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

The recent derivation of human trophoblast stem cells (hTSCs) provides a scalable in vitro model system of human placental development, but the molecular regulators of hTSC identity have not been systematically explored thus far. Here, we utilize a genome-wide CRISPR-Cas9 knockout screen to comprehensively identify essential and growth-restricting genes in hTSCs. By cross-referencing our data to those from similar genetic screens performed in other cell types, as well as gene expression data from early human embryos, we define hTSC-specific and -enriched regulators. These include both well-established and previously uncharacterized trophoblast regulators, such as ARID3A, GATA2, and TEAD1 (essential), and GCM1, PTPN14, and TET2 (growth-restricting). Integrated analysis of chromatin accessibility, gene expression, and genome-wide location data reveals that the transcription factor TEAD1 regulates the expression of many trophoblast regulators in hTSCs. In the absence of TEAD1, hTSCs fail to complete faithful differentiation into extravillous trophoblast (EVT) cells and instead show a bias towards syncytiotrophoblast (STB) differentiation, thus indicating that this transcription factor safeguards the bipotent lineage potential of hTSCs. Overall, our study provides a valuable resource for dissecting the molecular regulation of human placental development and diseases.


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Life sciences study design
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Sample size For CRISPR screen, RNA-seq, and ATAC-seq experiments, two replicates were used per sample; for TEAD1 CUT & Tag experiments, three replicates were used. These sample sizes are chosen because they are sufficient to assess reproducibility for the experiment in question. No statistical method was used to determine the sample size. For all other experiments in this study, no statistical method was used to determine the sample size, and the sample sizes are chosen because they provide sufficient confidence to assess the experimental results.
Data exclusions No data was excluded.

Replication
To ensure reproducibility, the CRISPR screen and the hTSC derivation experiments were conducted twice independently; the EVT and STB differentiation experiments were conducted at least three times independently. All other results reported in this study contained at least technical replicates. All replications yielded similar results and were successful.
Randomization No randomized experiment was performed.

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The BT5 hTSCs, K562 cells, and HEK293T cells were not authenticated; the H9 primed hPSCs were authenticated using STR profiling.

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