TY - JOUR
T1 - Targeted knock-in into the OVA locus of chicken cells using CRISPR/Cas9 system with homology-independent targeted integration
AU - Shi, Ming
AU - Kawabe, Yoshinori
AU - Ito, Akira
AU - Kamihira, Masamichi
N1 - Funding Information:
This work was supported in part by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (nos. 26289316 , 16K06874 , and 17H03470 ). The authors declare no financial or commercial conflicts of interest. The authors thank Edanz for editing the English text of a draft of this manuscript.
Publisher Copyright:
© 2019 The Society for Biotechnology, Japan
PY - 2020/3
Y1 - 2020/3
N2 - 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.
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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U2 - 10.1016/j.jbiosc.2019.09.011
DO - 10.1016/j.jbiosc.2019.09.011
M3 - Article
C2 - 31594694
AN - SCOPUS:85072826999
VL - 129
SP - 363
EP - 370
JO - Journal of Bioscience and Bioengineering
JF - Journal of Bioscience and Bioengineering
SN - 1389-1723
IS - 3
ER -