Orthogonal DNA double-strand break formation during meiosis

Lead Research Organisation: University of Aberdeen
Department Name: Biomedical Sciences

Abstract

Failure to generate sperm and egg cells during human reproduction causes an inability to conceive, miscarriages, and congenital disorders. Central to this process is a shuffling of genes within an individual's sex cells, achieved via a specialised cell division called meiosis. During meiosis chromosomes are re-assorted and recombined to produce haploid sex cells containing new chromosome configurations. This reshuffling involves the deliberate formation of DNA double-strand breaks by Spo11 and their subsequent repair into new configurations by meiotic recombination. Damaging hereditary material is potentially hazardous, but essential for chromosome segregation during meiosis, because it physically connects the equivalent maternal and paternal chromosomes during the reshuffling of the hereditary material.

Despite its implications for human health and animal well-being, meiotic recombination (the reshuffling of hereditary material) is still poorly understood on a mechanistic level. Nevertheless, this process is crucial for ensuring that traits of mating partners are distributed amongst the progeny, driving an increase in genetic diversity and fitness.

This project will make use of an orthogonal system, CRISPR-Cas9, to introduce DNA double-strand breaks circumventing the necessity of Spo11 function. Apart from Spo11 the inherent meiotic DNA breaking machinery requires a host of co-factors and the right (meiosis-specific) chromatin environment.

Using Schizosaccharomyces pombe as a model organism this project will ask the following research questions:

(1) Are CRISPR-Cas9-induced meiotic DNA breaks repaired differently from Spo11-induced breaks?

(2) Do crucially important co-factors of Spo11 influence the repair outcome of orthogonal DNA breaks?

(3) Does chromatin environment and chromosome organisation affect orthogonal DNA break formation and repair outcome?

These questions will be addressed by expressing various Cas9-constructs driven by a meiosis-specific promotor and targeting it to genetic reporters by appropriate single-guide RNAs. The Cas9-constructs envisaged are wild-type Cas9, nuclease-dead Cas9 fused to Spo11, and nuclease-dead Cas9 fused to chromatin modifiers. These experimental tools will enable us to understand:

(1) How Cas9 induces breaks and how these breaks are repaired (this will push our knowledge of how the clinically-relevant gene-editing tool Cas9 functions, and how its function is integrated into an orthogonal in vivo system).

(2) The contribution of Spo11 co-factors in downstream repair and recombination events, because it will be possible to induce DNA breaks by Cas9 and Cas9-Spo11 chimaeras without the help of Spo11 co-factors.

(3) How chromatin modifiers affect DNA break formation by targeting Cas9-chromatin modifier chimaeras to known hotspots of meiotic recombination and by targeting Cas9 and Cas9-Spo11 chimaeras to induce DNA breaks at repetitive genomic regions (such as centromeres and transposons) in wild type and in the absence of particular chromatin modifiers.

This project will deliver novel insight into how DNA double-strand breaks are repaired and how their repair affects meiotic recombination outcome, and will greatly expand our knowledge of meiosis. This will be delivered by employing an orthogonal DNA break machine (CRISPR-Cas9) in a genetically tractable model organism to specifically target DNA breaks and chromatin modification events; this will allow us to directly test how inherent factors important for DNA break formation itself affect downstream repair and recombination processes. This research will deliver important insight into the regulation and modulation of meiotic DNA break repair, and the mechanistic functionality of a state-of-art gene-editing tool, CRISPR-Cas9, orthogonally integrating into an in vivo system.

Publications

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Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M010996/1 01/10/2015 30/09/2023
1942702 Studentship BB/M010996/1 01/10/2017 30/09/2021 Samantha Jacqueline Mpaulo