CRISPR technology ready to take the next step

Breaking new ground 6. nov 2022 2 min Research director, professor Guillermo Montoya Written by Kristian Sjøgren

New CRIPSR technology is poised to take the next step towards clinical application. Researchers have determined the structure of a fundamental part of this new CRISPR system, which can transport much larger pieces of DNA than previous similar CRISPR systems. A researcher says that this will enable cures for several diseases to be addressed.

CRISPR technology (CRISPR-Cas9 and CRISPR-Cas12a) has revolutionised genetic engineering and research for curing many genetic disorders within the past decade.

However, CRIPSR technology is limited by the fact that the genetic scissors can only cut and paste relatively small pieces of DNA from and into the genome.

The next genetic tool in the form of the CAST (CRISPR-associated transposon) system can do some of the things that CRISPR-Cas cannot, and this could be in use soon.

Researchers have characterised the structure of the transposon part of CAST, and this paves the way for further developing the CRISPR system within research and in clinical practice.

“We have both characterised the structure of the transposon and introduced genetic changes so that we can improve the function by being able to insert large pieces of DNA into the genome we cut into. All this represents steps on the way to being able to use the technology on human cells at some point in the future,” explains a researcher behind the study, Guillermo Montoya, Professor, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen.

The research has been published in Nature Communications.

What are genetic scissors?

CRISPR technology works by making a double break on the DNA target.

A donor sequence is then introduced, which the body’s own repair mechanisms insert at the double break.

A genetic disorder could be the result of various mutations at a specific place on the genome, and CRISPR could be used to cut out the mutated part of the DNA and replace it with a functioning sequence.

The technology can also be used to manipulate DNA in other organisms and produce crops with higher yields or learn about the genetics of animals.

One challenge with the traditional form of CRISPR technology is that large pieces of DNA cannot be cut and pasted in and out of the genome. Researchers are limited to working with pieces of DNA comprising a few hundred base pairs.

The CAST system uses the transposon to transport pieces of DNA of up to 30,000 base pairs for the genetic cut-and-paste process.

“This enables us not just to insert parts of genes or small genes and also regulatory regions to control the genes. We could start to redesign cells, opening new avenues to synthetic biology,” says Guillermo Montoya.

Determined the structure of transposons

The researchers used cryoelectron microscopy to analyse the structure of the CAST transposase TnsB.

Cryoelectron microscopy can trap proteins in a vitrified sample, which enables researchers to determine the structure of the protein atom by atom.

This process has enabled the researchers to show, for the first time, what the transposon looks like and where it needs to be altered to make it change function.

Guillermo Montoya explains that since the CAST system has its origins in bacteria, the system needs to be developed before it can be used on human cells.

The insight into the transposon now enables researchers to identify how to mutate the protein before it can be used outside bacteria.

“We can also see how we can improve the ability to insert large pieces of DNA quickly and efficiently. In our experiments, we have already shown how small genetic changes can increase the speed and precision of the protein. This shows that the system can be changed and optimised for laboratory or clinical use,” explains Guillermo Montoya.

May pave the way for curing several types of disease

Guillermo Montoya is sure that the CAST system has an interesting future.

Once researchers develop the tool so that it can be applied to solving human problems, the possibilities are almost endless.

Guillermo Montoya envisions that it will enable treatments to be developed for more complex illnesses, which requires major structural changes in the genome.

In this scenario, large pieces of dysfunctional DNA can be cut out and replaced with DNA that has the correct sequence of bases.

“The technology will enable us to correct not just small errors but also major errors spread over a large part of the genome. The current CRISPR technology is amazing and very important because it can address treatment for many diseases, but it has some limitations, which CAST can overcome,” concludes Guillermo Montoya.

Structure of the TnsB transposase-DNA complex of type V-K CRISPR-associated transposon” has been published in Nature Communications. All authors are affiliated with the Novo Nordisk Foundation Center for Protein Research, University of Copenhagen.

The Montoya Group works to illuminate the molecular details of cellular processes. This knowledge is the basis for the understanding of diseases and t...

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