Department of Molecular Science and Technology, Ajou University, Suwon 16499, Korea.
S&K Therapeutics, Woncheon Hall 135, Ajou University, Suwon 16499, Korea.
Int J Mol Sci. 2021 Jan 1;22(1):397. doi: 10.3390/ijms22010397.
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 and . We found 22- and 6-fold upregulations in the mRNA level of by dCas9-VP192 and VP64-dCas9-VP64, respectively. Likewise, 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.
逆转所需基因的转录状态已被用于多种研究、治疗和生物技术目的。基于 CRISPR/dCas9 的激活子可以在被基因特异性 gRNA 引导后激活转录沉默的基因。在这里，我们通过同时靶向 和 ，在人胚肾细胞中对两种这样的激活子 VP64-dCas9-VP64 和 dCas9-VP192 进行了功能比较。我们发现 dCas9-VP192 和 VP64-dCas9-VP64 分别使 的 mRNA 水平上调了 22 倍和 6 倍。同样，使用 dCas9-VP192 和 VP64-dCas9-VP64 ， 分别上调了 4 倍和 2 倍。对于 POU5F1 蛋白水平，我们观察到 dCas9-VP192 和 VP64-dCas9-VP64 分别增加了 3.7 倍和 2.2 倍。同样，SOX2 的表达分别提高了 2.4 倍和 2 倍。dCas9-VP192 和 VP64-dCas9-VP64。我们还证实，只有在共转染激活子质粒和具有高特异性的多 gRNA 质粒时，才会发生激活，以参考基因。我们的数据表明，dCas9-VP192 比 VP64-dCas9-VP64 更有效地激活参考基因。
