Oxford Nanopore’s Plans for Pre-Packaged Cas9 Test Kits
By Allison Proffitt
September 29, 2016 | Oxford Nanopore Technologies is seeing more of a demand to focus on a subset of input materials, Clive Brown said this morning in his technical update. In addition to new pores, new chemistries, and updates on various devices, Brown dove into a Cas9 application that the company has been exploring and intimated that Oxford Nanopore will soon begin selling specific test kits for its mobile sequencers.
The idea prompted excitement from the webinar viewers.
Oxford Nanopore has been using disabled Cas9 to enrich sample on the front end, Brown said. Researchers set up a probe RNA molecule for a 20mer region of input sample—human sample, a 20mer virus region, or something else, he said.
The team used the new rapid 1D prep—“hopefully lyophilized, not cold chained”—and combined the Cas9 molecule to the 20mer target region.
“Molecules that don’t have [the attached Cas9] will effectively become invisible to the sequencer,” Brown said. The ONT motor enzyme will “whisk” those molecules through the pore. “The only molecules that survive to be sequenced are the ones with the stalled Cas9 on them.” The molecules with the Cas9 attached get “trapped on the pore,” Brown said; Cas9 is “kicked off by the pore” and only the targeting sequence fragments proceed.
Alternatively, the Cas9 could have a tether on it, and the other molecules wouldn’t, Brown said. “We get about a 20,000 fold enrichment just from that,” he said. He also mentioned using the Cas9 system as a sliding motor.
“In molecular space, from a complex soup, we can now just sequence the fragments of interest.”
In another example, Brown said that Oxford Nanopore has demonstrated using a Cas9 tag for “block counting.”
“On a different pore system, we can also not even bother with the existing sample prep. We can just detect Cas9 bound to duplex DNA,” he said. “As it transits the pore in various ways… it will generate a blip in the current flow. It’s very fast. And the point of that is you can measure millions of molecules in a few seconds. It’s a very powerful non-sequencing use of the device.”
There’s work to do on specificity, Brown admitted. And we can probably enhance the sensitivity even further either by using beads in addition to this, or by using multiple tethering strategies. But he’s extremely hopeful about the prospect.
“There’s nothing here you couldn’t do, actually, if you had access to a disabled Cas9 and all the other bits and bobs,” Brown told the listening group. “But it’s quite likely that we’re going to make this available in kit form within some reasonable time frame—I don’t know what it is, but quite reasonable.”
Setting up the probes is the “clever” part, Brown said. He predicted that Oxford Nanopore will supply a “standard set of tested Cas9 probes” for well-known tests that will then “allow call-down as part of your sample prep.”
In the case, Brown said, Oxford Nanopore will be able to sell pre-packaged tests developed specifically for the rapid mobile sequencers.
“That’s quite an exciting new venture for us,” Brown concluded.