Absent in the buzz surrounding therapeutic genome editing is acknowledgement of the time factor: Such treatments are years, if not decades, away. While Hollywood is unlikely to produce thriller docu-series about plant genome editing, the potential to realize tangible benefits by editing the essential life-code of plants has already been demonstrated.
The potential to realize tangible benefits by editing the essential life-code of plants has already been demonstrated.
One of the knocks against genetically modified plants is their ability to breed with organisms within their species, thereby spreading the mutant trait to “normal” plants. Maciej Maselko, a synthetic biologist at the University of Minnesota, has used CRISPR CAS9 gene editing to create genetically modified organisms that do not produce viable offspring when they breed with their unmodified counterparts. In essence Maselko has created a synthetic species that is identical to the original in every way but one.
“We’ve developed a method to engineer speciation events,” Maselko says.
Speciation occurs in nature when organisms like grizzly bears and polar bears are separated by geography. The other speciation mechanism is illustrated by post-zygotic isolation: When goats and sheep mate their offspring are not viable.
Engineering a speciation event requires two changes, Maselko explains. The first is a mutation to the gene’s promoter region—the regulatory area upstream of a coding sequence. “We then program in a second mutation for a transcriptional activator that looks for the promoter sequence of the original organism,” Maselko says. Only when it finds that specific promoter in the original organism, as would occur during inter-species reproduction, it drives lethal over-expression of actin, which kills the offspring.”
Maselko has demonstrated the method in yeast, a convenient test organism. “Our goal is to apply this to other organisms, particularly plants that produce high-value compounds like pharmaceuticals or biofuels.”
Plants that are conventionally genetically modified must be strictly isolated to prevent mutant genes from spreading or, in the case of food crops, from entering the food chain. By creating new plant species, these problems go away.
“What’s neat about our system is we can choose different promoter regions, meaning we could theoretically have an arbitrarily large number of mutually incompatible species, each of which is incompatible with each other, and with wild species. We can have 100 varieties of corn, each optimized to make their own compound of choice, grow them in proximity, and have absolutely zero gene flow between any of them,” Maselko says. “Engineered crops that are genetically incompatible allow us to revisit what is possible because the risk substantially goes down.”
Informatics: The back-story
Most of the discussion around genome editing involves reagents and chemistry but those represent only one aspect. Developers need first to identify genes responsible for desirable (or undesirable) traits, and determine the optimal means of enhancing, removing, or adding to them. Computational analytics, the “discovery” component of gene editing, is part of Edit, a new gene editing platform from Benson Hill Biosystems, which combines the analytical power of the company’s CropOS™ computational platform with its CRISPR-based reagents.
CropOS uses artificial intelligence to identify gene sequences conferring traits of interest while the CRISPR kits make precise changes to plant genomes to improve flavor, texture, nutrient content, and environmental sustainability.
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To counter the complex patent landscape surrounding zinc finger nuclease (ZFN), TALEN, or even CRISPR, Benson Hill aims to make Edit, including its CRISPR-based reagents, broadly available to smaller, innovative companies that lack sufficient resources to in-license alternative technologies, says Mohammed Oufattole, the company’s vice president of R&D. “Think of Edit as a complete ‘ecosystem’ for all things related to gene editing.”
Like other versions of CRISPR, Edit’s reagent components require only the synthesis of guide RNA specific to the application. Developers must create a new nuclease for each TALEN and ZFN study. “Changing the guide sequence is a lot easier than developing a new enzyme,” Oufattole adds. Gene editing holds another big advantage over conventional genetic engineering, which involves inserting one or more foreign genes into plants. “It taps genes the plant already possesses,” Oufattole says, “allowing users to leverage the natural genetic diversity of plants to improve our food system”. Similar to traditional breeding
For Syngenta, which has been working on genome editing since 2010, the emergence of CRISPR has lowered the cost of gene editing by a factor of ten, according to Michiel van Lookeren Campagne, who heads seed research at the company.
“Genome editing is one of many tools but not a panacea for plant breeding innovations.” Other available tools include genetic modification (of the traditional GMO type) and marker-assisted breeding. “CRISPR allows us to develop desired crop varieties in ways similar to traditional breeding, while more efficiently making deliberate and precise changes to the plant genome. By throwing all the technology we have at breeding, we hope to accelerate the development of new crop varieties with optimized plant health, nutritional, or yield characteristics.”
van Lookeren Campagne notes that all plant breeding relies on creating and exploiting genetic diversity to improve crops. “Genome editing allows us to generate this diversity, to fine-tune the function of genes. Most of these changes could also be obtained through classical breeding, however we can now do this faster, cheaper, and better.”
Texas A&M AgriLife Research, an agricultural research unit within the Texas A&M University System, is similarly taking a multi-pronged approach to plant genome manipulation by funding two new laboratories. Coupled with existing A&M facilities—arguably the world’s largest plant sequencing capabilities—the Crop Genome Editing Lab and the MultiCrop Transformation Lab will focus on creating a pipeline of genome-edited crops.
The University’s latest capabilities include single-guide RNA design, Cas9/sgRNA construct development, biolistic or Agrobacterium-based transformation, plant regeneration, and confirmation of gene-edited progeny.
Sticking with CRISPR
Texas A&M plant scientists mostly use the CRISPR-Cas9 editing platform, which has rapidly overtaken older ZFN and TALEN as the editing method of choice.
Prof. Michael Thomson, who heads the Crop Genome Editing Lab, notes that while all three major gene-editing methods have been used with plants, zinc finger and TALEN have an established intellectual property landscape, while CRISPR does not.
“Companies that already hold IP or licenses for those technologies tend to stick with them. But those methods are also more difficult to deploy than CRISPR. They require the design of a new TALEN or ZFN protein every time you target a new gene. With CRISPR you can just order the oligos you need to produce the guide RNA. That is why academic labs almost exclusively use CRISPR, in spite of the IP uncertainties.”
Thomson’s lab uses rice as a model species to understand which genes control traits of interest. Manipulating those genes requires first sequencing the gene, then determining which edit to make.
“In many cases a knockout is sufficient, other cases may require changing a few amino acids in the protein,” Thomson adds.
Knockouts are also the easiest editing transformation. “The guide RNA makes a double-strand break in a certain section of the gene. When the cell tries to repair the break it inserts a base pair or two, or deletes a base pair, which causes a frame shift in the coding sequence, which induces a premature stop codon that knocks out the gene’s function.”
Although most knockouts are deleterious, in some instances knocking out a gene induces desirable characteristics. Rice is normally a tall plant, but engineering shorter varieties improves yields by helping the plant to stand upright. That particular transformation involved knocking out the GA20 oxidase gene to produce semi-dwarf plants. “It’s rare to find knockouts that work that well,” Thomson notes. “In most cases you might need to replace an allele, which is harder to do than knockouts.”
Plants are more difficult to gene-edit because cell walls block entry of reagents, which is why the Texas A&M groups also rely on Agrobacterium as the transforming agent. But these organisms infect plants slowly. “And you still have to do the tissue culture and regeneration. We’re working on making that process more efficient but we’re not there yet.”
Image courtesy of Dreamstime.