CRISPR/Cas9-Mediated Hitchhike Expression of Functional shRNAs at the Porcine <i>miR-17-92</i> Cluster
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)....
Main Authors: | , , , , , , , , , , |
---|---|
Format: | Article |
Language: | English |
Published: |
MDPI AG
2019-02-01
|
Series: | Cells |
Subjects: | |
Online Access: | https://www.mdpi.com/2073-4409/8/2/113 |
id |
doaj-303c8ffd7ed2494fa6093a90102b7aca |
---|---|
record_format |
Article |
spelling |
doaj-303c8ffd7ed2494fa6093a90102b7aca2020-11-24T21:59:53ZengMDPI AGCells2073-44092019-02-018211310.3390/cells8020113cells8020113CRISPR/Cas9-Mediated Hitchhike Expression of Functional shRNAs at the Porcine <i>miR-17-92</i> ClusterChao Lu0Daxin Pang1Mengjing Li2Hongming Yuan3Tingting Yu4Peixuan Huang5Jianing Li6Xue Chen7Huping Jiao8Zicong Xie9Hongsheng Ouyang10Jilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Sciences, Jilin University, Changchun 130062, ChinaJilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Sciences, Jilin University, Changchun 130062, ChinaJilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Sciences, Jilin University, Changchun 130062, ChinaJilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Sciences, Jilin University, Changchun 130062, ChinaJilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Sciences, Jilin University, Changchun 130062, ChinaJilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Sciences, Jilin University, Changchun 130062, ChinaJilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Sciences, Jilin University, Changchun 130062, ChinaJilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Sciences, Jilin University, Changchun 130062, ChinaJilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Sciences, Jilin University, Changchun 130062, ChinaJilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Sciences, Jilin University, Changchun 130062, ChinaJilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Sciences, Jilin University, Changchun 130062, ChinaSuccessful 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 <i>miRNA-17-92</i> (p<i>miR-17-92</i>) 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 p<i>miR-17-92</i> cluster. Importantly, we confirmed that hitchhike expression of anti- classical swine fever (CSFV) shRNA had no effect on cell growth, blastocyst development and endogenous p<i>miR-17-92</i> 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.https://www.mdpi.com/2073-4409/8/2/113RNAishRNAmiRNA-17-92 clusterCRISPR/Cas9classical swine fever virus |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Chao Lu Daxin Pang Mengjing Li Hongming Yuan Tingting Yu Peixuan Huang Jianing Li Xue Chen Huping Jiao Zicong Xie Hongsheng Ouyang |
spellingShingle |
Chao Lu Daxin Pang Mengjing Li Hongming Yuan Tingting Yu Peixuan Huang Jianing Li Xue Chen Huping Jiao Zicong Xie Hongsheng Ouyang CRISPR/Cas9-Mediated Hitchhike Expression of Functional shRNAs at the Porcine <i>miR-17-92</i> Cluster Cells RNAi shRNA miRNA-17-92 cluster CRISPR/Cas9 classical swine fever virus |
author_facet |
Chao Lu Daxin Pang Mengjing Li Hongming Yuan Tingting Yu Peixuan Huang Jianing Li Xue Chen Huping Jiao Zicong Xie Hongsheng Ouyang |
author_sort |
Chao Lu |
title |
CRISPR/Cas9-Mediated Hitchhike Expression of Functional shRNAs at the Porcine <i>miR-17-92</i> Cluster |
title_short |
CRISPR/Cas9-Mediated Hitchhike Expression of Functional shRNAs at the Porcine <i>miR-17-92</i> Cluster |
title_full |
CRISPR/Cas9-Mediated Hitchhike Expression of Functional shRNAs at the Porcine <i>miR-17-92</i> Cluster |
title_fullStr |
CRISPR/Cas9-Mediated Hitchhike Expression of Functional shRNAs at the Porcine <i>miR-17-92</i> Cluster |
title_full_unstemmed |
CRISPR/Cas9-Mediated Hitchhike Expression of Functional shRNAs at the Porcine <i>miR-17-92</i> Cluster |
title_sort |
crispr/cas9-mediated hitchhike expression of functional shrnas at the porcine <i>mir-17-92</i> cluster |
publisher |
MDPI AG |
series |
Cells |
issn |
2073-4409 |
publishDate |
2019-02-01 |
description |
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 <i>miRNA-17-92</i> (p<i>miR-17-92</i>) 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 p<i>miR-17-92</i> cluster. Importantly, we confirmed that hitchhike expression of anti- classical swine fever (CSFV) shRNA had no effect on cell growth, blastocyst development and endogenous p<i>miR-17-92</i> 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. |
topic |
RNAi shRNA miRNA-17-92 cluster CRISPR/Cas9 classical swine fever virus |
url |
https://www.mdpi.com/2073-4409/8/2/113 |
work_keys_str_mv |
AT chaolu crisprcas9mediatedhitchhikeexpressionoffunctionalshrnasattheporcineimir1792icluster AT daxinpang crisprcas9mediatedhitchhikeexpressionoffunctionalshrnasattheporcineimir1792icluster AT mengjingli crisprcas9mediatedhitchhikeexpressionoffunctionalshrnasattheporcineimir1792icluster AT hongmingyuan crisprcas9mediatedhitchhikeexpressionoffunctionalshrnasattheporcineimir1792icluster AT tingtingyu crisprcas9mediatedhitchhikeexpressionoffunctionalshrnasattheporcineimir1792icluster AT peixuanhuang crisprcas9mediatedhitchhikeexpressionoffunctionalshrnasattheporcineimir1792icluster AT jianingli crisprcas9mediatedhitchhikeexpressionoffunctionalshrnasattheporcineimir1792icluster AT xuechen crisprcas9mediatedhitchhikeexpressionoffunctionalshrnasattheporcineimir1792icluster AT hupingjiao crisprcas9mediatedhitchhikeexpressionoffunctionalshrnasattheporcineimir1792icluster AT zicongxie crisprcas9mediatedhitchhikeexpressionoffunctionalshrnasattheporcineimir1792icluster AT hongshengouyang crisprcas9mediatedhitchhikeexpressionoffunctionalshrnasattheporcineimir1792icluster |
_version_ |
1725846697018392576 |