - It is anticipated that transgenic avian species will be used as living bioreactors for the production of biopharmaceutical proteins. Precise tissue-specific expression of exogenous genes is a major challenge for the development of avian bioreactors. No robust vector is currently available for highly efficient and specific expression. In recent years, genome-editing techniques such as the CRISPR/Cas9 system have emerged as efficient and user-friendly genetic modification tools. Here, to apply the CRISPR/Cas9 system for the development of transgenic chickens, guide RNA sequences (gRNAs) of the CRISPR/Cas9 system for the ovalbumin (OVA) locus were evaluated for the oviduct-specific expression of exogenous genes. An EGFP gene expression cassette was introduced into the OVA locus of chicken DF-1 and embryonic fibroblasts using the CRISPR/Cas9 system mediated by homology-independent targeted integration. For the knock-in cells, EGFP expression was successfully induced by activation of the endogenous OVA promoter using the dCas9-VPR transactivation system. The combination of gRNAs designed around the OVA TATA box was important to induce endogenous OVA gene expression with high efficiency. These methods provide a useful tool for studies on the creation of transgenic chicken bioreactors and the activation of tissue-specific promoters.

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