NEW YORK (GenomeWeb) – In the last half-year, two of the hottest trends in life science, optogenetics and CRISPR, have finally come together. Labs around the world have published papers showing light-activated Cas9 enzyme activity, which could create more powerful research tools and help nudge the technology closer to in vivo use.
The new techniques can allow either gene editing or activation and they all afford some measure of spatial or temporal control over the Cas9 enzyme.
Three recently published papers, in particular, illustrate technologies to control Cas9 activity with light.
One recent paper, authored by James Hemphill and Alex Deiters of the University of Pittsburgh and published in April in the Journal of the American Chemical Society, claimed to detail the first photo-activated Cas9 enzyme.
To make the standard Cas9 light-inducible, they re-engineered the protein to include a photo-caged lysine that would inhibit normal enzyme function near the guide RNA (gRNA) binding site. Exposure to UV light caused the nitrobenzyl protecting group to fall off and left what was functionally a wild-type Cas9.
The scientists were able to silence an endogenous gene by disrupting upstream regulatory elements and the amino acid sequence of transmembrane transferring receptor CD71 in HeLa cells.
"We saw complete inactivity before radiation," Deiters told GenomeWeb. A two-minute exposure to light restored enzymatic activity to wild-type levels, he said.
Deiters said that there's no way to turn off the enzymatic activity, except by natural degradation by the cell. "Currently we cannot reversibly switch it back off with light," he said. The ability to gain spatial and temporal control was important to demonstrate, he said.
"We'll see if we can use this approach to not just control gene editing but transcriptional activation and deactivation," he said, adding that he would like to tune the activation wavelength from UV to blue light or infrared.
Borrowing a leaf from the plant's notebook
When Hemphill et al. submitted their manuscript in December 2014 it was true that there wasn't a light-activated Cas9 yet. But Duke University's Lauren Polstein and Charles Gersbach were able to get their light-inducible Cas9 fusion in the literature first and were even able to get it to activate genes (although it didn't edit the genome). They published their paper online in February in Nature Chemical Biology.
Their system took a page out of plant biology to get more control over Cas9. "There are different protein pairs found in plants that will dimer in response to light," Polstein explained, with some that will respond to blue light and some to red. "The plant uses the mechanism to detect different seasons. In the spring there's more sunlight so it receives more blue light and induces flowering. We have taken advantage of that interaction and hijacked that system," she said.
She used a pair of proteins from Arabidopsis thaliana, CRY2 and CIB1, fusing them to the transactivation domain vp64 and to a toothless dCas9, respectively. With that system, they were able to activate endogenous genes in HEK293T cells.
"Right now there's a lot of synthetic biologists looking at gene circuits and they like light-inducible systems because it allows them to turn genes on or off in both space and time," Polstein said. "You can look and see how sender and receiver cells interact or you could have the sender cells release a protein in a dish by illuminating on one half of the dish and the other half would be not illuminated and not active," she said.
The system's most immediate use is as a research tool, she said, but it could be used in vivo in the same way scientists are thinking of using optogenetics for deep brain stimulation.
Cutting and interfering
The most recent paper, published June 15 in Nature Biotechnology, finally delivers on an optogenetic approach to genome editing. Somehow it didn't come out of the Broad Institute lab of Feng Zhang, who has worked to develop both technologies, but the University of Tokyo. Yuta Nihongaki and Moritoshi Sato led a team of scientists that designed a system that brings together split Cas9 fragments using dimerizing protein domains.
The challenge was in finding both the right place to split the Cas9 protein to still allow it to function as well as the domains that would allow them to tie it all together. They tried CRY2 and CIB1, but couldn’t get it to work; however, a system of photoswitchable proteins known as Magnets, published in 2014, was able to work.
They showed that the system, called photoactivatable Cas9 (paCas9), was able to induce genome edits through both non-homologous end joining and homology-directed repair pathways in HEK293T cells when exposed to blue light. They induced indel mutations in CCR5 and inserted a single-stranded repair template of HindIII into an edit at EMX1. The system worked when used with paired nickases to reduce off-target activity and even worked with a catalytically inactive dCas9 to enable gene silencing through CRISPR interference.
The scientists had spatial control over paCas9 and were able to turn off the activation of the enzyme by putting the cells in the dark.
There are other ways of controlling the Cas9 activation window. Researchers have also developed Cas9 enzymes that only work in the presence of small molecules like rapamycin and 4-hydroxytamoxifen, which also improves genome editing specificity. It allows spatio-temporal control in vivo, in places where light can't easily reach.
But you can't stop activity on a dime using small molecules and the molecules can diffuse into the tissue, perhaps disrupting biochemical pathways. Not so, with the Polstein and Nihongaki dimerizing systems, which could make them important tools for both research and clinical uses of CRISPR/Cas9.
"The spatiotemporal and reversible properties of paCas9 are well suited for the dissection of causal gene function in diverse biological processes and for medical applications, such as in vivo and ex vivo gene therapies," Nihongaki and his colleagues wrote.