Accelerating Target Discovery with Genomic and CRISPR Techniques
In November our Discovery series discussion group focussed on Genomic & CRISPR Techniques to Accelerate Target-Based Drug Discovery. Leading the conversation was Benedict Cross, Chief Technology Officer at PhoreMost, a biotech company based in Cambridge, UK. PhoreMost use their PROTEINi® and SITESEEKER® technology for novel target discovery and, according to Cross, employ “genomic/CRISPR approaches in different contexts to try and unpick mechanisms and identify dependencies in our experiments.”
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After gaining an understanding of these mechanisms, PhoreMost’s goal is to translate this knowledge into drug discovery programs. Cross said that they were mostly focussed on targeted protein degradation in this regard.
Before working at PhoreMost, Cross did his postdoc at Cambridge in the field of proteostasis and protein response. He then moved on to work for Horizon Discovery, a gene editing company based also in Cambridge. As Cross explained, Horizon were one of the early adopters of CRISPR based technologies for cell-line engineering and screening platforms.
Which ‘Flavours’ of CRISPR are of Interest?
The first topic that Cross introduced to the group concerned the uses and differences between the various forms of CRISPR methods. Cross commented on PhoreMost’s experience of the ‘flavours’ of the gene-editing tool: “For ten years now, we have had a lot of experience with using fully-nuclease active Cas9s to drive knockout.” This type of CRISPR was versatile enough to be performed in labs by professionals and amateurs and could be deployed at a very high rate.
“There are now lots of variants on that in terms of the different enzymes that are available,” Cross continued. He added that those other technologies offer different outcomes than is to be expected from silencing phenomena driven by CRISPRi or a gain function phenomenon driven by CRISPRa.
Cross said that nuclease active Cas9 was “still the most widely used and most powerful” approach at the moment. “Broadly speaking, knocking something out with a sledgehammer like Cas9 with a DNA double-strand break and hoping for the best with NHEJ repair is actually a remarkably powerful approach to driving phenotypic consequence,” Cross explained. “So, when it comes to functional genomics, that is a very powerful starting place.”
However, using such a powerful gene-editing tool can forfeit some desired nuance from a biological experiment. “This is both on the level of an unbiassed screen for perturbation and indeed, on the level of a target validation exercise,” Cross said.
Cross explained the three-fold reasons for this loss of nuance: “The assault on the cell from the knockout phenomenon; the kinetics which are sometimes very difficult to control or predict; and the fact that this kind of CRISPR knockout results in biological outcomes that might eliminate certain functions while not affecting others, for example scaffolding functions.”
Therefore, there is a need for a larger toolset of gene-editing technologies than simply Cas9. “Fortunately, that is provided by options like CRISPRi, alternative nucleases, Cas12a, or CRISPRa.
Degradation versus Genetic Knockout in Target Based Drug Discovery
One attendee brought up a question about the interplay between protein degradation and genetic knockout. dTAG is a protein degradation platform whereby a large FKBB tag is knocked in a target’s genome and by inducing proximity with a E3 ligase, the target is degraded. Although this method is not genetic knockdown, it does require CRISPR to knock in the tags, explained the participant.
As Cross explained, the dTAG platform introduces a binding site (FKBB12, a small E3 ligase) n-terminally or c-terminally into the locus of a particular gene of interest. “Then you can start to add in this ligand which causes its degradation.” Cross said that there is a direct chemical–genetic parallel to a functional genomics approach using a CRISPR knockout system, and asked: “What is better? To degrade your target of interest or to knock it out?”
There are also a number of opportunities that the union of these methods possesses. “They are also the perfect marriage, because degraders are not just tools, they’re also therapeutics—that’s obviously very exciting for lots of us,” he said. Cross argued that on a superficial level, protein degrading methods were “the perfect vehicle to exploit genetic data because you’re simulating the loss of function via the loss of a target in almost the closest way.”
Furthermore, protein degradation removes the issue of catalytic inhibition of complex metabolism: “you’re going straight to the root of the issue that was identified in a CRISPR-based knockout screen, recapitulating that with degradation,” Cross noted.
Coming back to target discovery and its challenges, Cross said that he was looking forward to seeing innovative ways of bringing the dTAG-based technology to a near genome-wide readout for loss of function.
Licencing and IP
One of the final topics that the discussion group participants considered was the intellectual property (IP) landscape for CRISPR technologies and whether it affected scientists’ freedom to operate. Cross said that IP did come into the conversation and that the field was becoming clearer on what was expected of in terms of IP. “We understand that the research and development opportunities presented by these technologies are generally open to all of us, and that is a good place to be.”
However, Cross noted that there were some situations, especially in product development, where the licencing conversation was “more difficult.“
“We’ll all doubtless operate via the best advice and means that we can to marry up the use of the perfect technology with our ability to use it legally. But it does have the unfortunate consequence of stifling our appetite and stifling our innovation”
Cross suggested that there would be more innovation and companies developing CRISPR technologies if the IP landscape was clearer than it is now. “I’m not a free-for-all champion, but if it was more clear; if everybody knew where they stood, then progress especially in the private sector might actually be faster,” said Cross.
There was some consensus from of the participants toward this assertion. One who had recently transitioned from academia to industry said that they were unfamiliar with the rights of licencing in an industrial setting. Another attendee mentioned that licencing issues to do with ambiguity and complexity had led some of their colleagues to seek out alternatives.
The hope for the future of CRISPR and gene editing tools is further collaboration and transparency will be able to drive the next generation of innovative target discovery methods and platforms.
Our Organ Modelling Discovery Congress addresses an important and expanding field which provides new understandings in organ functionality and novel approaches to drug development.
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