A genome-wide CRISPR-Cas9 knockout screen identifies essential and growth-restricting genes in human trophoblast stem cells. A genome-wide CRISPR-Cas9 knockout screen identifies essential and growth-restricting genes in human trophoblast stem cells

The recent derivation of bona fide human trophoblast stem cells (hTSCs) significantly improved our ability to study human placental biology and pathologies, but few studies have investigated the molecular regulators of hTSC identity. Therefore, we utilized a genome-wide CRISPR-Cas9 knockout screen to comprehensively define genes essential for or restricting the fitness of hTSCs. The resulting essential and growth-restricting genes include both well-established and potentially novel trophoblast regulators. We referenced our data to those of similar genetic screens performed in cancer cell lines and primed human pluripotent stem cells (hPSCs), as well as gene expression data of the early human embryos, to identify potential hTSC-specific and -enriched regulators. Among those are TEAD1, a gene previously reported to be dispensable for mouse placentation6. However, in the human trophoblast context, TEAD1 targets many hTSC regulators and plays a major role in its specification and maintenance. Overall, our study presents the first CRISPR/Cas9 knockout screen in a human extraembryonic lineage and provides a valuable resource for future trophoblast research. Overall design: CRISPR screening in hTSCs TEAD1 CUT&Tag in hTSCs ATAC-seq in naïve hPSCs

近期，纯正人类滋养层干细胞（human trophoblast stem cells, hTSCs）的成功建立，极大提升了我们研究人类胎盘生物学与病理特征的能力，但目前鲜有研究探索维持hTSC身份特征的分子调控因子。为此，我们采用全基因组CRISPR-Cas9敲除筛选技术，全面鉴定对hTSC存活至关重要或限制其增殖活力的基因。最终筛选得到的关键基因与增殖限制基因，既包含已被证实的滋养层调控因子，也涵盖潜在的新型调控靶点。我们将本研究数据与此前在癌细胞系、激活态人多能干细胞（primed human pluripotent stem cells, hPSCs）中开展的同类遗传筛选数据，以及早期人类胚胎的基因表达数据进行比对分析，以筛选出hTSC特异性及富集型调控因子。其中TEAD1基因此前被报道对小鼠胎盘形成非必需6，但在人类滋养层系统中，TEAD1可靶向调控诸多hTSC相关调控因子，并在hTSC的特化与维持过程中发挥核心作用。总体而言，本研究首次在人类胚外细胞谱系中开展CRISPR/Cas9敲除筛选，为后续滋养层研究提供了极具价值的参考资源。实验设计：在hTSCs中开展CRISPR筛选；在hTSCs中开展TEAD1的CUT&Tag实验；在初始态人多能干细胞（naïve hPSCs）中开展ATAC-seq实验