19 November 2020

Researchers map methods to get the most out of genome editing

Genome Editing

When researchers use genome editing such as CRISPR-Cas9, they do not always achieve what they expected. Therefore, they need sensitive technology to very accurately detect all the small changes that have occurred in the attempt to edit a genome. Now, researchers from the University of Copenhagen have mapped the most effective methods for just that.

DNA Helix
(Photo: Colourbox)

It was a revolution for modern science when it was discovered how to edit in the genomes of animal, plant and human cells. Indeed, the 2020 Nobel Prize in Chemistry was awarded to the inventors of the genome editing technology CRISPR-Cas9.

CRISPR-Cas9 and other types of genome editing have been called 'molecular scissors' as an illustration of its molecular precision. But contrary to what most people think, genome editing is not as accurate as you might wish.

‘The technology is quite accurate, but it intrinsically leads to both desired and undesired results. Therefore, all researchers working with genome editing must make a very detailed analysis of what actually happened in the cells where they edited the genomes, so that they can determine whether the desired result has been achieved. Our study maps the best methods for making that analysis’, says Eric Paul Bennett, Associate Professor at the Department of Odontology and employed at the Copenhagen Center for Glycomics.

The Older the Technology, the Poorer the Result

The research team wanted to find the best methods for performing follow-up analyses on genome editing. They mapped available technologies to uncover what has happened after the genome editing, down to the smallest detail, and tried to assess which ones were the best.

It is immensely important for researchers to know what has happened after a genome editing, because then they know if they can actually continue their experiments, or if they have to start over.

‘Just as you ensure that an operation has gone well before you send the patient home, these technologies must be used to ensure that the genome editing has happened the way it should – and that there have been no undesired effects. Our mapping shows that there are many really good tools on the market, but not all methods have been developed to be used in genome editing contexts. And in general, it can be said that the older they are, the poorer they are too’, explains Associate Professor Morten Frödin, Group Leader at the Biotech Research & Innovation Center, who is also the author of the new study.

At the same time, Eric Paul Bennett emphasises that even old technologies may still be used. It all depends on the researcher's purpose of the genome editing.

If the purpose is simply to document the removal of DNA building blocks – so-called bases – it does not require large sequencing resources. If, on the other hand, the goal is to change specific bases, one cannot get around the use of DNA sequencing methodologies, a very expensive and advanced technology.

Three Genome Editing Methods, All with the Same DNA Effect

There are many different notions about what researchers really want when they use genome editing. But a global survey in the journal Biotechniques last year showed that 80 percent of all researchers who use genome editing across scientific fields in both companies and at universities want to achieve a ‘genetic knock-out’.

In other words, they want a gene to stop working. For example, you may want to knock out a gene because in your experiment, you want to find out if it is related to a specific disorder. If you know which genes are particularly associated with specific diseases, such knowledge can improve the prevention of diseases.

Researchers may then choose from a variety of genome editing techniques. The three most commonly used are CRISPR-Cas9, ZFN and TALEN. The use of all three is described in the new study. But in reality, you achieve the same genome editing effect with all three, explains Morten Frödin.

‘No matter which kind of genome editing you use, roughly the same thing will happen in the cell, namely the formation of a double-stranded break of the DNA strand. How the cell itself repairs this intended damage varies depending on the DNA sequence around the place of fracture as well as other factors that the research has not yet fully clarified’, he says.

‘If the repair is done in a certain way, then the researchers do not achieve a “knock-out” of the genome, and then the experiment has failed. But in many of these experiments, the researchers will have tried to edit the genomes on a lot of cells at one and the same time. So, by analysing many cells, you can often find cells where what you wanted to happen has actually taken place – that is, if you have the right validation method in place’, explains Eric Paul Bennett.

He hopes that the new knowledge will make it easier for other researchers to choose the perfect validation method when they need genome editing in the future.

Read the full article: ‘INDEL detection, the “Achilles heel” of precise genome editing: a survey of methods for accurate profiling of gene editing induced indels’