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Programmable DNA-Cleaving Enzyme

Jesse Jenkins

Researchers have recently discovered a new method of cleaving DNA using an RNA-programmable DNA-cutting enzyme; a tool that may potentially offer genetic engineers a more efficient way to edit and research genomes.

According to new research at the Lawrence Berkeley National Laboratory, scientists have uncovered an RNA-based complex used by bacteria that directs a DNA-cutting enzyme called Cas9 to specific sites in the genomes of viruses.

Cas9 is a DNA endonuclease guided by two RNA molecules. Source: Science.

From this bacterial complex, the scientists at Lawrence Berkeley National Laboratory led by Jennifer Doudna, along with the Laboratory for Molecular Infection Medicine at Umeå University in Sweden, discovered that they can program the Cas9 protein with a single RNA to enable cleavage of essentially any DNA sequence.

Until now, researchers have had to design proteins that will recognize and bind to a particular DNA sequence when targeting a specific site in a genome; a time-consuming and expensive process.

However, Doudna says this new system could prove to be more efficient than current methods that rely on engineered new proteins to cleave DNA.

“What we think the Cas9 system potentially offers as an important advantage over the zinc finger nucleuses or TALEN proteins in that this is a single protein. We don’t need to make a new protein every time we’d like to target a different sequence in DNA. We simply have to redesign a very short RNA molecule and that will associate with Cas9 and allow it to cut the complementary sequence in DNA.”

According to a recent paper published in Science (1), the group’s study of the Cas9 protein during research on a bacterial immune system’s ability to destroy foreign DNA in bacterial cells led to the discovery. “The hypothesis that we wanted to test was whether this protein had enzymatic activity and whether it was able to cut DNA,’’ said Doudna. “If the answer was yes, could it then do so in a way that was guided somehow by RNA?”

Emmanuelle Sharpendee’s lab at Umeå University in Sweden determined that the Cas9 protein not only had enzymatic activity, but also required two different RNA molecules to cut DNA, one of which has a sequence that is directly complementary to the DNA target.

The researchers at Berkeley then tested different configurations of those RNA molecules by making changes to their sequence. By looking at the predicted interaction between the two RNA molecules that are required to guide the cas9 protein to a sequence in DNA for cleavage, the team found it could actually reengineer the two RNAs as a single RNA by introducing a linkage between the two molecules.

In the near future, scientists at Berkeley may explore the application of the Cas9 system in the research of biofuels and ethanol production, which requires the reengineering of various industrial strains of yeast or fungi. Doudna also sees potential impact in therapeutic drugs and engineering pathways into various organisms like bacterial systems and fungi.

However, Doudna said there are still initial proof of principle experiments to be done on the Cas9 system before it may fully be applied in these fields.

“One big question when you are working with a bacterial protein that operates on DNA is whether it can deal with chromatin in eukaryotic cells, and we just won’t know the answer to that until we test it,” said Doudna. “But we are encouraged by the fact that TALEN proteins are bacterial proteins and they deal with chromatin just fine. So, we have reason to think that in our system this will also be possible.”

1. Jinek M., K. Chylinski, I. Fonfara, M. Hauer, J.A. Doudna, and E. Charpentier E. 2012. A Programmable Dual RNA-guided DNA Endonuclease in Adaptive Bacterial. Science. [Epub ahead of print, June 28, 2012]

Keywords:  proteomics