For those of you who were thinking the CRISPR/Cas9 system was just limited to genome editing, think again because the story just got a new twist. Almost like combining the bullet speed of Superman with the hi-tech gadgets of Batman, Alan Tackett and colleagues come up with a new tool to look at all the proteins of a specific genomic region – the local epiproteome – by combining two techniques: CRISPR/Cas9 and ChAP-MS.
It is no secret that isolating a specific region of chromatin and identifying its component proteins and modifications can be extremely useful in studying transcription or epigenetic regulation. But it also comes with major hurdles: specific regions are by definition low-copy and require affinity-based enrichment. On top of that, one also needs to filter out all the non-specific proteins and pick out the true members of the local epiproteome family.
Using a tagged, catalytically inactive Cas9 and a custom-designed guide RNA (gRNA), Tackett and his colleagues target the yeast Gal1 promoter under conditions of active transcription. Then they use their previously published Chromatin Affinity Purification technique coupled to quantitative mass spectrometry (ChAP-MS) to purify the tagged Cas9 and identify the associated proteins. They find that:
- Cas9-gRNA can specifically bind to its target Gal1 promoter region.
- Using Cas9 alone without gRNA can be used to filter out proteins that non-specifically associate.
- Specific histone modifications associated with transcription were enriched.
However, just like Superman has his kryptonite, CRISPR-ChAP-MS is not without its weaknesses. Although the Cas9-gRNA has superior ability to specifically target and enrich the Gal1 promoter under transcriptionally active conditions, it’s unable to do so in a repressive context. Thus, the authors suggest testing target site enrichment before setting up a big proteomics experiment.
Considering the fast-evolving capabilities of the CRISPR/Cas9 system though, the future is bright for local epiproteome mapping.
Find out all the details: Epigenetics, July 2014