Fig. 3

CRISPR screening identifies key Etv2 upstream regulators. a Scheme for CRISPR screening. b FACS plot of the Etv2^high and PDGFRα⁺ cells from D3.5 TGET EB cells used for cell sorting after CRISPR library infection is shown. c Comparative reads analysis between TGET ES cells (24 h post infection, “lib”) and stabilized TGET ES cells (2 weeks selection post infection, “input”), which shows depletion of gRNAs against essential genes for pluripotency in stabilized TGET ES cells. d Comparative reads analysis between stabilized TGET ES cells (“input”) and PDGFRα⁺ cells, which shows depletion of gRNAs against essential genes for PDGFRα⁺ mesoderm differentiation. e Comparative reads analysis between PDGFRα⁺ and Etv2^high cells, which shows depletion of gRNAs against genes essential for Etv2^high cell generation. f Scheme for generation of the candidate gene list. Top 500 genes with depleted reads from PDGFRα⁺ to Etv2^high cells were compared to 10,402 genes expressed in mesoderm (with RPKM >1 in any of “pr”, “dp”, Etv2^int, or Etv2^highScl⁻), which yielded 215 genes. g Functional annotation of selected candidate genes is shown. Numbers at the end of each bar shows the P value of gene enrichment in the corresponding ontology item. h Validation of chosen candidate genes. The ratio of PDGFRα⁺ to Etv2^high cells by a candidate sgRNA depletion was normalized to that of empty vector. Refer to Supplementary Fig. 3b, c for more details. *P value <0.05 in Student’s t test, **P < 0.01, ***P < 0.001, and n = 3. Error bars are s.d