Main The function of WAKE is conserved in mammalsWe previously identified the clock-output molecule WIDE AWAKE (WAKE) from a forward genetic screen in Drosophila 4 . WAKE modulates the activity of arousalpromoting clock neurons at night, in order to promote sleep onset and quality 4,5 . The mammalian proteome contains a single ortholog, mWAKE (also named ANKFN1/Nmf9), with 56% sequence similarity and which is enriched in the core region of the master circadian pacemaker suprachiasmatic nucleus (SCN) 4,6 ( Fig. 1a, Extended Data Fig. 1a). To investigate whether the function of WAKE is conserved in mice, we generated a putative null allele of mWAKE (mWAKE (-) ) by CRISPR/Cas9 insertion of 8 base pairs (containing a stop codon and generating a downstream frameshift) in exon 4, which is predicted to be in all splice isoforms of mWAKE ( Fig. 1b). As expected, mWAKE expression, as assessed by quantitative PCR and in situ hybridization (ISH), was markedly reduced in mWAKE (-/-) mice, likely due to nonsense-mediated decay (Fig. 1c, 1d). Given mWAKE expression in the SCN, we first examined locomotor circadian rhythms and found that mWAKE (-/-) mice exhibit a mild but non-significant decrease in circadian period length (Extended Data Fig. 1b, 1c). These results are similar to findings from fly wake mutants and mice bearing the Nmf9 mutation (a previously identified ENU-generated allele of mWAKE) 4,6 .Because we previously demonstrated that WAKE mediates circadian regulation of sleep timing and quality in fruit flies 4,5 , we next assessed sleep in mWAKE (-/-) mice via electroencephalography (EEG). Under light:dark (L:D) conditions, there was no difference in the amount of wakefulness, non-rapid eye movement (NREM), or REM sleep between mWAKE (-/-) mutants and wild-type (WT) littermate controls (Extended Data Fig. 1d). In constant darkness (D:D), there is a modest main effect of genotype on wakefulness (P<0.05) and NREM sleep (P<0.05), and a mild but significant decrease in REM sleep in mWAKE (-/-) mutants (Fig. 1e). Although the amount of wakefulness did not appreciably differ in mWAKE (-/-) mutants compared to controls, there was a change in the distribution of wakefulness at night; mutants spent more daily time in prolonged wake bouts, and some mutants exhibited dramatically long bouts of wakefulness (Extended Data Fig. 1e, 1f).