Abstract
The 45S rRNA genes (rDNA) are among the largest repetitive elements in eukaryotic genomes. rDNA consists of tandem arrays of rRNA genes, many of which are transcriptionally silenced. Silent rDNA repeats may act as ‘back-up’ copies for ribosome biogenesis and have nuclear organization roles. Through Cas9-mediated genome editing in the Arabidopsis thaliana female gametophyte, we reduced 45S rDNA copy number (CN) to a plateau of ∼10%. Two independent lines had rDNA CNs reduced by up to 90% at the T7 generation, named low copy number (LCN) lines. Despite drastic reduction of rDNA copies, rRNA transcriptional rates, and steady-state levels remained the same as wild-type plants. Gene dosage compensation of rRNA transcript levels was associated with reduction of silencing histone marks at rDNA loci and altered Nucleolar Organiser Region 2 organization. Although overall genome integrity of LCN lines appears unaffected, a chromosome segmental duplication occurred in one of the lines. Transcriptome analysis of LCN seedlings identified several shared dysregulated genes and pathways in both independent lines. Cas9 genome editing of rRNA repeats to generate LCN lines provides a powerful technique to elucidate rDNA dosage compensation mechanisms and impacts of low rDNA CN on genome stability, development, and cellular processes.
Highlights
Ribosomal RNA genes are one of the most abundant repetitive elements in eukaryotic genomes and are typically clustered as tandem repeats containing hundreds or thousands of rRNA gene copies, known as rDNA and usually associated with the nucleolus
To determine the impact of reducing rDNA levels to their functional minimum in a model plant, we reduced the number of 45S rDNA copies in Arabidopsis thaliana using transgenerational Cas9 targeting of the 45S rDNA repeats (Figure 1)
This transgene cassette allows expression of Cas9 exclusively in the egg cell (EC) of the haploid female gametophyte, where we hypothesised that Cas9 activity across the 45S rDNA repeats would generate either large deletions or insertions of the repeats, through the subsequent activity of the error-prone Non-Homologous End Joining (NHEJ) DNA repair pathway (Figure 1C) (Cubbon et al, 2018)
Summary
Ribosomal RNA (rRNA) genes are one of the most abundant repetitive elements in eukaryotic genomes and are typically clustered as tandem repeats containing hundreds or thousands of rRNA gene copies, known as rDNA and usually associated with the nucleolus. Silenced 45S rDNA copies are involved in maintenance of genome stability and cell senescence (Kobayashi, 2014), highlighting their functional role(s) in the maintenance of cellular homeostasis. In mammals a role for silent rDNA copies in the maintenance of genomic stability is well established (Stochaj and Weber, 2020), where the reduction of 45S rDNA Copy Number (CN) (often coupled with amplification of 5S rDNA loci) has been associated with the onset of a range of cancers (Udugama et al, 2018; Xu et al, 2017)
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