Let there be light: Photoactivatable CRISPR-Cas9

It is safe to say that the gene editing technique CRISPR-Cas9 has shaken the science world. When something this great comes along, it pushes the field around it. Scientific progress is accelerated in a race to optimize the method and see just what it is capable of doing. There have already been newly engineered Cas9 proteins that can silence or activate genes. CRISPR-Cas9 libraries are also being used to do genome wide screens to find genes associated with drug resistance. In this post, we'll be looking at the next installment of innovation: light activated CRISPR-Cas9.

At the University of Tokyo, researchers have made a photo-activatable CRISPR-Cas9 construct. This tool, called paCas9, cuts the genome at a specified location when it is hit with a blue-light. To do this, researchers utilized a new pair of photo-switchable proteins called ‘Magnets'. The two proteins, name positive Magnet (pMag) and negative Magnet (nMag), respond to blue light by heterodimerizing to each other. The scientists cut the Cas9 protein in half, and attached the pMag to one half, and nMag to the other. When there is no blue light, the Magnets do not bind, and the Cas9 is inactive. When hit with blue light, the Magnets combine putting the Cas9 protein back together, making it active. If the two Magnet-Cas9 proteins are added along with the short guide RNA the reassembled Cas9 protein will be guided to make a double stranded break after blue-light exposure. This protocol is outlined in the diagram above to the right. With a few relatively simple modifications to the Cas9 protein, we now have location, time, and site specific gene-editing.

To show that their constructs did indeed work, they designed a cell line that would glow when the Cas9 successfully cut the genome. This cell line had a gene that produced a bioluminescent protein called luciferase. They put a premature stop codon in the middle of the luciferase gene, disrupting its transcription. Next, the Magnet-Cas9 proteins were put into the cells with a short guide RNA (sgRNA) to localize Cas9 construct to the stop codon in the luciferase gene. If the Cas9 successfully cut the gene at the right position, it would remove the stop codon, luciferase would be transcribed, and the cell would emit light. The researchers designed two separate sets of Cas9-Magnet constructs (Constructs 3 and 4) and, as a control, they designed two other sets of constructs (Constructs 1 and 2) using other, larger photo-activatable protein heterodimers. They observed that when they shine blue light on the cells, there is a dramatic increase in the amount of luciferase activity compared to the same cells kept in the dark. The control constructs and an unbound Cas9, labeled no contruct, showed no difference in luciferase levels whether they were exposed to blue light or kept in the dark. The data is shown in the graph to the left. This experiment demonstrated that their constructs were able to cut the genome, and do it only under blue light conditions.

Sometimes, mutating a gene isn't ideal. It may be better to just turn it off for a short period of time and look at the effects. In order to arrest transcription, but not mutate the gene, they did nearly the exact same experiment as above, but augmented the Cas9 protein slightly. By mutating the Cas9 at two amino acids, it is still able to localize to the target sequence using the sgRNA, but it sits there without cutting the gene. Attaching this mutated Cas9 protein (named dCas9) to the Magnets allowed the researchers to turn on and off gene activity. To test this, they used the luciferase reporter we saw before, but this time, there was no stop codon in the middle. When dCas9 is localized to the luciferase gene, it stops it's transcription, and thus, the activity of the luciferase protein. In the graph to the right, they show that cells initially exposed to light, with low luciferase activity caused by the dCas9, could recover back to normal if left in the dark as indicated by the dashed red and blue lines. If they continued blue light exposure, luciferase activity would continue to decrease. Using a photoactivatable, inactive form of Cas9, they could turn genes on and off with the flick of a switch.

Although relatively simple, this is a very important advancement in the gene editing field. There could be several advantages of the light-inducible gene-editing systems over its original counterparts. It may be easier to get all of the components into the cell when it is broken down into smaller parts. The halves of Cas9, even when attached to an nMag or pMag, are about 40% smaller than the whole Cas9 protein, making it easier to infect cells. Some of the biggest concerns of the CRISPR-Cas9 system stem from its off-target effects. But, by only activating Cas9 for a small amount of time, this could reduce the amount of off-target cuts because it reduces the chances of Cas9 finding and snipping the incorrect site. Using the gene silencing paCas9 mentioned above, we can reversibly and easily shut-off genes to test cyclic aspects of biology, like circadian rhythms, or different stages of mitosis. It will exciting to see what new experiments will be designed using photoactivatable Cas9, and it's especially fun to look out for the newest CRISPR-Cas9 modifications.

References:

Polstein, L.R., Gersbach, C.A. A light-inducible CRISPR-Cas9 system for control of endogenous gene activation. Nature Chemical Biology, 11, 198-200 (2015).

Nihongaki, Y., Kawano, F., Nakajima, T., Sato, M. Photoactivatable CRISPR-Cas9 for optogenetic genome editing. Nature Biotechnology, 33, 755-760 (2015).

Image credits:

All images are augmented from the Nihongaki et al. paper referenced above.