What’s next for gene editing? After ‘knock-out’, time for ‘knock-in’

Knocking out genes: to be replaced by 'knocking-in'?

Horizon Discovery says the gene editing tool CRISPR used to engineer its human cell lines is exploding the possibilities of screening for preclinical customers.

The technology allows Horizon to “knock-in” genes as well as perform more common gene “knock-outs,” creating superior patient disease models for screening drug candidates and validating drug targets, senior scientist Chris Thorne told Outsourcing-Pharma.com.

If you know from patient data that a high proportion of patients with a certain type of cancer have a certain mutation, you can recreate that mutation in a human cell line using CRISPR, and you have a model of the patient in a test tube. You can compare that with the original cell line which doesn’t have mutations to look for drug effectiveness.

The result is “very exciting” news for developers of personalized medicines with reduced toxicity and is a more flexible method than existing RNAi or zinc-finger nuclease tools, said Thorne.

‘Available to everyone’

CRISPR (clustered regularly interspaced short palindromic repeats) technology is more straightforward to use than existing gene editing tools which require design expertise or high safety precautions in the case of virus work, Thorne said.

The tool uses a short sequence of RNA to guide an endonuclease to the right point in the genome to cut and modify. “Because it’s a short sequence you can design pretty easily against any part of the genome, which makes it incredibly flexible and available to pretty much all of the research community.

Existing alternatives to CRISPR involve either Nobel-prize winning RNA Interference (RNAi) which removes the activity of a gene expression to study the consequences, or over-expression of a mutant, pathogenic form of a gene.

The problem with these two approaches is that they do not necessarily reflect what happens physiologically [in a patient] so you’re creating a false impression,” said Thorne. 

“What CRISPR gene editing lets you do is modify the genome of the organism itself and see what would happen in cells. Some people are using it in mice or in zebra fish to actually see in as true a way as possible what happens if you change the genes in situ.

CRISPR vs Zinc-finger nuclease

Unlike the zinc-finger nuclease method of gene editing, CRISPR does not engineer proteins to bind to DNA. It is easier to engineer a nuclease-recruiting scaffold connected to an RNA sequence, Thorne said. “You don’t need to go through the process of protein engineering; you simply have to know the sequence that you’re targeting.

With its flexibility, CRISPR will allow drug developers to identify novel drug targets and screen candidates on unprecedented scales, Thorne told us:

You can use it in a genome-wide way whereas it would be very, very challenging and very expensive to design a zinc-finger nuclease against every gene in the genome. But if you know that all you need to design is a short sequence of RNA for each gene then if you have the tools it’s easy. So suddenly with CRISPR you can create a targeting complex for every gene in the genome, and do very large-scale unbiased screens.

Whilst existing RNAi and overexpression models have provided tremendous insight into biological systems, they have also in some instances given off-target results as a consequence of the “artificial system,” Thorne told us, adding that many academic researchers believe CRISPR technology will soon be a requirement in published papers.

Now that this technology exists to relatively easily change the endogenous genes of human or mouse cells, there’s this argument, ‘why aren’t you doing it?’ It’s easy and it’s available and I think people will start to push for it.

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