CRISPR - shortcut to genome engineering

In order for a gene to be used, it needs to be read by RNA polymerase (right). If Cascade is programmed to bind at the gene or its promoter, the reading is blocked and the gene "switched off" (left).

Although the Earth’s ecosystems are dependent on bacteria, detailed understanding of the micro-organisms' own defence system did not emerge until the 21st century.  However, in 2007 researchers could show how the bacterial immune system was disabling viruses by cutting strands of DNA. The information was stored in the bacterial genome. The mechanism was named CRISPR, an acronym for Clustered Regularly Interspaced Short Palindromic Repeats, and considered the 2013 breakthrough of the year by the scientific journals Science and Nature.

“The human immune system requires a wide variety of cells communicating to learn how to identify viruses. It was hard to believe anything like this existed in single-celled bacteria, says Magnus Lundgren, microbiologist at the Department of Cell and Molecular Biology at Uppsala University. Unlike the human antibody-based system, the adaptive bacterial immune system recognizes virus DNA, saves it and then searches for similar sequences. As soon as the bacterium is infected by DNA containing a similar sequence, the DNA is destroyed”.

Viral defence becomes a tool
Magnus Lundgren shows an animation of the process on a computer screen at the Biomedical Centre. A virus attacks a cell and injects its DNA. This sends a signal to the cell to develop more viruses, something often causing cell death. But meanwhile the cell’s chromosome has incorporated short patches of the virus's genetic information. As these form part of the cell's genetic make-up, the immune system will use  proteins produced to detect new viruses. The system translates the virus-DNA to RNA-molecules, triggering the Cas proteins to find and eliminate invading DNA.

“What my group is focused on now is this step, the actual adaptation when the cell learns to recognize the virus. How does the cell find the virus, cut it off and stick its DNA in its own chromosome? There are many chemical steps to be performed by different proteins”.

The CRISPR mechanism is absent in people, plants, animals or cells similar to ours. But Magnus Lundgren's research group is testing whether there are any fundamental barriers to insert it in yeast cells that have the same characteristics and basic structure as human cells.

“Even if it may take a while before we can provide any details, we see indications of a functioning immune system. The system is so simple that scientists can easily access and reprogram what this system should adjust, which is basically anything”.

Their ambition is to further understand the potential use of bacterial defence mechanism to alter and control the genes in various organisms.

“If we add a piece of DNA at the same time as the cut up, the cell will use the patch for repair. Then we can simply do whatever DNA modification we want”.

More effective blocking with new method
Magnus Lundgren and his colleagues are now taking the findings a step further. In early December, his group published an article in the scientific journal Nucleic Acids Research, showing how the protein Cascade can be used to scan and switch off gene expression. The idea came two years ago, something he describes as an "in-the-shower insight", followed by numerous experiments and collection of data.

“As a geneticist and researcher, you want to be able to turn off genes and see what happens. But you don't want to remove an important gene, as it’ll kill the cell. So we simply transformed the system from cutting DNA to binding DNA at the place we wanted”.

In order for a gene to be activated, the protein RNA polymerase must recognize and read the gene. But they found that if the protein Cascade was inserted into the cell, it bound to the landing site of the RNA-polymerase and prevented the gene from being expressed.

“Our system is different from the one based on the protein Cas9. In that system, the protein Cas9 is modified which causes DNA to break down. Instead we have tested to remove Cas3, which has worked great”, says Magnus Lundgren.

“Our protein is much larger and more efficient in blocking RNA-polymerase. The Cascade system is also more specific, so it's easier to use in the right place.

Limitless potential
He believes the development of CRISPR-based tools can provide answer to the proteins behind certain diseases, such as the mutation causing cystic fibrosis. Inserting Cas9 into the mutated cells along with a piece of DNA corresponding to a healthy gene would replace the faulty gene and eliminate the disease, says Magnus Lundgren.

 “The trick is of course to successfully change enough cells. But previously we didn’t even know how to make this change. We now know it’s doable on cultured cells. And the latest breakthrough is the engineering part. Nothing says we won’t be able to enter and insert whatever DNA sequences we want, with boundless possibilities for identification”.

The lab work has taken place at BMC, where scientists have also used ScilifeLab’s technology platform BioVis for some of their analyses. The easy access to the lab has been highly appreciated by the group, according to Magnus Lundgren, but common lab equipment did most often suffice.

“This is a simple and straightforward tool and a typical example of the value of basic research. Ten years ago, if you’d said `let´s develop a great revolutionary genetic tool`, you’d never have succeeded, because nobody knew what to look for.
“There was no strategic financing; people simply stumbled upon CRISPR after much research and realized its potential”.

For more information, contact Magnus Lundgren

See article in Nucleic Acids Research

More about Magnus Lundgren´s research

The Linné lecturer Professor Jennifer A. Doudna will hold a lecture on CRISPR on February 26, 2015