Fig. 2. Validation of top candidates from the forward genetic screen.

a Top ten candidates ranked by MAGeCK enrichment score (lower rank indicates greater enrichment). The six guides for each candidate are ranked by individual enrichment. Enriched gRNAs with Benjamini–Hochberg corrected Wald test P < 0.001 are colored in red and summed in the first column. Candidates were tested by treating R26^Cas9-GFP; Myh7^YFP neonatal pups with the most highly ranked individual gRNA. AAV-Cre without gRNA served as negative control. Presence (column 2, yellow box) or absence (gray box) of persistent YFP expression at four weeks was assessed by imaging fixed, dissociated CMs. b Quantification of Myh7^YFP activation within transduced (GFP + ) CMs by flow cytometery. AAV-Cre was used as negative control. n = 3. P for all genes, except Poc1b and Setd6, was <0.0001. Setd6 P = 0.0111. c Quantification of mononucleation among dissociated, GFP + control CMs or YFP + candidate-depleted CMs. n = 3. Significant P: Rnf40, 0.0128; Thra, 0.0102; Taf3, 0.0027; Taf2, 0.0001. Error bars denote standard error. d–f Normalized projected area (d) and length:width ratio (e) of control and candidate-depleted CMs. Measurements from dissociated YFP + CMs were normalized to YFP- cells from the same heart. Significant P, area: Rnf40, Thra, Taf3, Rnf20, Taf2, and Ncl <0.0001. Significant P, length-to-width: Taf3, 0.007; Rnf20, 0.0042. f T-tubule transverse element density for control and candidate-depleted CMs as measured by AutoTT software-based quantification of CAV3 immunostaining. Significant P: Rnf40, Taf3, Rnf20, Taf2, Ncl < 0.0001; Zbtb7a, 0.0003. Bar plots show mean ± SD. At least 69 CMs from three mice were measured per group. Violin plots: shape indicates data distribution; point, median; whiskers, starts at quartile and extends 1.5 times the interquartile distance. Dunnett’s two-tailed t-test vs. Cre control: *P < 0.05, **P < 0.001. Source data are provided as a Source Data file.