While dead Cas9 (dCas9) has been coupled to optogenetic systems to enable on the fly effector domain action, the not so dead Cas9 has just gotten a very different form of light induction that works with genome editing. The designer genome editing system, developed by a talented team from the University of Pittsburgh and the University of North Carolina at Chapel Hill, utilizes an engineered CRISPR-Cas9 system by installing clever chemistry on choice lysine residues.
Previous light induction technologies use two plant proteins to enable on-the-fly fusions of a sequence recognizer (without nuclease activity) to an effector domain, but this isn’t as applicable to Cas9, since once it loads the sgRNA the catalytic fun starts. Thus, the current optogenetic technology can’t be modified to enable precise temporal and spatial control of CRISPR-Cas9 genome editing, since Cas9 is just one protein and won’t benefit from the current optogenetic inducible fusion approach. So the team went out and brought the light to Cas9 itself, by creating a system that uses a synthetic codon to incorporate the modified lysine residue, which is based off the approach of using a synthetic expanded genetic code.
Here’s what shines through:
- The team further examined the crystal structure and identified lysine residues close to where the sgRNA binds to Cas9 to stimulate catalytic activity.
- K866 is of special interest, as it undergoes a conformational change when binding sgRNA and appears to position it properly with the target DNA.
- The technology relies on site-specific installation of a photocaged lysine (PCK) amino acid to critical residues in Cas9.
- The cage leaves Cas9 inactivated until some UV light is shined to unleash it from its cage.
- The technology works beautifully and the upgrade does not seem to affect Cas9 function, while also enabling rapid activation.
- The system was shown off activating or deactivating exogenous (fluorescent reporters) and endogenous genes in human cells lines (HEK293T).
Ultimately, this research offers a solution to bringing the temporal control of genome editing out the dark ages, which is supported by the success of synthetic expanded genetic codes in model organisms and suggests that this system should work in vivo.
Learn to unleash the power of light-activatable Cas9 in the Journal of the American Chemical Society, April 2015