Functional remodelling of the Arabidopsis centromeres using CRISPR/Cas9

When cells divide, it is critical that each daughter receives a balanced set of chromosomes. Failure to segregate chromosomes accurately can cause genome instability and cancer. Each chromosome contains a dedicated region, called the centromere, that is required for correct segregation during cell division. The centromere acts to load a large complex of proteins called the kinetochore. The kinetochore is responsible for attaching the chromosomes to spindle fibres, called microtubules, that form during cell division. The microtubules act to pull the chromosomes to opposite sides of the cell and thereby achieve balanced segregation during division. Centromeres perform a deeply conserved cellular role across animals and plants. Despite this deep functional conservation, centromeres evolve extremely rapidly, and comprise divergent DNA sequences within and between species. This deep functional conservation, yet diverse sequence composition, is termed the centromere paradox. A further challenge to the study of centromeres, is that they are frequently composed of complex repeated sequences. Centromeric repeats have been impossible to correctly assemble with the previous generation of short-read sequencing technologies. However, new long-read sequencing methods allow the complete resolution of centromere repeats for the first time. For example, we have used long-read sequencing to assemble and study 330 centromeres from the model plant Arabidopsis, which has revealed extremely high levels of within-species sequence diversity. In this project, we aim to build on our foundational maps of the Arabidopsis centromeres, to functionally understand how their complex organization relates to their function during cell division. In recent years, the CRISPR/Cas9 system has emerged as a powerful system to direct pinpoint changes to the genome. For example, CRISPR has been used as 'molecular scissors' to delete target genes in a wide range of species. In this work, we will harness the CRISPR system in a novel approach that will target the centromere repeats. We have shown that this can cause dramatic deletions and rearrangements within the centromeres, which causes cell division defects. We will use CRISPR/Cas9 to dissect how centromeres remodelling influences their role in chromosome segregation. Beyond the DNA sequence, the centromeres are known to acquire epigenetic marks that are essential for their function, including DNA methylation. The CRISPR system can be further adapted to tether proteins of interest to target regions of the chromosome, including the centromeres. Therefore, we will use modified CRISPR systems to tether effectors of epigenetic information to the centromeres. This approach will allow us to test how epigenetic information influences centromere function during cell division, when the DNA sequence otherwise remains the same. A major theory for why centromeres evolve so fast, is that different centromere variants can selfishly compete. For example, during female reproductive development, it is common for an asymmetric cell division to form the egg cell. Selfish centromeres that can bias their inheritance into the egg cell have the potential to achieve an unfair transmission bias into the next generation. Consistent with this model, we have used sensitive fluorescently marked chromosomes to show that diverged European and African Arabidopsis centromeres show unequal transmission between generations. In the proposed work, we will use our remodelled centromeres to extensively test how they compete with both unmodified centromeres and between themselves. The proposed work will provide a comprehensive study of centromere genetic and epigenetic organization using a tractable model plant, and will reveal how this influences their role during cell division. Our work will have cross-cutting significance for centromeres across plant and animal species, with broad relevance for fertility and genome evolution.