CRISPR/Cas9-mediated mutagenesis of tim in either tim-positive or Mai179-positive neurons, by the co-expression of tim^{UAS.gRNAx3.pCFD6} and Spyo\Cas9^UAS.P2 under the control of Scer\GAL4^tim.PE or Scer\GAL4^Mai179, respectively, leads to complete loss of circadian rhythmic behavior, although flies are still rhythmic under LD cycles.

Mutagenesis of tim in Pdf-positive neurons (i.e. Scer\GAL4^P2.4.Pdf as driver of expression) leads to decreased circadian rhythmicity under constant darkness: although most individuals remain highly rhythmic, there is a significant decrease in the morning anticipation index, a significant increase in the evening anticipation index, and a small but significant increase in period, as compared to controls. Under LD cycles there are no obvious circadian phenotypes. There are no changes in the number of Pdf-positive neurons, as compared to controls.

Mutagenesis of tim in Mai179-positive, Pdf-negative neurons (expression under the combed control of Scer\GAL4^Mai179 and Scer\GAL4^P2.4.Pdf) does not have a major effect on circadian rhythmicity under constant darkness conditions: there is an increase in period, but rhythmic strength and both morning and evening anticipation indices are unaffected. There are no obvious circadian phenotype under LD cycles.

There are no circadian phenotypes when tim is mutagenized in the glia (i.e. Scer\GAL4^repo as driver of expression).