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The Best CRISPR-guided Transcriptional Activators

06/01/2016
Jeffrey M. Perkel, PhD

Researchers surveyed dCas9 variants to identify the best designs for boosting gene expression across genes and species. Learn more...


The dCas9-based SAM (synergistic activation mediator) system includes a dCas9-VP64 fusion and a modified single-guide RNA capable of recruiting both p65 and HSF1 transcriptional activation domains.

Credit: Nature 517, 583–588 (29 January 2015) doi:10.1038/nature14136.

The CRISPR/Cas9 system can do more than edit genomes. Using a nuclease-deficient (“dead”) Cas9, researchers can haul anything they like to defined locations within the genome. Researchers have used this approach to fluorescently label specific sequences, alter epigenomic status, and purify specific genomic regions.

Another popular application couples dCas9 to transcriptional activation domains to upregulate expression of targeted genes. At least eight such designs have been described since 2013, but because each study used different cell lines, guide RNAs, and targets, it wasn’t possible to directly compare them. Now it is.

Alejandro Chavez and Marcelle Tuttle in George Church’s lab at Harvard University and their colleagues surveyed seven “second-generation” activators and compared their performance in human, mouse, and fly cells to that of the “first-generation” design, which fused dCas9 to the viral transcriptional activation domain, VP64. [1]

Their conclusion: three activators, VPR, SAM, and Suntag, are the best of the lot, consistently outperforming VP64 and generally performing more or less equivalently (within an order of magnitude or so) across different species and gene targets, although the extent of transcriptional activation varied from gene to gene. (All tested activator systems are available from Addgene.)

That three designs should perform so comparably was unexpected. Also surprising, was that the team was unable to blend different approaches -- for instance, recruiting different activator domains to different parts of the complex -- to produce super-activators. “The only thing that increased activation was using multiple guide RNAs [per target],” said Chavez.

Stanley Qi, Assistant Professor of Bioengineering at Stanford University, said that as the first systematic comparison between engineered dCas9 activator systems, the study “will become a very useful resource for the choice of different activator systems for different purposes.”

The results also clearly demonstrate how far there remains to go, added Pablo Perez-Pinera of the University of Illinois at Urbana-Champaign, in that the transcriptional activation achieved by just one guide RNA remains highly variable. “This is currently a big limitation for studies that either require protein overexpression or for genetic screenings. We ultimately need systems to accomplish more robust, tunable, and sustained activation of native gene expression.”

Work to overcome such deficits is ongoing. In the meantime, Tuttle advised, “If you have the time and capability, try to pilot [test] all three [activators] and choose the one that works best for your particular application. But for the most part, they are pretty much interchangeable.”

Reference

Chavez, A., et al., “Comparison of Cas9 activators in multiple species,” Nat Methods, 23 May 2016. [doi:10.1038/nmeth.3871]