Using CRISPR/dCas9 to Dissect the Role of Ribosomal DNA in Cellular Growth and Aging in Saccharomyces cerevisiae
Ribosomal DNA (rDNA) plays a major role in cellular growth and aging, but dissecting this role is challenging due to its highly repetitive nature. In this study, a genomically integrated CRISPR/deactivated Cas9 (dCas9) tool was developed to probe the role of various transcribed and non-transcribed regions of the rDNA on growth and aging in the budding yeast S. cerevisiae, a model organism for studying cell physiology in eukaryotes. From constructed cell growth curves, among the 6 targets tested, none of the cell strains with dCas9 targeting a non-transcribed region of rDNA had any effect on cell growth rate, as expected. Surprisingly, however, among the strains with dCas9 targeting either one of the two genes of rDNA and their respective promoters, only the cell strain with dCas9 targeting the 35S rRNA transcribed region, not its promoter, had a significant effect on cell growth, slowing its doubling time from 100 minutes per cell division to 140 minutes. This was further confirmed with a spotting assay, which showed that only this strain’s colonies grew visibly more slowly than other strains’. Fluorescence microscopy showed an anti-correlation in this slow-growing strain between the abundance of dCas9-GFP localized in the nuclear region and the total abundance of a constitutively expressed reporter protein, Nhp6a-iRFP. These selective differences suggest the efficacy of this novel tool to probe the various regions of rDNA and may provide insight into the role of rDNA transcribed versus non-transcribed regions in cell growth and aging at the single-cell level.