CRISPR/Cas9-Mediated Gene Targeting for Crop Improvement

Abstract: CRISPR/Cas9 technology enables precise modifications of DNA sequences in vivo and offers a great promise for harnessing plant genes for crop improvement. However, the majority of the reported gene editing events in plants relied on error-prone non-homologous end joining (NHEJ) repair mechanism to generate mutations and gene knock-outs. Development of a technology that facilitates gene replacement rather than gene inactivation will greatly improve plant breeding by allowing the introduction of precise point mutations or enabling new functions. Homology-directed repair (HDR) of a DSB at specific locations generated by CRISPR/Cas9 potentially can provide a feasible approach to achieve gene replacement. Here, we employed CRISPR/Cas9-mediated in planta gene editing to simultaneously replace tryptophan 548 and serine 627 in the rice ALS gene with leucine and isoleucine, respectively. The precise substitutions of the two discrete amino acid residues in ALS were achieved by co-bombarding rice calli with a plasmid harboring Cas9/gRNAs expressing cassettes along with a repair template and free double-stranded DNA repair template fragment. We successfully recovered multiple T0 plants that were homozygous for the designed substitutions. The rice plants with the edited ALS gene were resistant to Bispyribac-sodium, an ALS inhibiting herbicide (Sun et al., 2016, Mol Plant, 9: 628-631). Meanwhile, simultaneous substitutions of two amino acid residues of wheat ALS are under way. Taken together, these results demonstrate that we can not only generate herbicide-resistant plants, but also precisely substitute amino acid residues in plant proteins through CRISPR/Cas9-mediated HDR, greatly expanding the ability to modify genes that confer agriculturally important traits in crops.