Knockout analysis of period and timeless and EGFP-based visualization of per-expressing clock cells in the cricket circadian clock

In the present study, we generated crickets with knockout of either period (per) or timeless (tim) gene by CRISPR/Cas9-based genome editing. We also identified a naturally occurring per- mutant lacking a large coding region including PAS domains. To examine possible synergistic effects, a per- and timKO double mutant was generated by applying genome editing to the per- crickets. Under constant darkness (DD), timKO crickets exhibited a locomotor rhythm with a free-running period of 23.06 ± 0.20 h (mean ± SD), which was significantly shorter than that of the parental strain (23.78 ± 0.12 h). By contrast, perKO and per- crickets showed basically similar phenotype of locomotor rhythm: they exhibited an arrhythmic pattern during the first two to three weeks after transfer to DD but subsequently showed a complex rhythmic pattern with one or multiple components with significantly longer free-running periods (33.35 ± 10.72 h). In the per-;timKO double mutants, approximately 60% of individuals became arrhythmic, while the remaining 40% exhibited complex rhythms with extremely longer free-running periods (37.0 ± 9.17 h) under DD. These results suggest the existence of an underlying oscillatory mechanism that is responsible for regulating locomotor rhythms independently of the canonical per/tim feedback loop. Furthermore, we generated a reporter line on a per− background by knocking egfp into exon 1 of the per gene, allowing egfp expression to report per transcription. 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 near the accessory medulla. These findings raise the possibility that these form part of the circadian clock network that governs circadian locomotor rhythms.