Journal
FEBS LETTERS
Volume 596, Issue 15, Pages 1865-1870Publisher
WILEY
DOI: 10.1002/1873-3468.14365
Keywords
CRISPR; Cas9; genome editing; promoter fusion; transcriptional gain-of-function; transgene-free; plant oil metabolism
Funding
- UK Biotechnology and Biological Sciences Research Council [BB/P012663/1]
- Rothamsted International
- DST-INSPIRE Faculty fellowship schemes
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Achieving gain-of-function phenotypes without inserting foreign DNA is an important challenge for plant biotechnologists. In this study, the researchers demonstrated a method using dual-guide CRISPR/Cas9 to bring a gene under the control of a promoter from an upstream gene by deleting the intervening genomic sequence. By fusing the promoter of a nonessential gene with DGAT2, the researchers were able to drive ectopic oil accumulation in leaves, greatly enhancing DGAT2 expression and triacylglycerol content. This deletion strategy offers a transgene-free approach for engineering traits that rely on transcriptional gain-of-function.
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.
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