CRISPR/Cas9-Mediated Hitchhike Expression of Functional shRNAs at the Porcine <i>miR-17-92</i> Cluster

Chao Lu; Daxin Pang; Mengjing Li; Hongming Yuan; Tingting Yu; Peixuan Huang; Jianing Li; Xue Chen; Huping Jiao; Zicong Xie; Hongsheng Ouyang

doi:10.3390/cells8020113

Cells (Feb 2019)

CRISPR/Cas9-Mediated Hitchhike Expression of Functional shRNAs at the Porcine <i>miR-17-92</i> Cluster

Chao Lu,
Daxin Pang,
Mengjing Li,
Hongming Yuan,
Tingting Yu,
Peixuan Huang,
Jianing Li,
Xue Chen,
Huping Jiao,
Zicong Xie,
Hongsheng Ouyang

Affiliations

Chao Lu: Jilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Sciences, Jilin University, Changchun 130062, China
Daxin Pang: Jilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Sciences, Jilin University, Changchun 130062, China
Mengjing Li: Jilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Sciences, Jilin University, Changchun 130062, China
Hongming Yuan: Jilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Sciences, Jilin University, Changchun 130062, China
Tingting Yu: Jilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Sciences, Jilin University, Changchun 130062, China
Peixuan Huang: Jilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Sciences, Jilin University, Changchun 130062, China
Jianing Li: Jilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Sciences, Jilin University, Changchun 130062, China
Xue Chen: Jilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Sciences, Jilin University, Changchun 130062, China
Huping Jiao: Jilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Sciences, Jilin University, Changchun 130062, China
Zicong Xie: Jilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Sciences, Jilin University, Changchun 130062, China
Hongsheng Ouyang: Jilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Sciences, Jilin University, Changchun 130062, China

DOI: https://doi.org/10.3390/cells8020113
Journal volume & issue: Vol. 8, no. 2
p. 113

Abstract

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Successful RNAi applications depend on strategies allowing stable and persistent expression of minimal gene silencing triggers without perturbing endogenous gene expression. In this study, we proposed an endogenous microRNA (miRNA) cluster as a novel integration site for small hairpin RNAs (shRNAs). We successfully integrated exogenous shRNAs at the porcine miRNA-17-92 (pmiR-17-92) cluster via a CRISPR/Cas9-mediated knock-in strategy. The anti-EGFP or anti-CSFV shRNAs could be stably and effectively expressed at the control of the endogenous promoter of the pmiR-17-92 cluster. Importantly, we confirmed that hitchhike expression of anti- classical swine fever (CSFV) shRNA had no effect on cell growth, blastocyst development and endogenous pmiR-17-92 expression in selected transgene (TG) porcine fetal fibroblasts (PFFs) clones. Moreover, these TG PFFs could inhibit the replication of CSFV by half and could be further used for generation of transgenic pigs. Taken together, these results show that our RNA interference (RNAi) expression strategy benefits numerous applications, from miRNA, genome and transgenic research, to gene therapy.

Published in Cells

ISSN: 2073-4409 (Online)
Publisher: MDPI AG
Country of publisher: Switzerland
LCC subjects: Science: Biology (General): Cytology
Website: https://www.mdpi.com/journal/cells

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