Deactivated Cas9 (dCas9) has quickly risen to the spotlight to show that transcriptional regulation is just as fashionable as the ever so popular genome editing. Now, a number of designer systems face-off in a competition to find out who is best at turning on a genome.
dCas9 provides the perfect combination of scaffold and targeting device to precisely localize an effector domain fused to its C-terminus. The first generation of genome seducer used VP64, which consists of four copies of VP16 (a viral protein sequence of 16 amino acids that are used for transcriptional activation). With the emergence of a number of second generation designer systems, the lab of George Church at Harvard University has provided some much needed comparative testing.
Church shares, “The possibility to selectively activate genes using various engineered variants of the CRISPR-Cas9 system left many researchers questioning which of the available synthetic activating Cas9 proteins to use for their purposes. The main challenge was that all had been uniquely designed and tested in different settings; there was no side-by-side comparison of their relative potentials. We wanted to provide that side-by-side comparison to the biomedical research community.”
Second Generation CRISPR Activators
Here are the 7 dCas9 systems they compared, which are illustrated in all their detail in the paper:
- The powerful activating trio VP64-p65-Rta (VPR): these three effector domains are separated only by short amino acid linkers.
- The Synergistic Activation Mediator SAM makes uses of not only VP64 but also sgRNA 2.0, which contains sequence to recruit a viral protein fused to even more effectors (p65-hsf1).
- RNA Scaffolds also utilizes sgRNA 2.0 and recruits 3 viral proteins fused to VP64.
- Suntag sports a protruding chain of 10 peptide epitopes that are recognized by an entourage of antibodies fused to VP64.
- The epigenetic editor p300 deposits activating H3K27ac.
- VP160, which is also known as CRISPR-on, has ten times the VPs of VP16.
- VP64-dCas9-BFP-VP64 makes use of that much neglected N-terminus.
Co-first author Marcelle Tuttle fills in, “We first surveyed seven advanced Cas9 activators, comparing them to each other and the original Cas9 activator that served to provide proof-of-concept for the gene activation potential of CRISPR-Cas9. Three of them provided much higher gene activation than the other candidates while maintaining high specificities toward their target genes.”
The top 3 activators could activate expression several orders of magnitude higher than dCas9-VP64 and they are: VPR, SAM, and Suntag. These activators were then taken to the next round of the competition. Here’s what went down in human embryonic kidney (HEK) 293T cells:
- SAM was most consistent at inducing gene expression.
- Suntag and VPR weren’t too far behind, staying within 5-fold of SAM, and tied for second.
- Each system has an upper limit for inducing gene expression, which is determined by its design.
- When it comes to turning on genes, the weakly expressed ones appear to be the most excitable.
- No designer system stood out in ability to multiplex or off-target effects.
The challenge for top activator didn’t end in HEK293T cells, as the cell-line showdown was up next. When tested for human cell line preference it was found that SAM is best in HeLa cells, while Suntag and VPR are more effective with U-2 OS and MCF7 cells.
Experiments in mouse (N2A and 3T3 cells) and Drosophila (S2R+) cells revealed that while all designer systems are better than dCas9-VP64, there is no particular star across all species, and the results vary by target gene. The experiments finally took a ‘Frankenstein’s monster’ approach by swapping around parts in a quest to make the ultimate hybrid, but this failed to yield a creation that outperformed the original systems.
Co-first author Alejandro Chavez shares that in, “In some cases, maximum possible activation of a target gene is necessary to achieve a cellular or therapeutic effect.” This obstacle was tackled in the final set of experiments, where Chavez fills in that, “We managed to cooperatively enhance expression of specific genes when we targeted them with … a top performing activator using three different guide RNAs.” So while recombining many different activation domains at a single target site didn’t help activation, stimulating multiple sites at the same time made a better turn on.
Go turn on your genes over at Nature Methods, May 2016