The phosphorylation of synaptic proteins is a significant biochemical reaction that controls the sleep–wake cycle in mammals1–3. Protein phosphorylation in vivo is reversibly regulated by kinases and phosphatases. In this study, we investigate a pair of kinases and phosphatases that reciprocally regulate sleep duration. First, we perform a comprehensive screen of protein kinase A (PKA) and phosphoprotein phosphatase (PPP) family genes by generating 40 gene knockout mouse lines using prenatal and postnatal CRISPR targeting. We identify a regulatory subunit of PKA (Prkar2b), a regulatory subunit of protein phosphatase 1 (PP1; Pppr1r9b) and catalytic and regulatory subunits of calcineurin (also known as PP2B) (Ppp3ca and Ppp3r1) as sleep control genes. Using adeno-associated virus (AAV)-mediated stimulation of PKA and PP1–calcineurin activities, we show that PKA is a wake-promoting kinase, whereas PP1 and calcineurin function as sleep-promoting phosphatases. The importance of these phosphatases in sleep regulation is supported by the marked changes in sleep duration associated with their increased and decreased activities, ranging from approximately 17.3 h per day (PP1 expression) to 4.3 h per day (postnatal CRISPR targeting of calcineurin). Localization signals to the excitatory post-synapse are necessary for these phosphatases to exert their sleep-promoting effects. Furthermore, the wake-promoting effect of PKA localized to the excitatory post-synapse negated the sleep-promoting effect of PP1–calcineurin. These findings indicate that PKA and PP1–calcineurin have competing functions in sleep regulation at excitatory post-synapses.
