CRISPR/Cas9 generated chd7 mutants recapitulate CHARGE syndrome related phenotypes. (A) Exon‐intron structure of zebrafish chd7 with CRISPR exon 9 and exon 16 targeted regions noted; amino acid alignment and conservation of Exon 9, 2nd chromodomain target across species, identified 7 bp deletion from mutagenesis; amino acid alignment of Exon 16, ATP‐helicase domain target, and identified 1 bp deletion from mutagenesis; predicted Chd7 protein domains in wild‐type (top), ATP‐helicase domain 1 bp deletion allele (chd7 ^{rdu1002/rdu1002} ), and chromodomain 7 bp deletion allele (chd7 ^{ncu101/ncu101} ), additional target regions from sgRNAs noted. (B) Survival rate (%) of chd7 ^+/+ (n = 19), chd7 ^ncu101/+ (n = 54), and chd7 ^{ncu101/ncu101} (n = 25) siblings during a 30‐day period. (C) qPCR analysis of chd7 mRNA in 5 dpf larvae (chd7 ^ncu101/, chromodomain allele), normalized to wild‐type siblings. Each point represents a biological replicate of pooled larvae (per pool, chd7 ^+/+ n ~ 12–13; chd7 ^ncu101/+ n ~ 20–21; chd7 ^{ncu101/ncu101} n ~ 13–14). (D) qPCR analysis of chd7 mRNA in 5 dpf larvae (chd7 ^rdu1002/, ATP‐helicase domain allele), normalized to wild‐type siblings. Each point represents a biological replicate of pooled larvae (per pool, chd7 ^+/+ n ~ 13–14; chd7 ^rdu1002/− n ~ 22–23; chd7 ^{rdu1002/rdu1002} n ~ 11–12) (mean ± SD, Ordinary one‐way ANOVA with Tukey's multiple comparisons, *p < 0.05, **p < 0.01, ***p < 0.001,****p < 0.0001). (E) Bright‐field images of 5 dpf larvae (chd7 ^+/+, chd7 ^ncu101/+, chd7 ^{ncu101/ncu101} ) with examples of morphological phenotypes including uninflated swim bladder (asterisk), pericardial edema (triangle), and craniofacial defects (solid triangle). (F) Examples of varying otolith defects in chd7 ^ncu101/, dashed orange lines represent morphology of the otic vesicle, and anterior and posterior otoliths.

Loss of chd7 induces a context‐dependent LLC phenotype in auditory driven behaviors. (A) Acoustic startle responses, average short‐latency c‐bend (SLC) frequency as acoustic stimulus intensity increases (chd7 ^+/+ n = 64, chd7 ^ncu101/+ n = 127, chd7 ^{ncu101/ncu101} n = 61) (mean ± SEM). (B) Short‐latency c‐bend sensitivity index, calculated by the area under the SLC frequency curves for individual larvae (mean ± SD, Ordinary one‐way ANOVA with Tukey's multiple comparison). (C) Long‐latency c‐bend (LLC) frequency as acoustic stimulus intensity increases (mean ± SEM). (D) Long‐latency c‐bend sensitivity index calculated by the area under the LLC frequency curves for individual larvae (mean ± SD, Kruskal‐Wallis with Dunn's multiple comparisons). (E) Short‐term habituation, average SLC frequency during 30 acoustic stimuli at highest intensity (chd7 ^+/+ n = 40, chd7 ^ncu101/+ n = 82, chd7 ^{ncu101/ncu101} n = 47) (mean ± SEM). (F) SLC half‐life calculated by nonlinear regression (one‐phase exponential decay) of SLC frequency curves for individual larvae (mean ± SD, Kruskal‐Wallis with Dunn's multiple comparisons). (G) Average LLC frequency during 30 acoustic stimuli at highest intensity (mean ± SEM). (H) LLC sensitivity index calculated by the area under the LLC frequency curves for individual larvae (mean ± SD, Kruskal‐Wallis with Dunn's multiple comparisons, *p < 0.05, **p < 0.01, ***p < 0.001,****p < 0.0001).

LLC deficit is independent of otolith morphology in chd7 chromodomain mutants. (A) Acoustic startle responses, average short‐latency c‐bend (SLC) frequency comparing chd7 ^ncu101/+ and chd7 ^{ncu101/ncu101} with or without otolith defects (chd7 ^+/+ n = 11, chd7 ^ncu101/+ w/o otolith defect n = 22, chd7 ^ncu101/+ with otolith defect n = 17, chd7 ^{ncu101/ncu101} w/o otolith defect n = 8, chd7 ^{ncu101/ncu101} with otolith defect n = 11) (mean ± SEM). (B) Short‐latency c‐bend sensitivity index, calculated by the area under the SLC frequency curves for individual larvae (mean ± SD, Ordinary one‐way ANOVA with Tukey's multiple comparison). (C) Long‐latency c‐bend (LLC) frequency as acoustic stimulus intensity increases (mean ± SEM). (D) Long‐latency c‐bend sensitivity index calculated by the area under the LLC frequency curves for individual larvae (mean ± SD, Ordinary one‐way ANOVA with Tukey's multiple comparison, **p < 0.01, ****p < 0.0001).

Hypo‐ and hyper‐activity phenotypes in chromodomain and ATP‐helicase domain mutants. (A) Chromodomain larval dark‐flash and (B) light‐flash response frequencies (chd7 ^+/+ n = 37, chd7 ^ncu101/+ n = 69, chd7 ^{ncu101/ncu101} n = 26) (mean ± SD, Ordinary one‐way ANOVA with Tukey's multiple comparison, **p < 0.01). (C) Chromodomain larval average total distance traveled plot during 9 h of recording, analyzed every 30 min, including 1 h of acclimation in the dark, 4 h in the dark, and 4 h in light (chd7 ^+/+ n = 44, chd7 ^ncu101/+ n = 70, chd7 ^{ncu101/ncu101} n = 26) (mean ± SEM). (D) Chromodomain larval area under the curve for individual distance plots during the dark or light phase (mean ± SD, Ordinary one‐way ANOVA with Dunnett's multiple comparisons, *p < 0.05). (E) ATP‐helicase domain larval average total distance traveled (chd7 ^+/+ n = 22, chd7 ^rdu1002/+ n = 42, chd7 ^{rdu1002/rdu1002} n = 20) (mean ± SEM) and (F) area under the curve during the dark or light phase (mean ± SEM (G) chromodomain larval total distance time plot during 18.5 min of recording (chd7 ^+/+ n = 46, chd7 ^ncu101/+ n = 67, chd7 ^{ncu101/ncu101} n = 29) (mean ± SEM). (H) Sum of total distance traveled for individual larvae, (I) swim (open bars) and turn (shaded bars) frequencies (mean ± SD, Kruskal‐Wallis with Dunn's multiple comparisons).

CRISPR guides targeting different chd7 Exons induce varying phenotypes. (A) Acoustic startle responses, average short‐latency c‐bend (SLC) frequency as acoustic stimulus intensity increases (Control n = 47, Exon 3 n = 66, Exon 9 n = 60, Exon 30 n = 54, Mix n = 45) (mean ± SEM). (B) Short‐latency c‐bend sensitivity index, calculated by the area under the SLC frequency curves for individual larvae (mean ± SD, Ordinary one‐way ANOVA with Dunnett's multiple comparisons). (C) Long‐latency c‐bend (LLC) frequency as acoustic stimulus intensity increases (mean ± SEM). (D) Long‐latency c‐bend sensitivity index calculated by the area under the LLC frequency curves for individual larvae (mean ± SD, Ordinary one‐way ANOVA with Dunnett's multiple comparisons). (E) Short‐term habituation, average SLC frequency (Control n = 48, Exon 3 n = 61, Exon 9 n = 59, Exon 30 n = 57, Mix n = 49) (mean ± SEM) during 30 acoustic stimuli at highest intensity with (inset) SLC half‐life calculated by nonlinear regression (one‐phase exponential decay) of SLC frequency curves for individual larvae (mean ± SD, Ordinary one‐way ANOVA with Dunnett's multiple comparisons). (F) Average LLC frequency (mean ± SEM) during 30 acoustic stimuli at highest intensity with (inset) LLC sensitivity index calculated by the area under the LLC frequency curves for individual larvae (mean ± SD, Ordinary one‐way ANOVA with Dunnett's multiple comparisons). (G) Average total distance traveled plot during 9 h of recording, analyzed every 30 min, including 1 h of acclimation, 4 h in the dark, and 4 h in light (Control n = 54, Exon 3 n = 27, Exon 9 n = 30, Exon 30 n = 31, Mix n = 31) (mean ± SEM). (H) Area under the curve for individual larvae distance plots during the dark or light phase (mean ± SD, Ordinary one‐way ANOVA with Dunnett's multiple comparisons, *p < 0.05, ***p < 0.001,****p < 0.000).

Correlation of mutation location and phenotype penetrance in a cohort of CHARGE patients. Heat map displaying the frequency of patients with a specified phenotype and a mutation within a particular CHD7 domain; domains were determined by amino acid alignments (n = 89, n = 30 patients excluded from the original pool of 119 participants because of insufficient information or mutation did not align to a domain). CHD7 domains are represented by columns and phenotypes are represented by rows. Frequencies were calculated by the number of patients with a reported phenotype and mutation in the respective domain over the total number of patients within the same domain (purples). Final row represents the overall average phenotype frequency for a specific domain (oranges). Patient data was obtained from the paper Phenotype and genotype analysis of a French cohort of 119 patients with CHARGE syndrome, Legendre et al. ¹⁶ Darker colors indicate a higher frequency, with ranges indicated in the legends. CNS, central nervous system; IUGR, intrauterine growth restriction; SCC, semicircular canal.