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SgRNA Expression of CRIPSR-Cas9 System Based on MiRNA Polycistrons as a Versatile Tool to Manipulate Multiple and Tissue-Specific Genome Editing
CRISPR/Cas9-mediated genome editing is a next-generation strategy for genetic modifications. Typically, sgRNA is constitutively expressed relying on RNA polymerase III promoters. Polymerase II promoters initiate transcription in a flexible manner, but sgRNAs generated by RNA polymerase II promoter l...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5517485/ https://www.ncbi.nlm.nih.gov/pubmed/28724960 http://dx.doi.org/10.1038/s41598-017-06216-w |
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author | Xie, Chen Chen, Yan-Lian Wang, Dong-Fang Wang, Yi-Lin Zhang, Tian-Peng Li, Hui Liang, Fu Zhao, Yong Zhang, Guang-Ya |
author_facet | Xie, Chen Chen, Yan-Lian Wang, Dong-Fang Wang, Yi-Lin Zhang, Tian-Peng Li, Hui Liang, Fu Zhao, Yong Zhang, Guang-Ya |
author_sort | Xie, Chen |
collection | PubMed |
description | CRISPR/Cas9-mediated genome editing is a next-generation strategy for genetic modifications. Typically, sgRNA is constitutively expressed relying on RNA polymerase III promoters. Polymerase II promoters initiate transcription in a flexible manner, but sgRNAs generated by RNA polymerase II promoter lost their nuclease activity. To express sgRNAs in a tissue-specific fashion and endow CRISPR with more versatile function, a novel system was established in a polycistron, where miRNAs (or shRNAs) and sgRNAs alternately emerged and co-expressed under the control of a single polymerase II promoter. Effective expression and further processing of functional miRNAs and sgRNAs were achieved. The redundant nucleotides adjacent to sgRNA were degraded, and 5′- cap structure was responsible for the compromised nuclease capacity of sgRNA: Cas9 complex. Furthermore, this strategy fulfilled conducting multiplex genome editing, as well as executing neural- specific genome editing and enhancing the proportion of homologous recombination via inhibiting NHEJ pathway by shRNA. In summary, we designed a new construction for efficient expression of sgRNAs with miRNAs (shRNAs) by virtue of RNA polymerase II promoters, which will spur the development of safer, more controllable/regulable and powerful CRISPR/Cas9 system-mediated genome editing in a wide variety of further biomedical applications. |
format | Online Article Text |
id | pubmed-5517485 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-55174852017-07-20 SgRNA Expression of CRIPSR-Cas9 System Based on MiRNA Polycistrons as a Versatile Tool to Manipulate Multiple and Tissue-Specific Genome Editing Xie, Chen Chen, Yan-Lian Wang, Dong-Fang Wang, Yi-Lin Zhang, Tian-Peng Li, Hui Liang, Fu Zhao, Yong Zhang, Guang-Ya Sci Rep Article CRISPR/Cas9-mediated genome editing is a next-generation strategy for genetic modifications. Typically, sgRNA is constitutively expressed relying on RNA polymerase III promoters. Polymerase II promoters initiate transcription in a flexible manner, but sgRNAs generated by RNA polymerase II promoter lost their nuclease activity. To express sgRNAs in a tissue-specific fashion and endow CRISPR with more versatile function, a novel system was established in a polycistron, where miRNAs (or shRNAs) and sgRNAs alternately emerged and co-expressed under the control of a single polymerase II promoter. Effective expression and further processing of functional miRNAs and sgRNAs were achieved. The redundant nucleotides adjacent to sgRNA were degraded, and 5′- cap structure was responsible for the compromised nuclease capacity of sgRNA: Cas9 complex. Furthermore, this strategy fulfilled conducting multiplex genome editing, as well as executing neural- specific genome editing and enhancing the proportion of homologous recombination via inhibiting NHEJ pathway by shRNA. In summary, we designed a new construction for efficient expression of sgRNAs with miRNAs (shRNAs) by virtue of RNA polymerase II promoters, which will spur the development of safer, more controllable/regulable and powerful CRISPR/Cas9 system-mediated genome editing in a wide variety of further biomedical applications. Nature Publishing Group UK 2017-07-19 /pmc/articles/PMC5517485/ /pubmed/28724960 http://dx.doi.org/10.1038/s41598-017-06216-w Text en © The Author(s) 2017 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Article Xie, Chen Chen, Yan-Lian Wang, Dong-Fang Wang, Yi-Lin Zhang, Tian-Peng Li, Hui Liang, Fu Zhao, Yong Zhang, Guang-Ya SgRNA Expression of CRIPSR-Cas9 System Based on MiRNA Polycistrons as a Versatile Tool to Manipulate Multiple and Tissue-Specific Genome Editing |
title | SgRNA Expression of CRIPSR-Cas9 System Based on MiRNA Polycistrons as a Versatile Tool to Manipulate Multiple and Tissue-Specific Genome Editing |
title_full | SgRNA Expression of CRIPSR-Cas9 System Based on MiRNA Polycistrons as a Versatile Tool to Manipulate Multiple and Tissue-Specific Genome Editing |
title_fullStr | SgRNA Expression of CRIPSR-Cas9 System Based on MiRNA Polycistrons as a Versatile Tool to Manipulate Multiple and Tissue-Specific Genome Editing |
title_full_unstemmed | SgRNA Expression of CRIPSR-Cas9 System Based on MiRNA Polycistrons as a Versatile Tool to Manipulate Multiple and Tissue-Specific Genome Editing |
title_short | SgRNA Expression of CRIPSR-Cas9 System Based on MiRNA Polycistrons as a Versatile Tool to Manipulate Multiple and Tissue-Specific Genome Editing |
title_sort | sgrna expression of cripsr-cas9 system based on mirna polycistrons as a versatile tool to manipulate multiple and tissue-specific genome editing |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5517485/ https://www.ncbi.nlm.nih.gov/pubmed/28724960 http://dx.doi.org/10.1038/s41598-017-06216-w |
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