Establishment of a CRISPR/dCas9 Activation Library for Screening Transcription Factors Co-Regulating OCT4 with GATA4...

OCT4 is a critical transcription factor for early embryonic development and pluripotency. Previous studies have shown that the regulation of OCT4 by the transcription factor GATA4 is species-specific in pigs. This study aimed to further investigate whether there are other transcription factors that co-regulate the transcription of OCT4 with GATA4 in pigs. A CRISPR activation (CRISPRa) sgRNA library was designed and constructed, containing 5056 sgRNAs targeting the promoter region of 1264 transcription factors in pigs. Then, a pig PK15 cell line was engineered with a single-copy OCT4 promoter-driven EGFP reporter at the ROSA26 locus, combined with the dCas9-SAM system for transcriptional activation. The CRISPRa sgRNA lentiviral library was used to screen for transcription factors, with or without GATA4 overexpression. Flow cytometry combined with high-throughput sequencing identified MYC, SOX2, and PRDM14 as activators and OTX2 and CDX2 as repressors of OCT4. In the presence of GATA4, transcription factors such as SALL4 and STAT3 showed synergistic activation. Functional validation confirmed that HOXD13 upregulates OCT4, while OTX2 inhibits it. GATA4 and SALL4 synergistically enhance OCT4 expression. These findings provide new insights into combinatorial mechanisms that control the transcriptional regulation of OCT4 in pigs.