Thursday, June 25, 2015

Photoactivatable #CRISPR-Cas9 systems!

There are a few recently developed light-activated CRISPR-Cas9 tools that have been reported lately.  I'm motivated to post this based on the most recent one, which demonstrated gene editing using modified "split" Cas9 protein halves that were conjugated to newly developed light-inducible dimerization domains named "Magnets".   This was a paper just published by Nihongaki et al. in Nature Biotechnology.   (PDF is as of the post only published online at this link). This system is nice in that it just requires expression of normal gRNA plus the two modified coding portions of Cas9, plus, blue light to activate dimerization and Cas9 targeting function and cleavage.   It's reversible too - removing the light stimulation lets the complex fall apart.  Neat!

Other groups in parallel have created very similar tools that allow light-inducible activation of Cas9 to allow targeting.  In a related paper Nihongaki and colleagues showed they could use this to activate transcription at CRISPR target genes using light, and Polstein and Gersbach have made very similar tools.  Both groups used the CRY2 and CIB1 light-induced dimerization domains from Arabidopsis.

Using a different strategy, Hemphill et al used a caged amino acid strategy to encode a light-activatable codon into Cas9.  This system is a bit more complex to set up, as it requires engineering a pyrrolysl tRNA synthetase into the cells being targeted - basically, re-engineering the genetic code to get a light-activated lysine into the guts of Cas9.  This seems very different mechanistically than the dimerization approach and so it maybe a good alternative for some applications, as it probably has some distinct wavelength and kinetic properties.  Always a good thing to have different tools in the toolkit.

No comments:

Post a Comment