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bioRxiv · 10.1101/722348

CRISPR/Cas9-mediated integration of large transgene into pig potential safe harbor

Li, G.·Zhang, X.·Wang, H.·Mo, J.·Zhong, C.·Shi, J.·Zhou, R.·Li, Z.·Yang, H.·Fang, W. Z.·Wu, L. D.
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Abstract

Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (Cas9) is a precise genome manipulating tool which can produce targeted gene mutations in various cells and organisms. Although CRISPR/Cas9 can efficiently generate gene knock-out, the gene knock-in efficiency mediated by homology-directed repair (HDR) remains low, especially for large fragment integration. In this study, we established an efficient method for CRISPR/Cas9-mediated integration of large transgene cassette carrying salivary gland-expressing multiple digestion enzymes ({approx} 20 kbp) in CEP112 locus in pig fetal fibroblasts. Our results showed that using homologous donor with a short left arm and a long right arm yielded the best CRISPR/Cas9-mediated knock-in efficiency, and the targeting efficiency in potential safe harbor CEP112 locus are higher than ROSA26 locus. The CEP112 knock-in cell lines were used as nuclear donors for somatic cell nuclear transfer to create genetically modified pigs. We found that knock-in pig (705) successfully expressed three microbial enzymes ({beta}-glucanase, xylanase, and phytase) in salivary gland, suggesting potential safe harbor CEP112 locus supports exogenous genes expression by a tissue-specific promoter. In summary, we successfully targeted CEP112 locus using our optimal homology arm system for precise modification of pigs, and established a modified pig model for foreign digestion enzyme expression in saliva.

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Li, G., Zhang, X., Wang, H., Mo, J., Zhong, C., Shi, J., Zhou, R., Li, Z., Yang, H., Fang, W. Z., Wu, L. D.. 2019-08-01. CRISPR/Cas9-mediated integration of large transgene into pig potential safe harbor. https://doi.org/10.1101/722348

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