For all the hoopla over CRISPR/Cas9 and its mystical powers, the system still hasn’t passed the most basic test of any magic talisman – waving it over a locked gate while chanting “open sesame”! Fortunately, a band of scientists led by Amira Barkal and Richard Sherwood have now vindicated CRISPR’s powers, using it to open locked chromatin.
Nuclease-dead dCas9 can perform many tasks, from transcriptional regulation to histone modification. However, it hasn’t been clear what, if anything, dCas9 does to closed chromatin. To test this, these modern-day Ali Babas designed sgRNAs targeting 16 regions with low chromatin accessibility in mouse embryonic stem cells (mESCs), finding that:
- DNase hypersensitivity increased at all targeted locations.
- Chromatin opening was strongest within 20 bp of the target site, and there was a significant increase in DNase sensitivity out to 100 bp away.
- Chromatin opening was strong enough to allow a previously excluded transcription factor (RAR) to bind an adjacent motif.
- When, and only when, both dCas9 and RAR targeted nearby locations, RAR was able to transcriptionally activate GFP.
The Cas9 chromatin effects were not huge – only 1.7-fold increase in DNase hypersensitivity and 1.5-fold increase in GFP expression – but they nevertheless reveal new capabilities and caveats for dCas9. First, dCas9 can be combined with transcription factors to activate genes buried away in closed chromatin, forming a transcriptional AND logic gate. Second, dCas9 significantly affects local chromatin structure, so genomic labeling studies should take this into account.
Despite the modest effects, DNase hypersensitivity and GFP expression were averaged over many cells, so it may be that some single cells see much greater effects than average, while others are not affected. Also, dCas9 chromatin opening might be enhanced by, e.g., a bulky protein fusion, or invoking “open poppy” instead of “sesame”!
In any case, you can look for treasures locked away in your own chromatin at PLoS ONE, 2016.