BALTIMORE – Fresh off a $7.9 million seed funding round, genome engineering company Napigen is advancing its mitochondrial and chloroplast gene editing technology toward agricultural applications with an eye toward future human health applications.
The Wilmington, Delaware-based startup has proved the concept of its proprietary organelle genome editing in yeast mitochondria and Chlamydomonas chloroplasts and is further applying the method in wheat and rice for hybrid seed production.
Founded in 2016, Napigen was established by its president and CEO Hajime Sakai, along with three other cofounders, including Jay Keasling, a bioengineering professor at the University of California, Berkeley.
When the company was created, CRISPR had already been around for several years, "but nobody could really edit organelle DNA well," said Sakai.
About two years after its inception, Napigen came up with a novel CRISPR-mediated organelle genome editing technique and successfully carried out proof-of-concept experiments in yeast mitochondria and Chlamydomonas chloroplasts.
According to Sakai, Napigen's core technology, which the company named Edit Plasmids and described in a peer-reviewed study published in 2020, deploys Cas9 and guide RNAs (gRNAs) to induce double-strand cut sites in the organelle genome and carries a piece of donor DNA for gene editing. Edit Plasmids are introduced into organelles through the biolistic microprojectile method.
Given the method's success in yeast and Chlamydomonas, Napigen tested the technology in plants, specifically using rice as a model. However, one of the biggest challenges the company faced was that there was no set of marker genes discovered for plant mitochondrial genome editing.
"We started from scratch," Sakai said, adding that the company has since established selectable marker genes that can be used for gene editing in plant mitochondria.
In addition to plants, Sakai said he believes the marker genes can also be used in other organisms, including mammalian cells. "We are very excited," he said. "With the combination of the selectable marker genes and our gene editing technology Edit Plasmids, we have now a more generalized method to engineer organelle DNA beyond plants."
On that front, Sakai said the company is also testing the method in human cells to investigate how applicable the technology is in mammalian mitochondrial genomes. Earlier this year, Napigen secured a $225,000 grant from the NIH Small Business Innovation Research (SBIR) program to enable mitochondrial transformation and gene editing in human cells.
However, the company considers agriculture a foremost business target at this point.
"Our near-term goal is definitely crop plants," said Sakai, who led genetic discovery at DuPont's agronomic trait R&D division for almost two decades before starting Napigen.
One "fairly clear target" for the company, he said, is to produce hybrid seeds for crops using the mitochondrial DNA editing technique, specifically in wheat and rice. A common way to create more efficient crop systems in modern agriculture, plant hybridization can effectively introduce the desired qualities — such as drought tolerance, disease resistance, and increased yields — to the offspring by cross-pollinating parent crop varieties with the preferred traits, according to Sakai.
However, because many crops, including wheat and rice, are self-pollinating, it has been "very difficult" to crossbreed them with other plants, he explained. To mitigate that challenge, scientists normally need to generate male-sterile plants, which are unable to produce or release functional pollen within themselves to enable hybridization.
Since there are already known mutations in plant mitochondrial DNA that are associated with male sterility, Sakai said the next milestone for the company is to edit the mitochondrial genome to achieve male-sterile crop plants that can be cross-pollinated to produce hybrid seeds.
To that effort, Sakai said Napigen will leverage the $7.9 million raised in seed funding to support research. "Since we are dealing with plants, it can be a little more intensive in resources," he said, adding that part of the funding will be devoted to helping with the research infrastructure such as growing space, incubators, and the greenhouse. Sakai said the company already has a greenhouse facility in Newark, Delaware that can house hundreds of plants at once.
In addition, he said the company also hopes to bring on more business development employees. The company, which is located in DuPont's incubator space called the Experimental Station, currently has 12 employees, many of whom were his former scientist colleagues at DuPont.
Sakai said the company also plans to further validate its technology. While company researchers have not yet thoroughly examined the method's off-target effects, they plan to do that with the ongoing rice experiments.
Napigen's Edit Plasmids technology competes with other mitochondrial genome editing methods, such as CRISPR and transcription activator-like effector nucleases (TALENs)-mediated approaches, but from a business standpoint, Sakai said he does not see serious competitors at the moment. He added that Napigen currently has four active patent applications covering its technology in the US and plans to file two more this year.
Beyond agriculture, Sakai is hopeful that Napigen's technology can eventually be harnessed for human healthcare applications. If the method proves to be safe and effective in human cells, he envisions it being used to directly fix disease-causing mitochondrial mutations in patients and to produce cell lines of various genotypes for mitochondrial disease drug screening.