Academic journal article Genetics

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

Academic journal article Genetics

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

Article excerpt

WHILE the role of ribosomal DNA (rDNA) in ribosome production is uncontested, there has been renewed interest in exploring the role that rDNA variation may play in cell cycle regulation, life span, and cancer (Wang and Lemos 2017; Parks et al. 2018). Because ribosomal content is one of the dominant components of cellular biomass, ribosomal RNA (rRNA) transcription imposes significant constraints on the speed with which cells can divide. The heavy transcriptional burden placed on the rDNA has necessitated large numbers of repeated units to cope with the demand for rRNAs. This burden, and the inherently repetitive nature of these sequences, can result in variation within an otherwise isogenic population. However, variation in the sequence or copy number of rDNA repeats has been difficult to assess experimentally (McStay 2016). Even though whole-genome sequencing (WGS) data are available for hundreds to thousands of different eukaryotes, the large sizes of the repeated, homogeneous, tandem structures make identifying and testing sequence and copy number variants challenging in most species. The copy number of repeats can be highly variable from individual to individual (Stults et al. 2008; Xu et al. 2017) and are often difficult to reliably determine when orthogonal methods, such as digital droplet PCR or quantitative PCR (qPCR), quantitative hybridization, and WGS readdepth measurements give variable estimations of copy number from the same DNA sample (Xu et al. 2017; Chestkov et al. 2018). Adding to the complexity of the problem is the fact that, in many eukaryotes, the rDNA is also found on multiple chromosomes. Directed mutational analysis, a technique so powerful for analyzing single-copy sequences, has not been a realistic option in the rDNA of most organisms because of the difficulty in mutagenizing all of the repeats simultaneously. Therefore the systematic testing ofindividual sequence or copy number variants has been genetically intractable.

In the budding yeast Saccharomyces cerevisiae, limited mutational analysis of the rDNA locus has been carried out by taking advantage of a recessive hygromycin-resistant mutation (T to C) at position 1756 of the 18S rRNA. When this variant rDNA repeat is introduced into yeast on a multicopy plasmid, cells can become hygromycin-resistant by deleting most or all of the chromosome XII copies of the rDNA (Chernoff et al. 1994; Wai et al. 2000), and relying on the plasmid copies of the rDNA for ribosome production. Using a plasmid-shuffle protocol or direct reintegration of sequences into chromosome XII, researchers can examine the consequences of mutated rDNA elements. This basic protocol has been used to explore transcriptional regulatory elements of the Polymerase I (PolI) promoter and enhancer (Wai et al. 2000), the role of expansion segments in ribosome fidelity (Fujii et al. 2018), the sequence requirements for replicationfork blocking at the 3'-end of the 35S transcription unit (the replication fork barrier, RFB) (Ganley et al. 2009; Saka et al. 2013), and on a more limited scale, the requirements for the rDNA origin of replication in copy number expansion (Ganley et al. 2009). While these experiments are elegant, there are problems with interpreting the resultant phenotypes; in particular, the introduction of altered rDNA sequences requires the simultaneous introduction of a selectable marker that becomes part of each of the amplified variant rDNA repeats.

Here, we edited the yeast rDNA locus directly without the introduction of a selectable marker by using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 (Doudna and Charpentier 2014). Our method to mutagenize rDNA was developed independently but is similar to one previously reported (Chiou and Armaleo 2018) which was intended for a different biological purpose. We altered the origin of replication that is found in each repeat by deleting it entirely, or by replacing the 11-bp A+T-rich origin consensus sequence with a G+C block or other functional origins. …

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