Gene Editing in Cereals

Gene editing can be an effective tool to precisely cleave a specific 20 bp DNA sequence in a genome that is then repaired by either error-prone nonhomologous end joining (NHEJ) or homology-directed repair (HDR). The specificity of this process offers significant advantages for deciphering gene function and improving crop traits. In this study, we explore in wheat two popular gene editing technologies, CRISPR and TALENs. In a pilot CRISPR experiment in rice we were able to effectively knock-out two closely-linked genes which modulate seed development with very high efficiency, with double mutants displaying expected seed phenotype. However, the same constructs targeting homologous genes in wheat failed to generate mutations. In contrast, two pairs of TALEN constructs were shown to efficiently modify wheat target loci. The first locus, a GUS transgene, was edited in 3 out of 9 transgenic lines. The second locus, an endogenous pseudogene of the Lr21 rust resistance gene was modified in 26 out of 40 transgenic lines produced. This latter psuedogene gene contains a one-base deletion that introduces a frame shift mutation in the gene ORF. TALEN modified alleles in which the gene ORF is restored by NHEJ are being sought and progeny of these plants are being tested for the rust resistance. Our results suggest that TALENs may be a better approach for wheat genome editing.