Format

Send to

Choose Destination
Genetics. 2019 Jul 10. pii: genetics.302351.2019. doi: 10.1534/genetics.119.302351. [Epub ahead of print]

Phenotypic and Genotypic Consequences of CRISPR/Cas9 Editing of the Replication Origins in the rDNA of Saccharomyces cerevisiae.

Author information

1
University of Washington.
2
University of Washington bbrewer@u.washington.edu.

Abstract

The complex structure and repetitive nature of eukaryotic ribosomal DNA (rDNA) is a challenge for genome assembly, and thus, the consequences of sequence variation in rDNA remain unexplored. However, renewed interest in the role that rDNA variation may play in diverse cellular functions, aside from ribosome production, highlights the need for a method that would permit genetic manipulation of the rDNA. Here, we describe a CRISPR/Cas9 based strategy to edit the rDNA locus in the budding yeast Saccharomyces cerevisiae. Using this approach, we modified the endogenous rDNA origin of replication in each repeat by deleting or replacing its consensus sequence. We characterized the transformants that have successfully modified their rDNA locus and propose a mechanism for how CRISPR/Cas9 mediated editing of the rDNA occurs. In addition, we carried out extended growth and life span experiments to investigate the long-term consequences that altering the rDNA origin of replication has on cellular health. We find that long-term growth of the edited clones results in faster growing suppressors that have acquired segmental aneusomy of the rDNA containing region of chr XII or aneuploidy of chromosomes XII, II, or IV. Furthermore, we find that all edited isolates suffer a reduced life span, irrespective of their levels of extrachromosomal rDNA circles. Our work demonstrates that it is possible to quickly, efficiently and homogeneously edit the rDNA locus via CRISPR/Cas9. It serves as a model for modifying other parts of the rDNA and, more generally, for editing other tandemly repeated sequences in higher eukaryotes.

KEYWORDS:

fitness; origins of replication; rDNA copy number; replicative lifespan; turbidostat

PMID:
31292210
DOI:
10.1534/genetics.119.302351
Free full text

Supplemental Content

Full text links

Icon for HighWire
Loading ...
Support Center