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 POU5F1 and SOX2. We found 22- and 6-fold upregulations in the mRNA level of POU5F1 by dCas9-VP192 and VP64-dCas9-VP64, respectively. Likewise, SOX2 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.
Keywords: CRISPR activator; CRISPR/dCas9; gene activation; reprogramming factor.