Gene editing technique enhanced thanks to optogenetics
Japanese scientists have developed optogenetic photoswitches, based upon Cas9 nuclease and 'Magnet' proteins, to enhance the control of the CRISPR gene editing system.
Professor Moritoshi Sato, and his colleagues from the University of Tokyo, managed to engineer a photoactivatable Cas9 (paCas9) to enable greater precision of the gene editing technique with higher spatial and temporal resolutions.
In the paper, recently published in Nature Biotechnology, they describe the processes of creating the light-activated Cas9 enzyme based upon previous research into the creation of photoswitching proteins called Magnets.
'Magnets' are pairs of proteins designed to come together when activated by light due to changes in electrostatic interactions. The researchers made paCas9 by splitting the Cas9 protein in to two fragments (rendering it inactive). They then added each fragment to one of the Magnet proteins in a pair.
The Magnets come together when hit with blue light enabling the Cas9 fragments to reconstitute and become active again. When the light is turned off, the paCas9 nuclease breaks apart, stopping activity. "Such an on/off-switching property of paCas9 is the most important breakthrough previously unattainable," said Professor Sato.
The CRISPR gene editing system
Watch the video below for an explanation of the CRISPR/Cas9 gene editing system:
Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins are found in many bacteria and archaea. In these organisms they help provide a form of acquired immune defence by breaking apart DNA from foreign pathogens (e.g. viruses).
One of the Cas genes, Cas9, is an endonuclease. Endonucleases are enzymes capable of cleaving polynucleotide chains (like DNA or RNA). Cas9 cuts double-stranded DNA molecules at specific sites depending on a guide RNA sequence. This sequence is complementary to a specific location on a DNA molecule.
By hijacking this process, researchers are able to edit DNA at almost any location by engineering a guide RNA sequence and delivering it into a target cell with Cas9. CRISPR gene editing is capable of silencing, enhancing, adding or modulating genes, creating a powerful tool for studying how genes affect physiology.
However, there are downsides to the traditional technique. The original Cas9 system does not allow modification of a small subset of cells, such as specific neuronal subtypes in the brain, and it often suffers from off-target effects due to uncontrolled activity.
Light-activated CRISPR gene editing overcomes these problems by controlling the temporal activity of the nuclease and by only turning the paCas9 enzyme on in certain cells (via a tightly focused beam of light).
Nihongako Y, Kawano F, Nakajima T, Sato M Photoactivatable CRISPR-Cas9 for optogenetic genome editing Nature Biotechonology (2015) doi: 10.1038/nbt.3245