Existing gene-editing technologies have led to significant advances in both medicine and food production. However, momentum appears to be slowing, particularly in health applications, as early hype is giving way to the realism of how difficult therapeutic development is turning out to be. Simply put, although major progress is being made, the field is struggling to put gene-editing tools into the healthcare market.
On the bright side, the safety issues that arise in medical research are not as large of a concern in the agricultural field, which continues to make steady progress. But scientists in both health and agriculture are delving deeper to uncover new solutions to tackle the tough issues. Building blocks such as recombinases, integrases, and transposases are being reexamined and enhanced with the use of AI to make them more specific and effective. New platforms are being introduced to insert larger DNA segments that potentially have a greater impact on both the human healthcare and agricultural fields.
Although the rate of progress may have slowed, new gene editing tool discoveries have the possibility to catapult the field into yet another growth spurt. As with all new scientific approaches it takes time and effort, but the results may be well worth it.
Mining for recombinases
The agriculture sector presents a particular set of needs. For example, traditional crop breeding is slow and requires screening of thousands of variants, but no high-throughput screening process exists. Decreasing the number of progeny would help expedite new trait creation.
Compared to therapy development, off-target effects in agricultural applications are not as limiting. More variation increases the chances of finding a new plant or trait. Once desired mutations are identified gene editing can be used to generate them more precisely. “We needed a technology that would allow us to efficiently and precisely insert large DNA cargoes into a ‘safe harbor’ region in the genome,” explained Kevin Zhao, PhD, co-founder and CTO, Qi Biodesign.
Prime Root editors are fundamentally based on recombinase technologies. While these enzymes allow large DNA insertions, they only recognize specific recombinase recognition sites. “These recombinase recognition sites rarely occur in nature where we want to insert our genes,” Zhao said. “So we need to first integrate these recombinase recognition sites into the targeted region then utilize recombinases for performing large DNA integration.”
The challenge is assembling the different pieces, the small recognition site and the large cargo, to generate a final gene edit. “The recombinase step limits Prime Root editors. We hypothesized that this was due to the recombinases themselves so we evolved these enzymes and also used protein structure-based AI predictions to mine for new ones to increase overall editing efficiency,” added Zhao.
Previous mining has traditionally been based on sequence similarity. Since the structure of a protein dictates its function, new proteins can be missed using this traditional sequence-based approach.
Recombinase efficiency increases allow Qi Biodesign to insert 20-30 kb (5 or 6 genes) at one time at one location across a variety of different crop genetics. In addition, a large team at Qi Biodesign works on plant transformation, another bottleneck in plant breeding innovations.
Gene-edited crops are regulated differently than traditional GMOs: the regulations are clearer and becoming more streamlined. Yet it remains a challenge to bring these crops to market. ”A lot of work is needed to get the world on the same page,” emphasized Zhao. “Agriculture products are commodities. Approval needs to be global.” Currently, Qi-Biodesign has 5 approvals in China and 1 in the U.S.
Re-inventing integrases
Despite remarkable advancements, existing genome engineering technologies integrate DNA randomly, require double-strand DNA breaks that risk unintended mutations, or are constrained by the size of DNA cargo they can insert.
KOMO Biosciences, under the leadership of its CEO Jennifer Manning, is pioneering a transformative solution to these challenges with its next-generation, precision genome-engineering platform.
KOMO’s innovation is its high-efficiency serine integrases that enable precise, site-specific integration, which offer a new standard for precision and reliability. Site-specific recombination enables precise modifications of the genome by facilitating the exchange of DNA segments between specific sequences.
This process is mediated by site-specific recombinases (SSRs), enzymes that recognize defined DNA sites, cleave the DNA backbone, exchange strands, and rejoin them. These natural systems play essential roles in bacterial genome replication, differentiation, and the movement of mobile genetic elements.
Among SSRs, serine integrases are particularly effective at inserting large DNA sequences into designated genomic sites. In addition, serine integrases function independently of cellular repair mechanisms, reducing the risk of unintended mutations. They do not require sequence homology between the donor and target DNA, making them highly versatile for genome engineering applications.
A key advantage of serine integrase-mediated recombination is that it does not induce DNA damage or rely on host cell factors, thereby avoiding the activation of error-prone repair pathways. This is particularly important for maintaining genomic integrity, as disruptions in pathways such as p53-mediated DNA damage response can lead to unintended selection for cells with impaired p53 function.
The first application of KOMO’s integrase technology is the development of rapid, stable, clonal cell lines, to be followed by genomically-directed serine integrases for in vivo cell therapy. “We believe that KOMO’s technology has the potential to significantly accelerate therapeutic development, improve the scalability and predictability of manufacturing, and reduce complex supply chains,” Manning explained. “This will improve access to and affordability of life-saving therapeutics.”
De novo on-off switches
“We are all painfully aware that most of the genome-editing tools we have today are great, but just not good enough,” said Jeff Graf, PhD, co-founder, Atelas Biosciences. “We still lack a tool that is both highly specific and highly efficacious. In addition, even the best of the second-generation tools, such as prime editing, cannot perform large DNA rewriting or large DNA insertions.”
Safety relies on the specificity and effectiveness of the tool. Although off-target edits, programmability, and targetability are all important, maintaining a high editing rate ranks higher because of its broad impact.
Atelas Biosciences is developing a technology that relies on existing building blocks. “Our technology ticks all the boxes,” said Graf. “It has high integration efficiency, is fully programmable, works in any tissue, and is specific. We incorporated all of the factors that influence safety to innovate a best-in-class genome editor that is also versatile and simple.”
The platform is based on well-characterized nucleases and transposases with the addition of another novel building block, a mechanism of control that activates and deactivates the transposition activity. “In the same system you have the functionality of the nuclease to go to the right place in the genome due to a RNA guide, and the activity of the transposase to insert large pieces of DNA,” said Luis Iniesta, co-founder, Atelas Biosciences.
The control mechanism is a unique AI-generated, de novo protein function that is inserted into the system and acts as an on-off switch for the transposase activity. This allows the transposase activity to be controlled by the nuclease activity in such a way that there are no unspecific transposition events. Activity only occurs when the nuclease has arrived at the correct location in the genome.
The high activity and large fragment insertion ability of the transposase is maintained and combined with nucleases that are specific and programmable, thus providing efficacious and specific DNA insertion. This targeted, highly-efficient, large insertion DNA gene-editing tool has many potential applications in both agriculture and human therapy development.