NEW YORK (GenomeWeb) – For most genome editing applications, CRISPR/Cas9 will do the trick. But when it doesn't, Integrated DNA Technologies hopes researchers will reach for its new Cpf1 genome editing system, launched this week.
"We're not intending for Cpf1 to replace Cas9," IDT CSO Mark Behlke told GenomeWeb. "But it's an add-on tool, part of building the tool box."
Specifically, IDT's new Alt-R System genome editing product uses a nuclease variant from the Acidominococcus family of bacteria (AsCpf1), which offers a few twists. The main benefit of Cpf1 as a genome editing technology is that it can access AT-rich genomic stretches that Cas9 can't. The AsCpf1 enzyme recognizes a protospacer-adjacent motif of "TTT" while Cas9 recognizes "NGG."
That could be useful in certain research areas like ag-bio and malaria. "There are several model plants including Arabidopsis, tobacco, potato, and tomato with either low GC content or regions with low GC content," IDT Product Manager Michel Cannieux said. And the AT-rich Plasmodium falciparum genome has been all but intractable to CRISPR/Cas9.
For many researchers in the private sector, IDT will provide their first shot at using Cpf1. Among academic labs, Cpf1 continues to be popular, according to Joanne Kamens, executive director at the plasmid repository AddGene, which does not offer its CRISPR plasmids to commercial labs. She said more than 1,000 labs have requested Cpf1 materials since they became available from Broad Institute researcher Feng Zhang in 2015 (other labs have also donated Cpf1 materials). A year and a half later, AddGene is still making Cpf1 shipments at an average clip of one to two per day.
IDT has licensed the technology from the Broad Institute, where Zhang discovered the alternative CRISPR system in several bacteria and demonstrated its ability to edit DNA. A representative of the Broad declined to disclose financial and other terms of the agreement.
Like the Alt-R Cas9 product, the Alt-R Cpf1 System will combine synthetic RNAs and recombinant protein, intended to be pre-complexed into RNP and then delivered. But there are several more differences between the two products that have technical implications.
The second major difference from Cas9 is that Cpf1 is naturally guided by a single guide RNA (gRNA). "It's a nice short one, too," Behlke said. Cas9 genome editing is often accomplished with a chimeric gRNA, fusing an RNA with the approximately 20-nucleotide target sequence and a longer "tracrRNA" important for interacting with the Cas9 enzyme. While a Cas9 gRNA is 100 nucleotides at minimum, IDT's Cpf1 gRNAs are only 41 nucleotides.
And where Cas9 typically makes a blunt double-strand break, AsCpf1 leaves a four- to five-nucleotide long five-prime overhang. Zhang has suggested that the overhang could be exploitable and Behlke said IDT has found the staggered cut makes detecting edits much easier by creating a larger damage site. Sequencing is the gold standard for evaluating CRISPR editing and the results are always far more accurate than T7 surveyor assays when using Cas9, he said, but for Cpf1, the mismatch assays give similar results to NGS.
But the differences are not all upside, as IDT's work has revealed a few quirks.
"Cas9 is really the gold standard, it's a fantastically active enzyme that works at most sites," Behlke said. "With Cpf1, you're going to have to do a little experimentation. If you're at a good site, can cleave with good efficiency, but it's active at fewer sites." A priori, Cpf1 only works at about one in three sites, he said, although IDT is working on an algorithm that will use experimental data to help predict activity.
And like the Alt-R Cas9 System, the Cpf1 product is designed to be pre-complexed into a ribonucleoprotein, then loaded into cells by either electroporation or microinjection. But IDT has found that the Cpf1-gRNA complex requires an electroporation enhancer to drag it into the cell. "Cas9 can get some benefit from an enhancer, but for Cpf1 it's almost essential," Behlke said. "Without that, the delivery is very, very low." For researchers looking to deliver Cpf1 via electroporation, IDT is supplying the enhancer as well.
Behlke showed GenomeWeb unpublished data suggesting that the AsCpf1 nuclease recognizes a TTTV PAM, where V is an A, C, or G. That differs from the TTTN PAM for AsCpf1 that two labs had reported in separate papers published last summer in Nature Biotechnology, but consistent with new in vivo data published in December in Nature Methods. "The fourth letter can't be a T," he said.
So far, several customers — mostly pharmaceutical companies — have taken IDT's Cpf1 for a test drive. Behlke declined to disclose which ones, but said they were itching to try it. "Anything new that's available, they want to try it," he said.
IDT chose AsCpf1 for its activity in mammalian cells, drawing on both internal experiments and a literature search. "Like Cas9, [Cpf1] should theoretically work in any cell," he said. In his own functional genomics technology development research, he's using it in human, mouse, and rat cells. "There's no reason to believe it wouldn't work in Caenorhabditis elegans, zebrafish, or in plants," he said.
While Zhang showed that several other Cpf1 "orthologs" could also edit genomes, Behlke said it wasn’t commercially viable to offer more than one.
IDT estimates the Cpf1 market has the potential to be only 10 percent to 20 percent of the Cas9 market. Because they don't expect to sell as much, they're charging slightly more for Cpf1 products than for Cas9.
And the toolkit doesn't have to end with AsCpf1. "If another variant comes along that works better than [AsCpf1], we'll certainly try to jump on that," Behlke said. "It's the beginning of a long story. We have a very active development program. There will be more products to come."