Fig. 2

FLCN KO hESC express naïve pluripotency markers. a Schematic representation of FLCN protein and location of CRISPR guide RNAs used. Sanger sequencing analysis of Elf1 2iL-I-F FLCN KO hESC reveals introduction of STOP codons in exons 3. b, c Generation of WIBR3 5iLA FLCN KO and Elf1 2iL-I-F FLCN KO line, and overexpression of FLCN-GFP fusion protein in wild type and FLCN KO background. d The pluripotency marker Oct4 is expressed in FLCN KO 2iL-I-F and 5iLA hESC. Scale bars represent 50 μm. e Expression pattern of genes up-regulated by FLCN KO compared to genes up-regulated in monkey pre- vs. post-implantation stage. Red: genes up-regulated in FLCN KO and pre-implantation, known ground state pluripotency markers are labeled. f tSNE analysis of naïve markers DNMT3L, TFCP2L1, and ESRRB in in vivo blastocysts from non-human primate, cynomolgus monkey (Macaca fascicularis³³). g, h RT-qPCR analysis of naïve markers DNMT3L and TFCP2L1 in naïve 2iL-I-F wild type (WT), FLCN KO, and rescue line (FLCN KO + OE FLCN-GFP) (g) and in naïve 5iLA WT and FLCN KO (h). S.e.m.; **p < 0.005, ***p < 0.001; two-tailed t-test, n = 3–10 biological replicates