We all know that the genome editing superhero CRISPR-Cas9 is a master of modifying the genome, but a recent paper from Zhang lab shows that it is more than just a one-trick pony. This latest research boosts the previously developed CRISPR-Cas9 transcriptional activators both in terms of the level of gene induction and in the number of endogenous loci that can be simultaneously overexpressed.
In order to improve this transcriptional activity of CRISPR-Cas9, Konermann et al. needed a few helpers. They took advantage of the reported nuclease-deficient Cas9 (dCas9) fused to the potent VP64 transactivation domain (dCas9-VP64), which is still able to bind guiding RNA (sgRNA) and to be targeted to chromatin through CRISPR. They made it more potent by appending to it an sgRNA able to bind the MS2 bacteriophage coat protein fused to p65, the NF-Kβ transactivating subunit, and to the activation domain of HSF1 (MS2-p65-HSF1).
The coexpression of these components produced a system that the authors named SAM (synergistic activation mediator). SAM was targeted using CRISPR’s usual friends, the RNA guides (sgRNA) to promoter elements to boost transcriptional activation.
With this tool the authors were able to show:
- At least a two-fold upregulation of all the genes tested, with a subset achieving up to 15-fold upregulation.
- An inverse correlation between gene expression and levels of induction.
- A higher overexpression with guiding RNAs annealing close to the TSS (up to –200 bp).
- Targeting of both non-coding RNAs and genes.
- Simultaneous activation of 10 genes with 10 guiding RNAs.
- Minimal off-target effects revealed by RNAseq.
This system performed well in a genome-wide activation screen. A stable A375 cell line expressing SAM was generated and then infected with lentiviruses carrying a library of guiding RNA (targeting every coding isoform of the RefSeq database with three RNAs). The cell lines generated were treated with the BRAF inhibitor PLX-4720.
After 14 days the team screened surviving cells and found overexpressed genes that could be implicated in mediating the resistance to the inhibitor including EGFR, which was previously known, and some new candidates like the ITG receptor family. The authors conclude with the idea that this system could be targeted to the epigenetic landscape soon, recruiting modifiers to defined regions.
To find out how CRISPR-Cas9 acquired another superpower head over to Nature, January 2015.