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

<i>OCT4</i> is a critical transcription factor for early embryonic development and pluripotency. Previous studies have shown that the regulation of <i>OCT4</i> by the transcription factor <i>GATA4</i> is species-specific in pigs. This study aimed to further investigate whether there are other transcription factors that co-regulate the transcription of <i>OCT4</i> with <i>GATA4</i> 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 <i>OCT4</i> 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 <i>GATA4</i> overexpression. Flow cytometry combined with high-throughput sequencing identified <i>MYC</i>, <i>SOX2</i>, and <i>PRDM14</i> as activators and <i>OTX2</i> and <i>CDX2</i> as repressors of <i>OCT4</i>. In the presence of <i>GATA4</i>, transcription factors such as <i>SALL4</i> and <i>STAT3</i> showed synergistic activation. Functional validation confirmed that <i>HOXD13</i> upregulates <i>OCT4</i>, while <i>OTX2</i> inhibits it. <i>GATA4</i> and <i>SALL4</i> synergistically enhance <i>OCT4</i> expression. These findings provide new insights into combinatorial mechanisms that control the transcriptional regulation of <i>OCT4</i> in pigs.