RamDA-seq analysis of CRISPRa-mediated RELA activation for proof-of-concept validation of CATaN

To test whether CATaN correctly recovers the transcriptional program driven by a specific TF, we activated RELA, a subunit of NF-κB, in Jurkat cells using CRISPR activation (CRISPRa; dCas9-VP64 with a single guide RNA targeting RELA) and compared it with a control guide RNA.

Transcriptomes were profiled by RamDA-seq. Transductions were performed in triplicate for each condition, and cells were harvested at five, six, and seven days post-transduction. Differential expression analysis showed concordant directional changes in RELA-regulated genes; CATaN applied to these data assigned higher CC1 loadings to RELA ChIP-seq samples than to other TFs.