Investigating the Roles for Essential Genes in the Regulation of the Circadian Clock in Synechococcus elongatus Using CRISPR Interference.

Circadian rhythms are found widely throughout nature where cyanobacteria are the simplest organisms, in which the molecular details of the clock have been elucidated. Circadian rhythmicity in cyanobacteria is carried out via the KaiA, KaiB, and KaiC core oscillator proteins that keep ~24 h time. A series of input and output proteins-CikA, SasA, and RpaA-regulate the clock by sensing environmental changes and timing rhythmic activities, including global rhythms of gene expression. Our previous work identified a novel set of KaiC-interacting proteins, some of which are encoded by genes that are essential for viability. To understand the relationship of these essential genes to the clock, we applied CRISPR interference (CRISPRi) which utilizes a deactivated Cas9 protein and single-guide RNA (sgRNA) to reduce the expression of target genes but not fully abolish their expression to allow for survival. Eight candidate genes were targeted, and strains were analyzed by quantitative real-time PCR (qRT-PCR) for reduction of gene expression, and rhythms of gene expression were monitored to analyze circadian phenotypes. Strains with reduced expression of SynPCC7942_0001, dnaN, which encodes for the β-clamp of the replicative DNA polymerase, or SynPCC7942_1081, which likely encodes for a KtrA homolog involved in K⁺ transport, displayed longer circadian rhythms of gene expression than the wild type. As neither of these proteins have been previously implicated in the circadian clock, these data suggest that diverse cellular processes, DNA replication and K⁺ transport, can influence the circadian clock and represent new avenues to understand clock function.