Developing Genome Editing Technologies for Crop Improvement

Genome editing using sequence-specific nucleases (SSNs) provides good opportunities for crop improvement. The precise genome modifications involved are generated by SSNs, namely zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regulatory interspaced short palindromic repeats/CRISPR-associated systems (CRISPR/Cas). SSNs enable precise genome engineering by introducing DNA double-strand breaks (DSBs) that subsequently trigger DNA repair by non-homologous end joining (NHEJ) or homologous recombination (HR). By exploiting NHEJ and HR, SSNs can be used to generate targeted genome modifications including mutations, insertions, replacements and chromosome rearrangements. To date, SSNs have been used to create gene knock-out plants in a variety of important crops, such as rice, maize, wheat, barley and soybean. However examples of real improvement of agronomic traits or the creation of excellent novel genotypes using SSNs are very limited. A successful example occurred in bread wheat. By knocking out all six alleles encoding the MILDEW-RESISTANCE LOCUS (MLO) protein with one pair of TALENs, we generated a mutant line with broad-spectrum resistance to powdery mildew, a devastating fungal disease. We provide another example of the use of SSN to disrupt biochemical pathways and create plants that accumulate valuable biosynthetic intermediates. We used TALEN technology to knockout OsBADH2 and obtain homozygous mutants with significantly increased content of the fragrant chemical 2AP; the level produced is similar to or even higher than in the fragrant rice cultivar Daohuaxiang. Our work provides plant breeders good examples of the use of SSNs for molecular breeding of crop plants.