With Europe moving towards a new regulatory framework for New Genomic Techniques (NGTs), DNA-free genome editing is receiving renewed attention. But at Hudson River Biotechnology, regulation was never the reason we chose this route.

We started developing our BioMaas workflow well before the 2018 ruling of the Court of Justice of the European Union. Our motivation was technical, not regulatory: introducing foreign DNA adds unnecessary complexity to the genome-editing process, making development slower, less predictable and harder to scale. Even if regulation had never become an issue, we would still have chosen to develop a DNA-free approach.

Our goal was to engineer a workflow that breeders could rely on repeatedly across projects, varieties and, ultimately, crops.

Why avoid using foreign DNA?

Many conventional plant genome-editing workflows rely on introducing foreign DNA in the form of transgenes that encode the editing machinery and allow it to be produced inside the plant cell. These constructs first need to be designed and assembled, often requiring adaptation to the species being edited. A promoter that efficiently drives expression of the editing machinery in one crop may not work nearly as well in another. For every new target, a DNA construct that encodes the appropriate RNA guide needs to be generated.

Our BioMaas platform takes a fundamentally different approach.

Instead of providing plant cells with the genetic instructions to produce the editing machinery, we deliver the editing machinery itself being a pre-assembled CRISPR RiboNucleoProtein (RNP) complexed with a guide RNA, that determines the editing location in the genome. There is therefore no need to develop and introduce a DNA construct for every editing procedure.

Once inside the cell, the RNP gets to work immediately. It scans the genome to find its target, makes a double strand break that is repaired by the plants genomic DNA repair machinery, causing the intended ‘edit’. Within a couple of days the RNP is degraded by the cell.

That temporary presence has an important advantage. When DNA encoding the editing machinery integrates into the plant genome, the machinery can continue to be produced until those transgenes are removed through subsequent breeding. Editing may therefore continue well beyond the initial transformation event, potentially creating additional or different editing outcomes over time.

With RNP delivery, the editing window is short and defined. Once the machinery is gone, editing stops.

If you cannot select edited cells, make editing efficient enough that you don't need to

- there is, however, a trade-off.

DNA-based transformation systems often include selectable marker genes, allowing researchers to select cells that have successfully received the construct. When you deliberately avoid introducing transgenes, you lose this convenient selection mechanism.

We knew from the beginning that this was an obstacle BioMaas would have to overcome.

The answer was editing efficiency.

After several rounds of optimisation, including mastering the production of highly pure CRISPR enzymes and efficient transfection of plant cells without cell walls, known as protoplasts, we achieved very high editing efficiencies.

The principle is surprisingly intuitive. An editing event requires the CRISPR machinery to encounter its target sequence in the genome during the limited period in which that machinery remains active. In that sense, genome editing has something in common with real estate: location, location, location!

By efficiently delivering high concentrations of active editing machinery directly into individual cells, we greatly increase the probability of that encounter.

High efficiency means we no longer need a selectable marker simply to find the occasional edited cell among thousands of unedited ones. It also opens doors to more ambitious editing strategies in the future.

With BioMaas, we can perform multiplex editing, targeting several genomic sites simultaneously, as well as pursue precise editing approaches in which a defined genomic sequence is replaced or altered.

One cell in, one genetically uniform plant out

Efficient editing is only one part of our BioMaas platform.

The other is regeneration.

After delivery of the editing machinery, we regenerate plants from individual protoplasts. Each regenerated plant therefore originates from a single edited cell.

This matters.

In transformation approaches where plants are regenerated from multicellular tissues or cell masses, different cells may carry different editing outcomes. Combined with editing machinery that remains active over time, this can result in genetically chimeric plants and create additional work before a stable genotype is obtained.

With BioMaas, editing and regeneration start at the single-cell level. The resulting plants are therefore genetically uniform from the outset.

After regeneration, plants are genotyped and those carrying the desired editing outcome can be selected immediately. Because no foreign DNA was introduced as part of the editing process, there is no transgene that subsequently needs to be crossed out.

The result is a remarkably direct workflow: isolate single cells, deliver the editing machinery, regenerate individual cells into plants, genotype them and select the desired edited plant.

Nothing needs to remain from the editing process except the intended genetic change.

DNA-free editing is only half the challenge

Of course, delivering CRISPR machinery efficiently does not automatically give you an edited plant.

The harder biological challenge is often convincing a single plant cell to become a complete plant again. Regeneration biology differs enormously between crops, and frequently even between varieties within the same crop.

This is where starting early was crucial for Hudson River.

Over the years, we have invested heavily in understanding and engineering single-cell regeneration. That has allowed us to establish the BioMaas platform across multiple species of crops including tomato, strawberry, white cabbage, cauliflower, lettuce and sugar beet, with others continuing to be developed.

For breeders, this distinction is important. A genome-editing tool demonstrated in a model plant is scientifically interesting, but a reproducible system that can take a commercially relevant variety, edit it and return genetically uniform plants is something very different.

That is the problem that our BioMaas platform was designed to solve.

… then regulation caught up

Which brings us back to Europe.

The emergingNGT framework of the EU is intended to create a more proportionate regulatory pathway for certain genetically edited plants carrying changes that could also arise through conventional breeding or naturally. For European breeders, this could substantially change the practical possibilities for using genome editing in commercial variety development.

Interestingly, this also makes many of the technical choices Hudson River made years ago increasingly relevant.

BioMaas was not designed to make plants fit a particular regulatory definition. We designed it to avoid unnecessary biological and technical complexity. But a workflow that introduces no foreign DNA, leaves behind only the intended genetic change and produces genetically uniform plants directly is naturally well suited to a breeding environment where the final genetic outcome matters.

That distinction is important. Regulation can change - and it has, but good technology should still make sense when it does.

Climate change, emerging diseases, pressure to reduce agricultural inputs and rapidly changing consumer and market demands require breeders to move faster than conventional breeding alone often allows. Genome editing gives breeders an extraordinary tool to introduce targeted genetic variation, but its value depends on whether that tool can be translated into actual varieties efficiently and repeatedly.

We believe genome editing should not be slowed down by unnecessary technical complexity.

That is why we built BioMaas: not because regulation demanded it, but because modern plant breeding did.