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

Abstract

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.

Applications and prospects of genome editing in plant fatty acid and triacylglycerol biosynthesis

“…For example, DGAT2 UPSTREAM GENE 1 ( DUG1 ) which exists upstream of DGAT2 has a higher expression in leaves than DGAT2 . Therefore, they deleted the 5′UTR region of DUG1 to the 5′UTR region of DGAT2 in Arabidopsis sdp1 mutant using CRISPR/Cas9 so that DGAT2 was controlled by the DUG1 promoter ( Bhunia et al, 2022 ). As a result, total lipid content (% cell dry weight) in leaves increased 2-fold and TAG content (% CDW) increased 30-fold compared to sdp1 mutant ( Bhunia et al, 2022 ).…”

“…Therefore, they deleted the 5′UTR region of DUG1 to the 5′UTR region of DGAT2 in Arabidopsis sdp1 mutant using CRISPR/Cas9 so that DGAT2 was controlled by the DUG1 promoter ( Bhunia et al, 2022 ). As a result, total lipid content (% cell dry weight) in leaves increased 2-fold and TAG content (% CDW) increased 30-fold compared to sdp1 mutant ( Bhunia et al, 2022 ). This method can be used only if the promoter direction of the upstream gene is appropriate ( Bhunia et al, 2022 ).…”

“…The scissor shape represents CRISPR/Cas9. By removing from the 5′UTR of the B gene to the 5′UTR of the A gene, the B gene can be controlled by the A promoter ( Bhunia et al, 2022 ). In addition, the transcription level can be regulated by deleting the cis -regulatory elements of the B gene.…”

“…As a result, total lipid content (% cell dry weight) in leaves increased 2-fold and TAG content (% CDW) increased 30-fold compared to sdp1 mutant (Bhunia et al, 2022). This method can be used only if the promoter direction of the upstream gene is appropriate (Bhunia et al, 2022). We think that the function of the upstream gene is irrelevant to plants since it will be eliminated by CRISPR/Cas9.…”

“…. By removing from the 5′UTR of the B gene to the 5′UTR of the A gene, the B gene can be controlled by the A promoter (Bhunia et al, 2022).…”