“Our research team targeted the gene TcNPR3 because we learned from earlier studies that it acts as a molecular ‘brake’ on the plant’s natural defense system,” Guiltinan said. “NPR3 proteins — the family to which TcNPR3 belongs — are negative regulators of plant immunity, essentially preventing plants from mounting robust defenses against pathogens when they’re not immediately under attack.”
Think of NPR3 as a security system that’s set to “standby mode,” he explained. Disrupting the gene turns on its “high alert” mode, increasing the plant’s natural defenses and making the plant less susceptible to pathogen attacks.
Perhaps more novel than using CRISPR-Cas9 technology to precisely mutate the TcNPR3 gene, Guiltinan said, was using traditional plant breeding to eliminate the foreign DNA sequences associated with the gene-editing machinery.
The result — cacao plants that contain only the desired genetic modifications without any transgenic elements — establishes a significant regulatory precedent, Guiltinan said, because the U.S. Department of Agriculture (USDA) has determined that these edited plants are not subject to biotechnology regulations since they contain no foreign genetic material — only precise edits to native cacao genes. After review of extensive data in the study manuscript, the USDA officially stated that it does not consider the genome-edited cacao lines to meet the same regulation requirements as genetically modified plants. However, the plants may still come under regulation by the U.S. Food and Drug Administration, Guiltinan said, “but that is down the line.”
This regulatory clarity removes a major barrier to adoption, Guiltinan suggested, adding that next the team will have to test the lines outside on research stations in tropical areas.
“We need to assess the plants’ performance outside of our greenhouses,” he said. “If successful, our hope is that someday soon, farmers and consumers can benefit from these disease-resistant plants to improve their livelihoods and protect the environment.”
Guiltinan and his team are assessing additional targets to increase disease resistance, as well as exploring new methods of genome editing, with the goal of developing a second generation of genome-edited cacao lines in the coming years.
“We’re not just creating better cacao plants — we’re exploring how modern biotechnology can work within existing regulatory frameworks to address real-world agricultural challenges,” Guiltinan said. “Traditional breeding approaches are slow, often taking decades to develop new resistant varieties. For the millions of farmers who depend on cacao cultivation, and the billions who enjoy chocolate, this research offers hope for a more sustainable and secure future — one precise genetic edit at a time.”
Other members of the team contributing to the research were: Lena Landherr, assistant research professor in plant science; Siela Maximova, research professor of plant biotechnology and co-director of the Endowed Program in the Molecular Biology of Cocoa at Penn State’s Huck Institutes of the Life Sciences; Dante DelVecchio, graduate student in plant science; Aswathy Sebastian, bioinformatics analyst at the Huck Institutes; and Istvan Albert, research professor of bioinformatics at the Huck Institutes.
This research was supported by the Huck Institutes of the Life Sciences and the Penn State Endowed Program in the Molecular Biology of Cacao.