Scientists at the Department of Plant Science and Huck Institute of Life Sciences, Pennsylvania State University, have successfully applied the ‘CRISPR’ gene-editing technique to cocoa for the first time.
Use of the powerful gene-editing tool CRISPR-Cas9 could help to breed cocoa that exhibits desirable traits such as enhanced resistance to diseases, such as cocoa black pod (shown here), they believe.
In a paper entitled ‘Transient Expression of CRISPR/Cas9 Machinery Targeting TcNPR3 Enhances Defense Response in Theobroma cacao,’ they presented the first application of genome editing technology in cocoa, using Agrobacterium-mediated transient transformation to introduce CRISPR/Cas9 components into cocoa leaves and cotyledon cells.
“As a first proof of concept, we targeted the cocoa Non-Expressor of Pathogenesis-Related 3 (TcNPR3) gene, a suppressor of the defence response,” they explained. “After demonstrating activity of designed single-guide RNAs (sgRNA) in vitro, we used Agrobacterium to introduce a CRISPR/Cas9 system into leaf tissue and identified the presence of deletions in 27% of TcNPR3 copies in the treated tissues.”
The edited tissue exhibited an increased resistance to infection with the cocoa pathogen Phytophthora tropicalis and elevated expression of downstream defence genes.
Analysis of off-target mutagenesis in sequences similar to sgRNA target sites using high-throughput sequencing did not reveal mutations above background sequencing error rates.
The results confirm the function of NPR3 as a repressor of the cocoa immune system and demonstrated the application of CRISPR/Cas9 as a powerful functional genomics tool for cocoa.
Several stably transformed and genome edited somatic embryos were obtained via Agrobacterium-mediated transformation, and ongoing work will test the effectiveness of this approach at a whole plant level.
Their paper was published in Plant Science 9:268. doi: 10.3389/fpls.2018.00268