CRISPR Screening Reveals a Novel Role for FOXH1 in Regulating Pluripotency of Porcine Embryonic Stem Cells

pESCs hold significant potential for applications in regenerative medicine and stem cell-based breeding programs. However, the regulatory mechanisms governing pluripotency in pESCs, including aspects of cellular adaptability and the maintenance of a pluripotent identity, are not yet fully understood.To distinguish these two features of pESCs, we conducted two types of CRISPR-Cas9 knockout screenings targeting all known transcription factors and epigenetic regulators in the porcine genome. Through the CRISPR-Cas9 screenings, we identified 900 essential genes, including the species-conserved gene PRMT1 and the pig-specific gene NASP, which were experimentally validated for their roles in preserving the survival of pESCs. In addition, we found that FOXH1 is indispensable for maintaining pESCs pluripotency. pESCs deficient in FOXH1 displayed a flatter and more dispersed clonal morphology, accompanied by downregulation of pluripotency genes and upregulation of lineage-specific genes. In parallel, FOXH1 knockdown significantly impaired blastocyst formation during early pig embryogenesis, demonstrating an unrecognized role of FOXH1 that is not conserved in hESCs. At the molecular level, FOXH1 exhibits a dual role by regulating chromatin accessibility at pluripotency gene loci and H3K4me3 modifications at the promoters of developmental genes. The resources developed through this study not only lay the groundwork for more extensive CRISPR screenings but also provide crucial theoretical support for the establishment of pPSC cell lines with higher differentiation potential. This study employs multiple high-throughput sequencing technologies, including NGS, ATAC-seq, ChIP-seq, and RNA-seq, to comprehensively investigate the dynamics of sgRNA loss or enrichment during the library screening process, and to assess the effects of FOXH1 knockout on chromatin accessibility, transcription factor binding, and gene expression in pEPSCs. The dataset comprises samples from cell mutation libraries across different generations and pEPSCs under a variety of conditions, such as wild-type and gene knockout (e.g., FOXH1).

猪胚胎干细胞（porcine Embryonic Stem Cells，pESCs）在再生医学及基于干细胞的育种项目中具备重大应用潜力。然而，调控猪胚胎干细胞多能性的分子机制，包括细胞适应性与多能身份维持等维度，尚未得到完全阐明。为区分猪胚胎干细胞的这两项核心特征，我们开展了两类CRISPR-Cas9基因敲除筛选，靶向覆盖猪基因组中所有已注释的转录因子与表观遗传调控因子。通过本次CRISPR-Cas9筛选，我们共鉴定出900个必需基因，其中包含物种保守基因PRMT1与猪特异性基因NASP，二者在维持猪胚胎干细胞存活中的功能已通过实验验证。此外，我们发现FOXH1对维持猪胚胎干细胞多能性不可或缺：FOXH1缺陷的猪胚胎干细胞呈现出更扁平、更分散的克隆形态，同时伴随多能性基因表达下调及谱系特异性基因表达上调。与此同时，FOXH1敲除显著抑制了猪早期胚胎发生过程中的囊胚形成，揭示了FOXH1存在未被认知的、在人类胚胎干细胞中不保守的生物学功能。在分子层面，FOXH1发挥双重调控作用：既可调控多能性基因位点的染色质开放性，又可修饰发育基因启动子区域的H3K4me3组蛋白修饰。本研究构建的研究资源不仅为更广泛的CRISPR筛选奠定了坚实基础，也为建立具有更高分化潜能的猪多能干细胞（porcine Pluripotent Stem Cells，pPSCs）细胞系提供了关键理论支撑。本研究采用多种高通量测序技术，包括下一代测序技术（Next-Generation Sequencing，NGS）、转座酶可及性测序（Assay for Transposase-Accessible Chromatin with high-throughput Sequencing，ATAC-seq）、染色质免疫共沉淀测序（Chromatin Immunoprecipitation Sequencing，ChIP-seq）及RNA测序（RNA Sequencing，RNA-seq），全面解析了文库筛选过程中单导RNA（single guide RNA，sgRNA）的缺失与富集动态，并评估了FOXH1敲除对猪胚胎多能干细胞（porcine Embryonic Pluripotent Stem Cells，pEPSCs）染色质开放性、转录因子结合及基因表达的影响。本数据集涵盖不同世代的细胞突变文库样本，以及多种培养条件下的猪胚胎多能干细胞样本，例如野生型及基因敲除（如FOXH1敲除）样本。