RNA-guided transcriptional activation via CRISPR/dCas9 mimics overexpression phenotypes in Arabidopsis.
Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR associated protein 9 (Cas9) system allows effective gene modification through RNA-guided DNA targeting. The Cas9 has undergone a series of functional alterations from the original active endonuclease to partially or co...
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2017-01-01
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| Series: | PLoS ONE |
| Online Access: | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0179410&type=printable |
| _version_ | 1826578780871720960 |
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| author | Jong-Jin Park Emma Dempewolf Wenzheng Zhang Zeng-Yu Wang |
| author_facet | Jong-Jin Park Emma Dempewolf Wenzheng Zhang Zeng-Yu Wang |
| author_sort | Jong-Jin Park |
| collection | DOAJ |
| description | Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR associated protein 9 (Cas9) system allows effective gene modification through RNA-guided DNA targeting. The Cas9 has undergone a series of functional alterations from the original active endonuclease to partially or completely deactivated Cas9. The catalytically deactivated Cas9 (dCas9) offers a platform to regulate transcriptional expression with the addition of activator or repressor domains. We redesigned a CRISPR/Cas9 activation system by adding the p65 transactivating subunit of NF-kappa B and a heat-shock factor 1 (HSF) activation domain to dCas9 bound with the VP64 (tetramer of VP16) activation domain for application in plants. The redesigned CRISPR/Cas9 activation system was tested in Arabidopsis to increase endogenous transcriptional levels of production of anthocyanin pigment 1 (PAP1) and Arabidopsis thaliana vacuolar H+-pyrophosphatase (AVP1). The expression of PAP1 was increased two- to three-fold and the activated plants exhibited purple leaves similar to that of PAP1 overexpressors. The AVP1 gene expression was increased two- to five-fold in transgenic plants. In comparison to the wild type, AVP1 activated plants had increased leaf numbers, larger single-leaf areas and improved tolerance to drought stress. The AVP1 activated plants showed similar phenotypes to AVP1 overexpressors. Therefore, the redesigned CRISPR/Cas9 activation system containing modified p65-HSF provides a simple approach for producing activated plants by upregulating endogenous transcriptional levels. |
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| id | doaj.art-cdacdd8c83d44038a5a457e9fc156cae |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2025-03-14T14:07:50Z |
| publishDate | 2017-01-01 |
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| series | PLoS ONE |
| spelling | doaj.art-cdacdd8c83d44038a5a457e9fc156cae2025-02-27T05:38:45ZengPublic Library of Science (PLoS)PLoS ONE1932-62032017-01-01126e017941010.1371/journal.pone.0179410RNA-guided transcriptional activation via CRISPR/dCas9 mimics overexpression phenotypes in Arabidopsis.Jong-Jin ParkEmma DempewolfWenzheng ZhangZeng-Yu WangClustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR associated protein 9 (Cas9) system allows effective gene modification through RNA-guided DNA targeting. The Cas9 has undergone a series of functional alterations from the original active endonuclease to partially or completely deactivated Cas9. The catalytically deactivated Cas9 (dCas9) offers a platform to regulate transcriptional expression with the addition of activator or repressor domains. We redesigned a CRISPR/Cas9 activation system by adding the p65 transactivating subunit of NF-kappa B and a heat-shock factor 1 (HSF) activation domain to dCas9 bound with the VP64 (tetramer of VP16) activation domain for application in plants. The redesigned CRISPR/Cas9 activation system was tested in Arabidopsis to increase endogenous transcriptional levels of production of anthocyanin pigment 1 (PAP1) and Arabidopsis thaliana vacuolar H+-pyrophosphatase (AVP1). The expression of PAP1 was increased two- to three-fold and the activated plants exhibited purple leaves similar to that of PAP1 overexpressors. The AVP1 gene expression was increased two- to five-fold in transgenic plants. In comparison to the wild type, AVP1 activated plants had increased leaf numbers, larger single-leaf areas and improved tolerance to drought stress. The AVP1 activated plants showed similar phenotypes to AVP1 overexpressors. Therefore, the redesigned CRISPR/Cas9 activation system containing modified p65-HSF provides a simple approach for producing activated plants by upregulating endogenous transcriptional levels.https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0179410&type=printable |
| spellingShingle | Jong-Jin Park Emma Dempewolf Wenzheng Zhang Zeng-Yu Wang RNA-guided transcriptional activation via CRISPR/dCas9 mimics overexpression phenotypes in Arabidopsis. PLoS ONE |
| title | RNA-guided transcriptional activation via CRISPR/dCas9 mimics overexpression phenotypes in Arabidopsis. |
| title_full | RNA-guided transcriptional activation via CRISPR/dCas9 mimics overexpression phenotypes in Arabidopsis. |
| title_fullStr | RNA-guided transcriptional activation via CRISPR/dCas9 mimics overexpression phenotypes in Arabidopsis. |
| title_full_unstemmed | RNA-guided transcriptional activation via CRISPR/dCas9 mimics overexpression phenotypes in Arabidopsis. |
| title_short | RNA-guided transcriptional activation via CRISPR/dCas9 mimics overexpression phenotypes in Arabidopsis. |
| title_sort | rna guided transcriptional activation via crispr dcas9 mimics overexpression phenotypes in arabidopsis |
| url | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0179410&type=printable |
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