NEW YORK (GenomeWeb) – CRISPR is no longer an upstart technology — it is now a genome-editing regime that has almost completely taken over in research. But there are a few well-carved out niches left for non-CRISPR technologies, notably in clinical and agricultural biotechnology, where companies are on the cusp of bringing new products to market using transcription activator-like effector nucleases (TALENs) and zinc finger nucleases (ZFNs).
"In academic research in general, pretty much everybody is using CRISPR/Cas9," Dan Voytas, a professor at the University of Minnesota and a pioneer in genome editing in plants, told GenomeWeb. He's developed both TALENs and CRISPR/Cas9 for plant biology, and while his lab used to supply TALEN assembly kits to other labs, demand has completely dried up.
"We haven't sent out a kit in more than a month, and we used to send out lots," he said.
"[TALENs] actually work super well and are not that different from CRISPR in terms of efficiency, but they're a little harder to make, so academics tend to prefer the cheap and easy route, so that's why they go to CRISPR/Cas9," Voytas said. "I think the only domain where you'll see TALENs being used is commercially, and that's for intellectual property reasons."
In addition to running a lab at the University of Minnesota, Voytas is also Chief Science Officer at Calyxt, an ag-bio firm working on gene editing that has IP rights to TALENs in the space. For companies like Calyxt, its parent company, Cellectis, which is using TALENs for clinical gene-editing applications, and others with rights to non-CRISPR IP, it's far from the end.
Clinical gene editing is more crowded with competing technologies than ag-bio, but within those niches, several companies are well on their way to bringing new products to market. For example, Sangamo Biosciences is in the process of putting together clinical trials for several diseases. Bluebird bio is actively investing in development of its meganuclease/TALENs hybrid gene editing platform for T-cell editing.
And new research on single-base editing enzymes is proving that Cas9, while dominant, has not entirely snuffed out other technologies.
Data compiled by Addgene, the Cambridge, Massachusetts-based plasmid repository, backs up Voytas' anecdote about the CRISPR takeover in research. Demand for TALENs peaked in 2013 and has dropped steadily ever since. In that year, Addgene shipped around 2,800 TALENs kits. In 2015 alone, Addgene shipped more than 20,000 CRISPR plasmids. The number of ZFN kits shipped by Addgene peaked in 2011 and has similarly tailed off to barely a trickle.
Companies including Thermo Fisher Scientific offer TALEN kits, though they declined to share data on demand. "Thermo Fisher has been working with a number of leading organizations and researchers to provide TALEN products to accelerate discoveries that may lead to new therapies for disease treatment, more robust crops, and new biofuels," VP and General Manager of Synthetic Biology Helge Bastian said in an email. "Despite the growing interest in our CRISPR products, TALEN technology is often chosen because of its proven effectiveness and exceptional precision."
ZFNs and TALENs are often the underlying technology in recent headline-making gene editing breakthroughs, like the infant whose leukemia was put in remission after an experimental, edited T cell therapy and hornless cattle developed by St. Paul, Minnesota-based ag-bio firm Recombinetics.
While it's no guarantee, the head start provided to TALENs and ZFNs means that those technologies bring new products to market first. And the messy fight for the CRISPR patent between the University of California and the Broad Institute is even more reason to pursue other technologies, if one can.
"Pretty much everything at Calyxt is TALENs," Voytas said, even though the firm holds some IP for CRISPR in plants. The firm has several food-related products in its pipeline and is already planning the 2018 commercial launch of a soybean variety Voytas says will make a trans fat-free cooking oil.
"Soybean oil in the past has been chemically treated and that makes trans fatty acids," he explained. "We went in and changed the fatty acid composition so you don't have to do that."
The firm has specifically pursued a strategy of knock-out editing. "It's been a challenge in plants, but that's where the potential is, in the ability to make a subtle modification to a genome. You want to be able to change an amino or two in a genome or alter transcriptional control," he said.
While ZFNs offer a similar ability to precisely knock out genes, its future in ag-bio is uncertain. Back in 2008, Sangamo licensed the technology to Dow AgroSciences; however, Dow Chemical recently announced a merger with DuPont, which has acquired a strong position in CRISPR/Cas9 IP through its subsidiary DuPont Pioneer.
But in clinical gene editing, ZFNs may be the most mature technology. In December 2015, the firm announced that the US Food and Drug Administration approved an investigational new drug application for a hemophilia B treatment called SB-FIX, which will insert a therapeutic gene into the albumin gene locus. It is enrolling patients in a related Phase I clinical trial.
The firm has already completed a Phase I clinical trial for CCR5-edited T cells for an HIV/AIDS therapy and is developing a Phase I trial for CCR5 editing in hematopoietic stem and progenitor cells.
As interest in developing T cell therapies intensifies, Bluebird bio is continuing to invest in its megaTAL platform, which fuses a meganuclease to the genome-targeting part of a TALEN. Jordan Jarour, a scientist at Bluebird, recently presented data on the technology at last month's American Society of Hematology Workshop on Genome Editing in Washington, DC.
"Pretty much anything we could dream of editing, we can do" with megaTAL, he said. The firm is working on expanding the multiplex editing capability of the technology, as well as working on the ability to insert genes using homology directed repair, in addition to knocking out genes.
While CRISPR/Cas9 leaves a blunt end and Fok1 nucleases used in many TALENs technologies leave a 5' overhang, megaTALs leave a 3' overhang. This biochemical difference gives Bluebird scientists more influence over which DNA repair process — non-homologous end joining leading to indels or HDR incorporating gene templates — is used, Jarjour said. "We control which [DNA repair pathway] we go down," he said.
Another technology that has Voytas and others excited is single base editing. In April, scientists led by David Liu of Harvard University published a study describing a method to introduce C to T point mutations by using a cytidine deaminase.
And last week in Science, Japanese scientists led by Kobe University's Akihiko Kondo published a paper describing a similar enzyme, found in a sea lamprey.
"These base editors are going to be very interesting in plants and elsewhere," Voytas said, adding that he's seen unpublished data from the Japanese team that suggests these cytidine deaminases also work in plants. He's already begun using them in his Minnesota lab.
"They're highly efficient and they give you a degree of precision that NHEJ knockouts don't," he said. "You can't change every base you want, but for the bases you can change, I like them a lot."
Of course, the nucleotide flippers aren't useful unless they're brought to a specific target. And what was Liu and Kondo's genome-targeting system of choice? CRISPR/Cas9. The nuclease-null enzyme wasn't doing the cutting, but it was CRISPR/Cas9 all the same.