A donor-DNA-free CRISPR/Cas-based approach to gene knock-up in rice.

Structural variations (SVs), such as inversion and duplication, contribute to important agronomic traits in crops1. Pan-genome studies revealed that SVs were a crucial and ubiquitous force driving genetic diversification2-4. Although genome editing can effectively create SVs in plants and animals5-8, the potential of designed SVs in breeding has been overlooked. Here, we show that new genes and traits can be created in rice by designed large-scale genomic inversion or duplication using CRISPR/Cas9. A 911 kb inversion on chromosome 1 resulted in a designed promoter swap between CP12 and PPO1, and a 338 kb duplication between HPPD and Ubiquitin2 on chromosome 2 created a novel gene cassette at the joint, promoterUbiquitin2::HPPD. Since the original CP12 and Ubiquitin2 genes were highly expressed in leaves, the expression of PPO1 and HPPD in edited plants with homozygous SV alleles was increased by tens of folds and conferred sufficient herbicide resistance in field trials without adverse effects on other important agronomic traits. CRISPR/Cas-based genome editing for gene knock-ups has been generally considered very difficult without inserting donor DNA as regulatory elements. Our study challenges this notion by providing a donor-DNA-free strategy, thus greatly expanding the utility of CRISPR/Cas in plant and animal improvements.

The identification of an R2R3-MYB transcription factor involved in regulating anthocyanin biosynthesis in Primulina swinglei flowers.

Primulina genus is an ideal wild ornamental flower and emerging model for studying biosynthesis, diversity, and evolution of flower pigment. However, the molecular mechanism underlying anthocyanin biosynthesis and regulation in Primulina remains unknown. Here, changes in anthocyanin content and the expression profiles of anthocyanin biosynthetic structural genes were examined in developing Primulina swinglei flowers and three other organs. Seventy-three R2R3-MYB transcription factor genes were identified from transcriptome of P. swinglei flowers, two of which, PsMYB1 and PsMYB2, are candidate regulators of anthocyanin biosynthesis according to clustering analysis. Furthermore, transient over-expression studies using tobacco leaves showed distinct pigment accumulation following co-infection with PsMYB1 and MrbHLH1 (a previously confirmed anthocyanin regulator from Morella rubra). Additionally, dual luciferase assays showed that PsMYB1 trans-activated the PsANS promoter, with the addition of MrbHLH1 resulting in a 5-fold increase in the intensity of this interaction. PsMYB1 did not, however, have any effect on the PsF3H promoter. The expression profile and dual luciferase assays showed that PsMYB2 plays no roles in anthocyanin regulation. Therefore, PsMYB1 is proposed to be the transcription factor gene regulating anthocyanin biosynthesis in P. swinglei.