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 crops 1 . Pan-genome studies revealed that SVs were a crucial and ubiquitous force driving genetic diversification 2 – 4 . Although genome editing can effectively create SVs in plants and animals 5 – 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, promoter Ubiquitin2 ::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.