Meiotic recombination in plants-controlling the transition of DNA double-strand breaks to genetic crossovers – MEIOREC

The main objective of this project is to develop an in vivo system to analyze the formation of meiotic double strand breaks (DSB). The methods used will be based on the ability of the CRISPR dCas9 system to deliver proteins of interest to any location of the genome. We will take advantage of this characteristic to deliver to a target reporter gene the DSB forming machinery known in plants (in whole or in part) and test the appearance of DNA lesions at the target site.

As part of the start of the MEIOREC project, all the required tools have been generated and preliminary analyzes were carried out to demonstrate the feasibility of the system (delivery of an active endonuclease to the chosen target reporter gene). We are now in a position to move on to the meiotic DSB proteins themselves.

The ability of the dCas9 to deliver a nuclease protein complex to a given DNA locus in P. patens cells having now been demonstrated, we have in hand a powerful tool for in vivo testing of the functionality of any nuclease complex. We will therefore use the second part of the MEIOREC project to analyze the possible nuclease activity of the meiotic SPO11s / MTOPVIB complex.

Demonstration of the functionality of a modular Cas9 platform for editing of the APT locus in P. patens :
publication
Guyon-Debast A, Alboresi A, Terret Z, Charlot F, Berthier F, Vendrell-Mir P, Casacuberta JM, Veillet F, Morosinotto T, Gallois JL, Nogué F. A blueprint for gene function analysis through Base Editing in the model plant Physcomitrium (Physcomitrella) patens. New Phytol. 2021 Jan 9. doi: 10.1111/nph.17171. Epub ahead of print. PMID: 33421132.

Genetic variation generated through the process of homologous recombination (HR) during meiosis underpins plant breeding and efforts to deliver the rapid improvements in crops that will be required to ensure Food Security into the foreseeable future. HR is initiated by the programmed formation of DNA double-strand breaks (DSBs) by the SPO11 complex. DSBs are processed by components of the HR pathway and are repaired as crossovers (COs), which recombine the homologous parental chromosomes, or non-crossovers (NCOs), where only short stretches of DNA are exchanged. In plants most DSBs (90%+) are repaired as NCOs1. This limits the genetic variation that is generated in each meiotic division. Moreover, the distribution of COs, notably in cereal crops, is skewed towards particular chromosomal regions. The extensive work and collaborative efforts of the MEIOREC investigators have led to significant progress in understanding the basis of these limitations and have made headway in addressing them1. Nevertheless, full understanding of how the transition of a DSB to yield a CO is controlled at a particular genomic locus and how this can be optimized remains a major challenge. We believe that the combined expertise of MEIOREC consortium provides a real opportunity to make substantial progress in addressing this important problem. Controlled formation of meiotic COs involves a coordinated programme whereby DSBs are channelled through a series of transitions to form either CO or NCO products.
The aim of MEIOREC is to decipher the DSB to CO transitional steps and evaluate strategies to manipulate CO formation using model species thereby laying the foundation for subsequent translation of the most promising into crops. This will be implemented by a set of workpackages (WPs) with specific scientific objectives (SOs): WP1: Factors controlling DSB formation; WP2: DSB processing and CO resolution; WP3: Relationship of chromosome remodelling and CO formation.