Life is full of challenges, and when that challenge is epigenome editing even a rock star like CRISPR/Cas9 stands to benefit from helping a hand. Epigenome editing with CRISPR/Cas9 involves using a dead Cas9 (dCas9) that confers the precision of the genome editing system without nuclease activity. The standard approach involves utilizing a fusion protein of dCas9 and effector domain, which allows an effect of choice in the regulation of gene expression, placement of epigenetic marks, or genomic visualization.
However, designer approaches have also emerged that involve a genetically engineered epigenome editing system. One design approach is known as ‘sgRNA 2.0’ and it involves using viral sequences that form stem loops and are recognized by respective RNA binding proteins that can also be fused to effector domains. Overall, sgRNA 2.0 opens up new avenues to overcome the limited powers of a single effector domain in activating gene expression, particularly at enhancers, and also provides new multiplexing opportunities.
By forging dCas9’s genomic targeting to Pumilio’s unique designer pairing the team created Casilio. The sgRNAs of Casilio have PUF binding sites (PBS) inserted into their 3’ end that can be bound by a PUF-effector domain fusion.
Here’s what went down in a human embryonic kidney cell line (HEK293T):
- The hybrid system works!
- A number of distinct designer modules are possible, as the sgRNA will only bind to the activator with the proper pairing of PUF and PBS.
- By borrowing the p65HSF1 activator from the sgRNA 2.0 of SAM they found that it was 3 times more potent at activation than VP64.
- Testing their new designer dCas9/PUFa-p65HSF1 against a more ‘standard’ dCas9-p65HSF1 direct fusion approach revealed a much higher activation rate on stem cell genes (OCT4 and SOX2).
Expanding past transcriptional activation, the team:
- Examined the ability to repress gene expression by using a KRAB domain and found that they could simultaneously activate OCT4 and repress SOX2.
- Turned to a histone acetyltransferase (HAT) effector domain and by targeting different promoter and enhancer elements of OCT4, they found that the Casilio system did a better job at activating enhancer elements.
- Showed off the power to label genomic loci in live cells by examining telomeres and centromeres, which demonstrated that Casilio can be used to label multiple loci.
Overall, the Casilio system provides a number of advantages to the standard dCas9-effector domain direct fusion approach. Casilio enables multiplexing by offering a number of diverse and independent modules that can be easily programmed. Casilio also offers up multimerization by enabling a number of PBS to be engineered into a single sgRNA without altering their function and thus making transcriptional regulation and visualization easier. Interestingly, while not demonstrated yet, the Casilio system also offers the potential feature of complex formation with a single sgRNA containing diverse pairings of effector domains.
Go learn more about the benefits of Casilio’s ‘hybrid vigor’ in Cell Research, January 2016