Investigating the roles for essential genes in the regulation of the circadian clock in Synechococcus elongatus using CRISPR interference

Abstract

Circadian rhythms are approximately 24-hour cycles that allow organisms to anticipate predictable environmental changes such as light intensity, temperature, and humidity. Circadian rhythms are found widely throughout nature where cyanobacteria are the simplest organisms known to carry out circadian rhythmicity. Circadian rhythmicity in cyanobacteria is carried out via the KaiA, KaiB and KaiC core oscillator proteins that keep ~24h time. A series of input and output proteins; CikA, SasA and RpaA, regulate the clock by sensing environmental changes, and time 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 Reverse Transcriptase (RT)-PCR for reduction of gene expression and rhythms of gene expression were monitored to analyze circadian phenotypes. We present data that reduced expression of SynPCC942_0001, which encodes for the {beta}-clamp of the replicative DNA polymerase, or SynPCC7942_1081, which likely encodes for KtrA involved in K+ transport, displayed a long circadian period compared to 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.