Fig. 4: TADA2B KOs and OTUD5 KOs have opposing fitness phenotype.

a Schematic of TADA2B including location of gRNA targeting and sequences of knockouts (KOs). Boxes indicate TADA2B exons, and filled gray boxes indicate the coding sequence of TADA2B. b Western blot of TADA2B confirming KOs in HUES8 iCas9 WT1, WT2, WT3, and HUES8 iCas9 TADA2B^-/- KO1, KO2, and KO3 clonal lines. Representative from 2 independent experiments. c Schematic of OTUD5 targeting and KOs. d Western blot of OTUD5 confirming KOs in HUES8 iCas9 WT2, WT3, and HUES8 iCas9 OTUD5^-/- KO1, and KO2 clonal lines. Representative from 2 independent experiments. e Growth curve and area under growth curve (AUC) of WT and TADA2B KOs in E8 maintenance conditions and (f) E6 challenge conditions. 3 WT and 3 KO lines analyzed per experiment. n = 3 independent experiments. Data represented as mean. Error bars indicate s.d. Statistical analysis was performed by unpaired two-tailed Student’s t test. P values for significant comparisons indicated. g Growth curve and AUC of WT and OTUD5 KOs in E8 maintenance conditions. 2 WT and 2 KO lines analyzed per experiment. n = 5 independent experiments. Data represented as mean. Error bars indicate s.d. Statistical analysis was performed by unpaired two-tailed Student’s t-test. No significant comparisons were found. h Growth curve and AUC of WT and OTUD5 KOs in E6 challenge conditions and (i) after plating without Y-27632 (ROCKi), a standard component of hESC culture. 2 WT and 2 KO lines analyzed per experiment. n = 3 independent experiments. Data represented as mean. Error bars indicate s.d. Statistical analysis was performed by unpaired two-tailed Student’s t test. P values for significant comparisons indicated. j Cell competition assay WT vs. OTUD5 KOs. Individual cell lines were labeled with LARRY barcodes¹⁰⁵, and pooled. Pooled cells were expanded in E8 conditions for 28 days. Pools were collected and sequenced for representation of barcodes every 7 days. WT parental line and KO1, KO2 lines analyzed per experiment. n = 5 independent experiments. Data represented as mean. Error bars indicate s.d. Statistical analysis was performed by unpaired two-tailed Student’s t test. P values for significant comparisons indicated. Source data are provided as a Source Data file.