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Church Lab Scientists Compare CRISPR/Cas9-Based Gene Expression Activators


NEW YORK (GenomeWeb) – For scientists looking to induce targeted gene expression, a trio of CRISPR/Cas9-based activation systems stand above the rest, according to a study published today.

The VP64-p65-Rta activator (VPR, pronounced "viper), synergistic activation mediators (SAM), and the SunTag peptide array "are all pretty good choices," Marcelle Tuttle, a post-graduate fellow at the Wyss Institute for Biologically Inspired Engineering told GenomeWeb. The three technologies are all based on a nuclease-null Cas9 enzyme (dCas9) fused to proteins help that promote gene expression.

Tuttle and Alejandro Chavez, a postdoc in George Church's lab at Harvard University, recently led a study comparing eight different CRISPR/Cas9-based gene activation systems. They published their results today in Nature Methods. Church and Jim Collins, of the Massachusetts Institute of Technology, were co-senior authors.

VPR (pronounced "viper"), SAM, and SunTag are so-called next-generation CRISPR activator systems, based on the idea of fusing dCas9 to the VP64 transcription activator domain independently described by several groups, including those led by Duke University's Charles Gersbach, Massachusetts General Hospital's Keith Joung, and Church.

Chavez and Tuttle contributed to the development of VPR in the Church Lab, which added two more transcription activator domains to the mix. Scientists from Feng Zhang's lab at the Broad Institute and MIT developed SAM, which includes the dCas9-VP64 fusion as well as a modified single guide RNA (sgRNA) featuring MS2 aptamers and engineered multi-activation domain helper proteins. SunTag, developed by Jonathan Weissman's lab at the University of California, San Francisco, fuses that peptide array to dCas9 and recruits up to 10 antibodies fused to VP64 to drive expression.

Following her work on VPR, Tuttle wondered how the different activator systems compared head to head, in the same system, at the same dosing, and on the same genes.

"We wanted to play around with the different parts being published," she said. "We wanted to know which one worked best. If we took the time to flesh it out, it could be a useful resource for the field."

First, the authors performed a pilot study, comparing not only the dCas9-VP64 fusion and the three eventual winners, but also VP160, p300, VP64-dCas9-BFP-VP64, and Scaffold sgRNA technologies.

Based on two experiments targeting two separate genes, the scientists chose to move forward with VPR, SAM, and SunTag, although in almost all cases, the protein fusions increased relative RNA expression.

Next, they compared the three activator systems in multiple human cell lines — HEK293T, HeLa, U-2 OS, and MCF7 — and Drosophila and mouse cell lines. Different activators performed better or worse depending on the context. SAM was more potent in HeLa cells, while SunTag and VPR were more potent in U-2 OS and MCF7 cells.

"The biological basis for these cell-line-specific differences remains to be determined," the authors wrote. Tuttle offered some practical advice for scientists trying to pick one: just try them all and see what works best. Alternatively, use the one you're most familiar with. "The all work about equivalently," she said. Finding the best would depend on both the target and the model system.

For research purposes, plasmids containing the respective systems can be obtained from Addgene. "If you already have a plasmid with the normal [sgRNA], you would have to order the SAM guide RNA," she said, adding that VPR was a two-component system, while the others have three, so would require fewer rounds of selection.

For the new Nature Methods study, the scientists also attempted to use multiple activator systems to target the same gene at the same time. Accomplishing this with several different sgRNAs achieved "additive or greater levels of gene activation," they wrote.

They noted that studies using CRISPR activation have already revealed mechanisms of cell death and tumor resistance to targeted therapy. This comparison study may help other researchers more easily compare technologies and enrich this vein of research.