breeding-design-for-functional-rice-with-genome-editing

Breeding by Design for Functional Rice with Genome Editing Technologies

The conventional approaches to crop breeding, which rely predominantly on time-consuming and labor-intensive methods such as traditional hybridization and ...

Bellagen Biotechnology Co. Ltd

Shandong Normal University

Amylose content (AC), which is regulated by the Waxy (Wx) gene, is a major indicator of eating and cooking quality (ECQ) in rice (Oryza sativa). Thus far, only a limited number of mutations in the N-terminal domain of Wx were found to have a major impact on the AC of rice grains and no mutations with such effects were reported for other regions of the Wx protein. Here, nucleotide substitutions in the middle region of Wx were generated by adenine and cytosine base editors. The nucleotide substitutions led to changes in 15 amino acid residues of Wx, and a series of novel Wx alleles with ACs of 0.3%-29.43% (wild type with AC of 19.87%) were obtained. Importantly, the waxy allele showed a "soft rice" AC, improved ECQ, favorable appearance, and no undesirable agronomic traits. The transgenes were removed from the waxy progeny, generating a promising breeding material for improving rice grain quality.

Novel Wx alleles generated by base editing for improvement of rice grain quality.

The microRNA miR396 directly represses  () and has been implicated in regulating rice yield and in nitrogen assimilation. Overexpressing the miR396 targets  and  improves rice yield via increased grain size and panicle branching, respectively. Here, we used CRISPR/Cas9 to assess the function of miR396 genes in rice. Knockout of  ( and ), but not other isoforms, enhanced both grain size and panicle branching, resulting in increased grain yield. Importantly, under nitrogen-deficient conditions,  mutants showed an even higher relative increase in grain yield as well as elevated above-ground biomass. Furthermore, we identified  as a new target of miR396, in addition to the known targets  and . Disruption of the miR396-targeting site in  was sufficient to both enlarge grain size and elongate panicles. Our results suggest that rice-seed and panicle development are regulated by miR396ef-GRF4/6/8-GIF1/2/3 modules and that miR396ef are promising targets of genome editing for breeding environmentally friendly rice varieties that require less nitrogen fertilization.

Disruption of  and  improves rice yield under nitrogen-deficient conditions.

The CRISPR/Cas type V-I is a family of programmable nuclease systems that prefers a T-rich protospacer adjacent motif (PAM) and is guided by a short crRNA. In this study, the genome-editing application of Cas12i3, a type V-I family endonuclease, was characterized in rice. We developed a CRIPSR/Cas12i3-based Multiplex direct repeats (DR)-spacer Array Genome Editing (iMAGE) system that was efficient in editing various genes in rice. Interestingly, iMAGE produced chromosomal structural variations with a higher frequency than CRISPR/Cas9. In addition, we developed base editors using deactivated Cas12i3 and generated herbicide-resistant rice plants using the base editors. These CRIPSR/Cas12i3-based genome editing systems will facilitate precision molecular breeding in plants.

Jinshan Zhang

Bellagen Biotechnology Co. Ltd

Breeding by Design for Functional Rice with Genome Editing Technologies