Functional Comparison between VP64-dCas9-VP64 and dCas9-VP192 CRISPR Activators in Human Embryonic Kidney Cells
Reversal in the transcriptional status of desired genes has been exploited for multiple research, therapeutic, and biotechnological purposes. CRISPR/dCas9-based activators can activate transcriptionally silenced genes after being guided by gene-specific gRNA(s). Here, we performed a functional compa...
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MDPI AG
2021-01-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/22/1/397 |
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| author | Nasir Javaid Thuong L. H. Pham Sangdun Choi |
| author_facet | Nasir Javaid Thuong L. H. Pham Sangdun Choi |
| author_sort | Nasir Javaid |
| collection | DOAJ |
| description | Reversal in the transcriptional status of desired genes has been exploited for multiple research, therapeutic, and biotechnological purposes. CRISPR/dCas9-based activators can activate transcriptionally silenced genes after being guided by gene-specific gRNA(s). Here, we performed a functional comparison between two such activators, VP64-dCas9-VP64 and dCas9-VP192, in human embryonic kidney cells by the concomitant targeting of <i>POU5F1</i> and <i>SOX2</i>. We found 22- and 6-fold upregulations in the mRNA level of <i>POU5F1</i> by dCas9-VP192 and VP64-dCas9-VP64, respectively. Likewise, <i>SOX2</i> was up-regulated 4- and 2-fold using dCas9-VP192 and VP64dCas9VP64, respectively. For the POU5F1 protein level, we observed 3.7- and 2.2-fold increases with dCas9-VP192 and VP64-dCas9-VP64, respectively. Similarly, the SOX2 expression was 2.4- and 2-fold higher with dCas9-VP192 and VP64-dCas9-VP64, respectively. We also confirmed that activation only happened upon co-transfecting an activator plasmid with multiplex gRNA plasmid with a high specificity to the reference genes. Our data revealed that dCas9-VP192 is more efficient than VP64-dCas9-VP64 for activating reference genes. |
| first_indexed | 2024-03-10T13:32:53Z |
| format | Article |
| id | doaj.art-60486c25cda64cacbfb80bb2bc062ea8 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T13:32:53Z |
| publishDate | 2021-01-01 |
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| series | International Journal of Molecular Sciences |
| spelling | doaj.art-60486c25cda64cacbfb80bb2bc062ea82023-11-21T07:44:34ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-01-0122139710.3390/ijms22010397Functional Comparison between VP64-dCas9-VP64 and dCas9-VP192 CRISPR Activators in Human Embryonic Kidney CellsNasir Javaid0Thuong L. H. Pham1Sangdun Choi2Department of Molecular Science and Technology, Ajou University, Suwon 16499, KoreaDepartment of Molecular Science and Technology, Ajou University, Suwon 16499, KoreaDepartment of Molecular Science and Technology, Ajou University, Suwon 16499, KoreaReversal in the transcriptional status of desired genes has been exploited for multiple research, therapeutic, and biotechnological purposes. CRISPR/dCas9-based activators can activate transcriptionally silenced genes after being guided by gene-specific gRNA(s). Here, we performed a functional comparison between two such activators, VP64-dCas9-VP64 and dCas9-VP192, in human embryonic kidney cells by the concomitant targeting of <i>POU5F1</i> and <i>SOX2</i>. We found 22- and 6-fold upregulations in the mRNA level of <i>POU5F1</i> by dCas9-VP192 and VP64-dCas9-VP64, respectively. Likewise, <i>SOX2</i> was up-regulated 4- and 2-fold using dCas9-VP192 and VP64dCas9VP64, respectively. For the POU5F1 protein level, we observed 3.7- and 2.2-fold increases with dCas9-VP192 and VP64-dCas9-VP64, respectively. Similarly, the SOX2 expression was 2.4- and 2-fold higher with dCas9-VP192 and VP64-dCas9-VP64, respectively. We also confirmed that activation only happened upon co-transfecting an activator plasmid with multiplex gRNA plasmid with a high specificity to the reference genes. Our data revealed that dCas9-VP192 is more efficient than VP64-dCas9-VP64 for activating reference genes.https://www.mdpi.com/1422-0067/22/1/397CRISPR/dCas9gene activationreprogramming factorCRISPR activator |
| spellingShingle | Nasir Javaid Thuong L. H. Pham Sangdun Choi Functional Comparison between VP64-dCas9-VP64 and dCas9-VP192 CRISPR Activators in Human Embryonic Kidney Cells International Journal of Molecular Sciences CRISPR/dCas9 gene activation reprogramming factor CRISPR activator |
| title | Functional Comparison between VP64-dCas9-VP64 and dCas9-VP192 CRISPR Activators in Human Embryonic Kidney Cells |
| title_full | Functional Comparison between VP64-dCas9-VP64 and dCas9-VP192 CRISPR Activators in Human Embryonic Kidney Cells |
| title_fullStr | Functional Comparison between VP64-dCas9-VP64 and dCas9-VP192 CRISPR Activators in Human Embryonic Kidney Cells |
| title_full_unstemmed | Functional Comparison between VP64-dCas9-VP64 and dCas9-VP192 CRISPR Activators in Human Embryonic Kidney Cells |
| title_short | Functional Comparison between VP64-dCas9-VP64 and dCas9-VP192 CRISPR Activators in Human Embryonic Kidney Cells |
| title_sort | functional comparison between vp64 dcas9 vp64 and dcas9 vp192 crispr activators in human embryonic kidney cells |
| topic | CRISPR/dCas9 gene activation reprogramming factor CRISPR activator |
| url | https://www.mdpi.com/1422-0067/22/1/397 |
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