Transcriptomic profiling of interferon responses induced by IVT sgRNAs in CRISPRi HEK293T cells

In this study, we investigated the transcriptional responses induced by in vitro–transcribed (IVT) sgRNAs in a CRISPR interference (CRISPRi) system. A total of 906 sgRNAs were generated using a commercially available IVT workflow and delivered to HEK293T cells stably expressing dCas9-KRAB-MECP2. RNA sequencing was performed to profile genome-wide transcriptional changes following sgRNA transfection. Compared with mock-transfected controls, sgRNA-transfected cells exhibited a strong and consistent upregulation of interferon-stimulated genes (ISGs), with prominent enrichment of interferon-α response pathways across the majority of sgRNAs. While most sgRNAs induced robust interferon signaling, a small subset showed a more limited transcriptional response. IFIT1 and IFIT2, two canonical markers of interferon activation, were consistently upregulated and were selected as representative genes for downstream validation. This dataset provides a systematic resource for assessing innate immune activation triggered by IVT sgRNAs in CRISPRi-based perturbation experiments.