Eukaryotic circadian oscillators share a common circuit architecture, a negative feedback loop in which a positive element activates the transcription of a negative one that then represses the action of the former, inhibiting its own expression. While studies in mammals and insects have revealed additional transcriptional inputs modulating the expression of core clock components, this has been less characterized in the model Neurospora crassa, where the participation of other transcriptional components impacting circadian clock dynamics remains rather unexplored. Thus, we sought to identify additional transcriptional regulators modulating the N. crassa clock, following a reverse genetic screen based on luminescent circadian reporters and a collection of transcription factors knockouts, successfully covering close to 60% of them. Besides the canonical core clock components WC-1 and WC-2, none of the tested transcriptional regulators proved to be essential for rhythmicity. Nevertheless, we identified a set of 23 transcription factors that when absent lead to discrete, but significant, changes in circadian period. While the current level of analysis does not provide mechanistic information about how these new players modulate circadian parameters, the results of this screen reveal that an important number of light and clock-regulated transcription factors, involved in a plethora of processes, are capable of modulating the clockworks. This partial reverse genetic clock screen also exemplifies how the N. crassa knockout collection continues to serve as an expedite platform to address broad biological questions.

A timely and dynamic response to strong temperature fluctuations is paramount for organismal biology. At the same time, inducible promoters are a powerful tool for fungal biotechnological and synthetic biology endeavors.