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Multidimensional CRISPR/Cas mediated engineering of plant breeding

Periodic Reporting for period 2 - CRISBREED (Multidimensional CRISPR/Cas mediated engineering of plant breeding)

Reporting period: 2019-04-01 to 2020-09-30

The basic principle of classical plant breeding is to combine advantageous traits in crops like disease resistance and high yield and at the same time eliminate unfavorable traits like bitter taste. For this purpose, the breeder is crossing an elite cultivar of a crop with close relatives. The genetic information coding for all traits is coded in genes, which are linearly packed like beads on a string on a smaller number of individual chromosomes. By crossing, an exchange of traits between parental chromosomes can be achieved. However, in many cases individual traits cannot be combined due to their specific position on a chromosome, especially if they are close to each other. Thus, it is often not possible for the breeders to obtain crops resistant to certain pests and the farmer has to use pesticides to protect the harvest. Also due to global warming, the heat and salt resistance of various crop plants needs to be improved, to avoid deleterious effects in future. These very important challenges for our global agriculture could be achievable by the development of specific technologies that allow controlled changes of plant chromosomes. Thus, it should be possible to change the structure of individual chromosomes as well as to the exchange segments between different chromosomes. Till now, the use of molecular scissors like CRISPR/Cas was restricted to the change of an individual and or a few genes. The current project is aiming to establish now novel CRISPR/Cas based technologies for plant chromosome engineering. Thus, we hope to enable the development a new kind of breeding to cope with the challenges of the 21th century.
The first 30 months of the project were dedicated to demonstrate that heritable CRISPR/Cas mediated chromosome engineering is possible¬ and to define which experimental conditions and which tools are best to achieve these changes most efficiently. It turned out that the best conditions for genome engineering in the model plant Arabidopsis are obtained if we use the molecular scissors CRISPR/Cas specifically in egg cells for cutting. The broken ends of the chromosomes are ligated by the use of the “non homologous end joining” pathway, mostly without loss of information. If we perform our experiments in absence of a factor that keeps the broken ends together in the correct orientation, we were even able to increase the frequency of the formation of chimeric chromosomes. Applying this technology, we were already able to achieve two major breakthroughs: For the first time we were able to obtain plants in which by the use of CRISPR/Cas arms were exchanged between different chromosomes. Thus, it is possible to exchange traits between chromosomes so that either favorable novel combinations of traits can be formed or unfavorable combinations can be resolved. Both outcomes are important for breeding.

On the other hand, we were able to reverse the orientation of a longer segment of an Arabidopsis chromosome that was inverted during evolution. This original inversion had the consequence that all traits inside this segment were located like beads on a string in the opposite orientation and thus not accessible for a genetic exchange, anymore. After the reversion, we could show that these traits can be exchanged now again between the parental genomes. As many crop varieties carry inversions on various chromosomes this technology should enable breeders to combine and break genetic linkages between traits, that were not accessible for breeding before.
We were already able to achieve a decisive goal within the first 30 month of the project. For the first time worldwide, we were able to establish heritable plant chromosome engineering. Thus, we definitely went already beyond the current state of art of plant genome engineering. In future, we want to develop the technology further quantitatively but also qualitatively. Quantitatively by bringing the unlinked chromosome ends together that should be rejoined or making them more accessible for the machinery involved in the rejoining process. Qualitatively by inducing novel major chromosomal rearrangements within but also between chromosomes. We are also in the process to test whether we are able to induce recombination between parental chromosomes in regions that are not accessible for classical breeding. Moreover, we started to develop a novel technology based on CRISPR/Cas for genome haploidization, the use of which would also be highly beneficial for breeding.
Last but not least, we began to transfer our knowledge obtained in the model plant Arabidopsis to the crop plant tomato to transfer a disease resistance gene from a wide relative into the crop plant.