Cas Endonuclease Technology—A Quantum Leap in the Advancement of Barley and Wheat Genetic Engineering

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.

Site‐directed mutagenesis in bread and durum wheat via pollination by cas9/guide RNA‐transgenic maize used as haploidy inducer

“…Site‐directed mutagenesis facilitates the experimental validation of gene function and can speed up plant breeding by producing new genetic variability or by reproducing previously known gene variants in other than their original genetic backgrounds. However, its application is challenging in wheat owing to high genomic redundancy and highly genotype‐dependent DNA transfer methods (Koeppel et al ., 2019). In wheat, large chromosomal regions are hardly amenable to meiotic recombination, which limits the potential for trait improvements.…”

Conversion of hulled into naked barley by Cas endonuclease-mediated knockout of the NUD gene

“…With the emergence of RNA-guided Cas endonucleases, the number of examples of site-directed genome modification has been strongly increasing in crop plants [13]. The technology is currently well established in barley, including both reverse genetics studies and improvement of agronomically important characteristics [14].…”

Engineering Smut Resistance in Maize by Site-Directed Mutagenesis of LIPOXYGENASE 3

“…Upon cleavage at the genomic target motif, the resultant DNA double-strand break is recognized and processed by the cells endogenous repair mechanisms, which, in the case of the error-prone non-homologous end-joining, entails the formation of random nucleotide insertions and/or deletions (Nishitoh et al, 2002;Puchta and Fauser, 2014). This novel principle of genetic engineering has been well established in the majority of important crop plants (Kumlehn et al, 2018;Koeppel et al, 2019). Here, Cas9 endonuclease-triggered mutagenesis of maize LOX3 is reported.…”