0042 Melanopsin Crispants in Zebrafish Display Difference in Sleep Latency

Abstract Introduction Melanopsin (OPN4) is a blue light sensitive opsin type G protein couple receptor. It is highly expressed in the photsenstive ganglion cells which mediate responses to light including regulation of sleep, circadian photoentrainment and pupillary light responses. Mutations in OPN4 were shown to affect responses to light ultimately affecting the regulation of circadian rhythms and sleep patterns. Sleep homeostasis was altered in homozygous knockout of Opn4, gene encoding for melanopsin, in mice. Human melanopsin variants which alter function significantly may contribute to sleep disturbance, circadian dysfunction and visual impairment. In order to further dissect the role of melanopsin in sleep in a diurnal vertebrate model, we created an F0 knockout model of opn4 gene in zebrafish and assessed sleep/wake phenotype. Methods In CRISPR/Cas9 screening, single cell-stage zebrafish embryos were injected with injection cocktail containing Cas9 protein and crRNAs targeting opn4a and opn4b, homologous genes of human OPN4. Control groups were negative control crRNA injected embryos. Zebrafish larvae were individually pipetted into each well of a 96 well plate. Larval zebrafish were raised on a 14 hour/10 hour light/dark cycle at 28.5◻C until sleep phenotyping starts. Sleep phenotyping was carried out between days 5-8 post injection. Results Longer sleep latency at night was observed in opn4 CRISPR knockout larvae. This is in line with prior literature. Additional tests by exposing opn4 crispants and control zebrafish larvae to white light and dark pulses at different times were performed however the activity and sleep latency did not change across groups. Conclusion Sleep/wake phenotyping of opn4 (melanopsin) crispants in zebrafish displayed significant difference in sleep latency parameter compared to controls. Although additional tests carried out using pulses of white light need to be repeated, current results highlight the importance of OPN4 mediated pathways in affecting sleep latency. Support (if any)