A native promoter–gene fusion created by CRISPR/Cas9‐mediated genomic deletion offers a transgene‐free method to drive oil accumulation in leaves

Achieving gain‐of‐function phenotypes without inserting foreign DNA is an important challenge for plant biotechnologists. Here, we show that a gene can be brought under the control of a promoter from an upstream gene by deleting the intervening genomic sequence using dual‐guide CRISPR/Cas9. We fuse the promoter of a nonessential photosynthesis‐related gene to DIACYLGLYCEROL ACYLTRANSFERASE 2 (DGAT2) in the lipase‐deficient sugar‐dependent 1 mutant of Arabidopsis thaliana to drive ectopic oil accumulation in leaves. DGAT2 expression is enhanced more than 20‐fold and the triacylglycerol content increases by around 30‐fold. This deletion strategy offers a transgene‐free route to engineering traits that rely on transcriptional gain‐of‐function, such as producing high lipid forage to increase the productivity and sustainability of ruminant farming.