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

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