Abstract
Domestication and breeding have created productive crops that are adapted to the climatic conditions of their growing regions. Initially, this process solely relied on the frequent occurrence of spontaneous mutations and the recombination of resultant gene variants. Later, treatments with ionizing radiation or mutagenic chemicals facilitated dramatically increased mutation rates, which remarkably extended the genetic diversity of crop plants. However, a major drawback of conventionally induced mutagenesis is that genetic alterations occur simultaneously across the whole genome and at very high numbers per individual plant. By contrast, the newly emerging Cas endonuclease technology allows for the induction of mutations at user-defined positions in the plant genome. In fundamental and breeding-oriented research, this opens up unprecedented opportunities for the elucidation of gene functions and the targeted improvement of plant performance. This review covers historical aspects of the development of customizable endonucleases, information on the mechanisms of targeted genome modification, as well as hitherto reported applications of Cas endonuclease technology in barley and wheat that are the agronomically most important members of the temperate cereals. Finally, current trends in the further development of this technology and some ensuing future opportunities for research and biotechnological application are presented.
Highlights
In cases where genetic modifications are intended to be performed in genotypes other than those with reference genomes, it is recommended to countercheck pre-selected target motifs for their presence and integrity, because even single nucleotide polymorphisms typically result in a dramatic drop in guide RNA (gRNA)/Cas efficiency
After selection-free plant regeneration, mutants were identified with a frequency of more than four percent, which is on a par with the efficiency of conventional wheat transformation
Current utilization of Cas endonuclease technology is still largely limited to random alterations of the DNA sequence at the user-defined genomic sites. This comparatively simple approach of site-directed mutagenesis relies on the error rate of the DNA repair mechanism, called non-homologous end-joining (NHEJ), which is predominant in plant cells
Summary
For more than 10,000 years, plants have been cultivated to feed humans and their livestock, as a source of raw materials and to generate energy. The rate of spontaneous mutations is too low to keep up with the demands for crop improvements. It is in the nature of spontaneous mutations that they are unpredictable in terms of both position and resultant nucleobase sequence. Induced mutagenesis breeding has so far produced over 3000 approved varieties [7] and for many further cultivars it has not been documented which induced mutations they have inherited from germplasms they derive from Using this technology, thousands of mutations occur at different sites in a single plant’s genome at a time. Already in 2012, a detailed draft of the barley genome was published [15], which was recently complemented by much improved data, including genomic sequences of a large number of representative gene bank accessions [16,17]
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