Dissecting circadian clock machinery by genome-editing in the cricket Gryllus bimaculatus

In the present study we have generated crickets with knockout of either per or tim gene by CRISPR/Cas9 based genome-editing. We also obtained naturally occurred per ⁻ mutant lacking a large coding region including PAS domains. To investigate synergistic effects, a per ⁻ and tim ^KO double mutant was produced by applying genome editing to the per ⁻ crickets. Under constant darkness (DD), tim ^KO crickets showed a locomotor rhythm with a free-running period significantly shorter than that of the parental strain. The per ^KO and per ⁻ crickets showed basically similar phenotype of locomotor rhythm: they exhibited arrhythmic pattern first two to three weeks after transfer to DD but subsequently showed a complex rhythmic pattern with a single or multiple components with significantly longer free-running periods. In the per ⁻ ; tim ^KO double mutants, approximately 60% of individuals became arrhythmic, while the remaining 40% exhibited complex rhythm with extremely longer free-running periods under DD. These results suggest the existence of underlying oscillatory mechanism that is responsible for regulating locomotor rhythms independently of the canonical per/tim feedback loop. Furthermore, we generated per ⁻ reporter line with egfp knocked in exon 1 of the per gene. EGFP expression was detected in three distinct clusters of cells within the optic lobe: two located along the dorsal and ventral boundaries between the lamina and medulla neuropils, and one situated in the proximal medulla neuropil near the accessory medulla. These findings suggest that these specific cells constitute a circadian clock network that governs circadian locomotor rhythms.