We knew it was coming. No, not another Jurassic Park sequel. No, not another installment of Star Wars. More inevitable than the revival of the Terminator franchise: we now have photoactivatable CRISPR/Cas9.
Yes, as surely as the sun rises, scientists have developed a way to use that light (or at least the blue part) to activate Cas9 gene editing. The new photoactivatable Cas9, developed by Yuta Nihongaki and colleagues in the lab of Moritoshi Sato at the University of Tokyo, builds on another recent photoactivated Cas9, enabling gene editing in specific cells at specific times.
The Dawn of paCas9
We may have known photoactivatable Cas9 was coming, but that doesn’t mean it was easy. Previously, catalytically dead dCas9 was made into a photo-induced transcriptional activator by using a photo-dimerization system (CRY2/CIB1) to make it reversibly bind activator domains. That let Cas9 turn genes on, but not edit them. In other recent work, fully active Cas9 was made photoactivatable by replacing a key lysine with photocaged lysine. However, that required a special genetic code-expanding, amber-suppressing tRNA, which could potentially cause side effects for genes no longer stopping at the TAG codon.
In the new paper, a split Cas9 was generated that reversibly dimerizes when hit with light, activating its full gene-editing ability. This took a bit of optimizing:
- First, the team screened potential split sites using a rapamycin-induced dimerization system, finding a good fragment pair
- Next, they fused this pair to the photo-dimerizing Cry2/CIB1 domains, producing… nothing
- Then the team tried another (smaller) photo-dimerization pair: the Magnet system, which worked!
- But there was still some background gene cutting in the dark, so they tried another version of Magnets, which was just as active in the light, but this time stayed off in the dark
paCas9, Hey, What’s it Good For?
Ok, so we have photoactivatable CRISPR. Does it do everything the regular version can? Fortunately, Nihongaki and colleagues performed a tour-de-force of demonstrations, including:
- Light-induced mutation by non-homologous end joining (NHEJ)
- Light-induced gene replacement by homologous recombination
- Light-induced nicking
- Reversible Cas9 activity
- Light-induced (and reversible) transcriptional repression
- Gene editing in a narrow illuminated stripe
- Gene editing in multiple cell lines (HEK293T and HELA)
The system isn’t quite perfect; it’s only about 60% as active as regular Cas9, but it extends the precision of CRISPR to space and time. As a bonus, the Magnet-CRISPR fragments are small, making them easier to shove into a virus for more efficient delivery.
Read more in Nature Biotechnology, June 2015