Gene editing receives press coverage unseen since the early days of biotechnology. Thirty years later, therapeutic biotech is huge. But 2015 annual global sales of $85 billion, projected to reach $140 billion by 2024, are still just 15 percent of anticipated 2024 worldwide pharmaceutical sales of $1.12 trillion. Point: Ground-shaking science, especially that related to human health, moves fast, but tangible results take a very long time.
So it will be with genome editing, whose discovery stage, amazingly, continues even as biologists improve and turn basic science into practical methods. While such discoveries as new enzymes, editing platforms, and even targets will emerge, by far the hardest work will occur out of sight and out of news reports.
As recently as the late 1990s, scientists still used the term “junk DNA” to describe the 97 percent of the genome that did not code for proteins. Now we know that many of these genes act as switches, regulators, and signposts for both cellular and genetic events. These discoveries led scientists to recognize that the genetic code is much more mysterious than previously imagined and may harbor more secrets that gene editing could one day exploit.
While studying the divergent functions of six varieties of the human actin protein, University of Pennsylvania professor Anna Kashina surmised that what controlled functional differences was not amino acid sequences but subtle differences in the genes coding for them, which Kashina calls the silent genetic code.
For example, mice deficient in beta actin die at the embryonic stage, while those lacking gamma actin are stunted but survive to adulthood. Although beta and gamma actin differ by only four amino acids, the encoding genes show 13 percent dissimilarity, wherein lurks the silent code.
Using the nearly ubiquitous CRISPR gene-editing method, Kashina edited the mouse beta actin gene to produce gamma actin but to retain most of its original genetic sequence. Although the mice produced no beta actin, they survived, suggesting that given the correct instructions, the gamma actin performed the functions of beta actin.
“Researchers have traditionally held that the genetic code relates only to a protein’s amino acid sequence,” Kashina says. “Our research implies that a lot more is encoded than that. The proteins may be essentially the same and the amino acid sequences nearly identical but they perform entirely different functions.”
Editing the silent genetic code vastly expands the potential for tomorrow’s gene-editing efforts. When the code is fully understood, scientists and clinicians will be able to enhance the effects of proteins by changing their coding sequences without—or by minimally—affecting the amino acid sequence. “You can improve the gene without changing the protein,” Kashina says.
The future of gene editing may well depend on understanding additional silent codes. Cedric Wu, senior R&D director at GenScript (Piscataway, NJ), mentions a paper in Science describing RNA editing using the newly discovered CRISPR-Cas13 system, which opens new options for human therapy.
“Current gene editing relies mostly on inducing DNA double-stranded breaks, which are not always specific,” Wu says. “CRISPR-Cas13 targets ephemeral RNAs instead of DNAs to avoid unintended and permanent changes in the genome. I think they open a lot of options to accurately and efficiently treat human diseases with few safety concerns.”
NOVEL TOOLS, OPEN ACCESS
A great deal of innovation in genome editing will depend on newly discovered editing platforms and reagents. In December 2017, Inscripta (Boulder, CO) released MAD7, a unique CRISPR enzyme that all academic and commercial researchers may use freely, with no licensing fees or royalties. Very specific commercial use will entail a royalty that, according to the company, will be “far below the standard industry terms.” Inscripta anticipates that providing the enzyme on such generous terms will expedite testing, improvement, and adoption of MAD7.
To Inscripta CEO Kevin Ness, the future of genome editing rests on accessibility and optimization of editing methodologies, which the current intellectual property landscape impedes. “The promise of CRISPR can only be realized through research, but innovators lack access to the right enzymes—the molecular scissors that cut and thus enable gene editing. Many great ideas are not being tested because of this.”
Inscripta hopes that users will develop and refine MAD7 to the point where it enjoys wide, unrestricted usage. Ness explains the business model: “Our ultimate goal is to build the best tools for genome editors, which will include a full suite of reagents, software, and instrumentation, as well as enzyme engineering services.”
In early 2018, Synthego (Redwood City, CA) launched Inference of CRISPR Edits (ICE), an analysis tool that allows gene editors to validate their work through Sanger sequencing. Scientists previously relied on much cruder agarose gel assays, unreliable online analysis tools, or costly nextgeneration sequencing (NGS) for authenticating their work. ICE is free and open-source and, according to Synthego, “the only CRISPR analysis software featuring free, reliable batch processing of Sanger data.”
“When researchers plan to knock out or knock in a gene, they first assess the targeted region of the genome for ease of editing,” explains Kevin Holden, PhD, Synthego’s chief scientist. Until recently, scientists used NGS to sequence genes for specific evidence of insertions or deletions— telltale signs that the CRISPR edit is feasible. ICE gives them the option of employing the more familiar and accessible Sanger sequencing technology and analyzing multiple experiments simultaneously, as a batch.
“NGS is expensive enough, but when you have more than a few samples it becomes prohibitively so for many labs. We showed, in a paper whose publication coincided with the release of ICE, that data from Sanger sequencing can be as statistically reliable as NGS results,” says Holden.
Issues arise from the inherent variability of gene editing. Among a population of cells successfully undergoing gene editing, one edit will predominate. “It was originally thought that NGS was needed to tease out those numbers,” says Holden. “Turns out you don’t need NGS to gain a good understanding of those edits.”
With the ability to process up to 700 samples at a time and obtain results in minutes, ICE removes a bottleneck in gene editing, thereby accelerating and enabling the development and optimization required to move the field forward.
PLANT GENOME EDITING
Plant genome editing receives very little coverage, yet applications in this area are likely to overcome ethical, regulatory, and scientific hurdles years before therapeutic gene editing. Plant genomes hold the secrets to the low-priced manufacture of drugs, biopharmaceuticals, biofuels, and countless other value-added products. A huge challenge is the tendency of genetically modified plants to breed with their wild-type counterparts. Since many production-worthy GMO plants are also food crops, segregating mutants from the original plants is imperative.
Maciej Maselko, PhD, a synthetic biologist at the University of Minnesota, has used the CRISPR-Cas9 platform to turn the phrase “safe sex” on its head. He has edited the promoter region of yeast to create a synthetic species that is identical to the original in every way but one—the ability to reproduce sexually.
Using this technique may eliminate the risk of genes either escaping from the population of engineered organisms or infiltrating it. Ideally, that means no need to isolate or segregate synthetic species. “One could theoretically select different promoter regions and have one hundred varieties of corn growing in proximity, each producing a different product,” Maselko says. “Reproductively incompatible but otherwise identical crops will cause us to revisit what is possible with gene editing.”
In his work with CRISPR, Maselko uses a version of Cas9, the most common CRISPR-associated RNA-guided endonuclease, which possesses no catalytic activity. “So while you can target it for any location within the genome, it no longer cuts DNA,” he says. Instead, it makes breeding with wild-type organisms impossible, an advance that Maselko calls “revolutionary.”
Applications aside from breeding also abound. “As we learn more about what occurs transcriptionally, we can begin to think about novel tissue types that don’t yet exist,” Maselko says. Late last year, ag-tech firm Benson Hill Biosystems (Saint Louis, MO) launched Edit, the first complete genome-editing platform for crops. At the time, CEO Matt Crisp noted that genome editing “can help level the playing field” for innovators to upgrade food crops for desirable qualities.
Edit combines Benson Hill’s CropOS™ computational platform with a portfolio of CRISPR genome-editing nucleases to help optimize plants for flavor, nutrients, and environmental sustainability. CropOS uses artificial intelligence and cloud data to identify sequences in the plant genome responsible for various traits.
Mohammed Oufattole, PhD, the company’s VP of R&D, calls Edit an “ecosystem” for gene editing: “When people talk about genome editing, they usually mention reagents and chemistry. Computational analytics, the other component, helps us understand which genes cause which traits. Edit identifies genetic codes responsible for desired phenotypes.”
CRISPR-Cas9 currently dominates gene editing. The method has a reputation for being relatively unencumbered by IP issues, but that is actually not entirely true. MIT’s Broad Institute and the University of California are engaged in a patent battle over ownership of key aspects of the technology, so in a sense CRISPR may be riskier than TALEN or ZFN.
Regardless, Benson Hill aims to make its portfolio of genome-editing nucleases broadly available, including to smaller, innovative companies that “lack the resources to obtain what would otherwise be an expensive license,” Oufattole says. The new reagents use a new chemical locator and are smaller than typical CRISPR enzymes. But being based on CRISPR, they offer the usual advantages over TALEN and ZFN—namely, the need to only change the guide RNA instead of the nuclease.
In the future, plant genome editing has a lot of near-term upside compared with mammalian genome editing or gene therapy, both of which are heavily regulated. “There are tons of new product ideas, covering the range of healthier foods to more sustainable crops that may bear fruit in less than five years,” Oufattole says. The most optimistic timeline for approving gene therapies are at least twice as long.
“Gene editing taps into the genetic diversity already existing in the crop of interest, without needing to insert a foreign gene,” Oufattole says.