"Cas endonuclease-triggered genome modification in cereals"

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Thursday 28 March 2019, 11:00 - 12:30


Site-directed genome modification induced by RNA-guided Cas endonucleases offers unprecedented opportunities for the elucidation of gene functions and the improvement of crop performance. To increase the efficiency of platform establishment, extension and application, a modular and versatile vector system has been developed that relies on the ability of type IIS restriction enzymes to cut DNA outside of their recognition site thereby facilitating seamless DNA recombination procedures. This vector system allows not only for the simultaneous expression of multiple guide RNAs (gRNAs), but also, any newly emerging system components such as Cas derivatives with improved or novel functionality can be readily tested and utilized. The functionality of such vectors has already been confirmed in monocot and dicot species. In barley, for example, resistance to Bymoviruses, a useful grain phenotype and improved malting quality have been achieved. In further approaches, the architecture of barley and wheat was modified towards increased yield potential. However, the application potential of customizable endonuclease technology will be fully tapped only if elite germplasm can be used directly to generate the desired genetic modifications. Although there is a broad range of transformation methods at our disposal, the aspect of genotype-dependency still remains a major limitation in applied genetic engineering. A new concept for the delivery of RNA-guided Cas endonucleases involves the use of plant lines capable as male parent of inducing the formation of haploid progeny. There are a number of plant species in which the formation of maternal haploids can be achieved via cross-pollination followed by uniparental genome elimination, with wheat pollinated by maize being the most broadly utilized example. Upon genetic transformation of haploidy-inducing lines using gRNA and Cas endonuclease expression units, transgene-derived transcript and ribonucleoprotein are produced and transferred in their sperm via pollination and fertilization to (wheat x maize) hybrid zygotes formed on the female parent. While the wheat genome contributed by the mother plant is subjected to targeted modification, the transgene-carrying genome derived from the haploidy inducer (maize) is being completely eliminated in the context of the mitoses taking place in the early zygotic embryo that thereby becomes haploid. Resultant haploid progeny can be chemically induced to undergo whole genome duplication, by which entirely homozygous plants, so called doubled haploids, are being generated. Cross-pollination of wheat with gRNA/Cas9-transgenic maize was demonstrated to result indeed in site-directed mutagenesis in the wheat genome. In addition, our expectation was thereby confirmed that no gRNA-, Cas9- or selectable marker-encoding transgenes be present in the successfully modified wheat plants. This concept bears quite a number of particular advantages over current methods, with the potential reduction in genotype-dependency being the most important one. While site-directed mutagenesis has been exemplified in many crop species and is now being routinely used world-wide to produce knock-out lines, precise genome editing is still difficult to achieve and thus has been exemplified in only a few cases. Using homology-directed repair of site-directed DNA cleavage, we showed that GFP can be converted into YFP, albeit, for the time being, only at the cellular level. In conclusion, targeted genome modification technology holds great promise to be of high value in future breeding programs, provided that Europe will once enjoy a knowledge-based legal framework for plants generated by the use of customizable endonucleases.

Location Jozef Schell Seminar Room
Contact Dr Jochen Kumlehn
Leibniz Institute of Plant Genetics and Crop Plant Research (IPK)