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Figure 3 CRISPR-generated dmist mutants sleep less and are hyperactive at night. (A) CRISPR/Cas9 targeting of the first exon of dmist resulted in an 8 bp insertion (dmisti8) (grey line) within the coding sequence, leading to an early stop codon (red line with *). Guide RNA target sequence and PAM sequence are shown as black bars. The sequence that is deleted in the mutant is indicated with a red bar. (B) Predicted Dmisti8 peptide sequence lacks most of the N-terminal signal peptide sequence (magenta) and the full C-terminus. (C, D) Representative 48 hr traces of mean ± SEM sleep (C) and waking activity (D) shows decreased sleep and increased waking activity at night for dmisti8/i8 fish compared to dmisti8/+ and dmist+/+ siblings. n = number of fish. (E–H) Analysis of sleep/wake architecture of the experiment depicted in (C, D) indicates that dmisti8/i8 larvae sleep less at night (E) due to fewer sleep bouts (F). Sleep bout length is unchanged (G). Waking activity is also increased in dmisti8/i8 fish (H). The black line represents the mean ± SEM except for (G), which is the median ± SEM. *p<0.05, **p<0.01, ***p<0.001; one-way ANOVA, Tukey’s post hoc test. (I) Combining five independent experiments with a linear mixed effects model reveals dmisti8/i8 fish sleep less at night due to fewer sleep bouts and also show increased waking activity at night. Plotted are the genotype effect sizes (95% confidence interval) for each parameter relative to wild type. Shading indicates day (white) and night (grey). p-Values are assigned by an F-test on the fixed effects coefficients from the linear mixed effects model. *p<0.05, **p<0.01, ***p<0.001, ns p>0.05. (J) Adult dmisti8/i8 fish have a higher mean swim speed compared to their wild-type siblings at night. Data in (J) is quantified at night in (K). (J, K) show mean ± SEM. *p<0.05, one-way ANOVA. (L) Cumulative probability distribution of all night-time swim bout speeds in adult fish. The dashed lines show the half max (0.5 probability) for each curve. *p<0.05 for dmisti8/i8 fish compared to wild-type siblings; Kolmogorov–Smirnov test.