Clean cut genetics

Wellcome Trust-funded researchers are helping to show how new 'genome editing' techniques could be used to transform the future of genetics research.

Reading sequences of genetic code has become faster and cheaper over recent years, in part fuelled by the needs of the Human Genome Project but continuing to accelerate ever since. Now, tools are being developed that allow us to accurately and efficiently rewrite DNA as well as read it. At the Wellcome Trust Sanger Institute, researchers are already adopting these 'genome editing' techniques and extending their potential applications.

The latest genome-editing tool to cause a buzz is called CRISPR-Cas. It uses a DNA-cutting enzyme borrowed from bacteria with a short piece of RNA to guide it. Together, they can make a clean break at a specific point in a cell's DNA, just like snipping a piece of thread with a pair of scissors. It’s only a few years since the mechanism of CRISPR-Cas was first revealed, and researchers around the world have begun successfully using it to alter the genes of mice and other model organisms – and even those in human cells.

"CRISPR-Cas technology is revolutionising how we study the behaviour of cells," said Dr Kosuke Yusa, a member of the Sanger Institute Faculty.

Screen test

Faced with CRISPR-Cas's potential, Yusa and his colleagues decided to see if they could use it in genome-wide screening experiments to identify genes associated with particular cellular traits.

When a gene is cut using CRISPR-Cas, the cell's DNA repair mechanisms kick into action. If you give the cell a template of the sequence you want it to have, it will copy that sequence – this is the ultimate aim of genome editing. In the absence of a template, however, the cell will just try to rejoin the two loose ends. This mechanism is not perfect and mistakes can creep in during the repair. These tiny mutations can change the activity of a gene, sometimes even resulting in a complete loss of function. Studying a cell in which one gene has been mutated in this way can reveal which processes in the cell that gene influences.

Before Yusa and his team could use CRISPR-Cas, they had to make the short pieces of RNA to guide the cutter. They made almost 90,000 'guide RNAs' β€“ at least two for almost every gene in the mouse genome. Combining CRISPR-Cas with the guide RNAs, they were able to generate a bank of mouse embryonic stem cells, each cell containing mutations in different genes.

This library was then used to screen for responses to a bacterial toxin called Clostridium septicum alpha-toxin. When it was put on the cells, some of them had different responses depending on the genetic mutations they had. The team could therefore identify which genes were important for determining whether a cell was particularly vulnerable or resistant to the toxin. They found genes already known to be involved in this behaviour, but they also uncovered several genes previously not known to have an effect on how cells respond to this toxin.

"Dr Yusa and his team have used the CRISPR-Cas technique in a very innovative and creative way," said Professor Allan Bradley, Director Emeritus of the Sanger Institute. "This study highlights the power of the technology and illustrates some potential applications it can have."

CRISPR-Cas future

While this work was, essentially, a proof-of-principle study to demonstrate that you can use CRISPR-Cas to set up genome-wide screening studies, the team say that other researchers will now be able to use their library of mouse genome guide RNAs. Their own plan is to create mutations in cancer cell lines and determine which genes are involved in the significant problem of tumours acquiring resistance to cancer drugs.

Commenting on the potential application of the work, Dr Chris Lord, a senior research scientist at the Institute of Cancer Research in London, said: "These libraries have very clear potential in allowing us to understand the genetic basis of cell behaviour, disease and drug resistance."

The next step will be to generate a library of guide RNAs for the human genome, building on work by other groups already using CRISPR-Cas in human cells. This will allow many more scientists and clinicians to study disease and issues like drug resistance at the genetic level and, hopefully, to develop treatments for millions of people with devastating diseases.

CRISPR-Cas is not the only technology capable of such an approach, but it does seem to be the simplest, fastest and cheapest way yet developed to do it.

Yilong Li, who worked on the Sanger study and is supported by the Wellcome Trust PhD programme, summed up its potential: "The true power of CRISPR-Cas comes from the fact that when you want to target any site in the genome, all you need is a small piece of RNA."