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Harvard Scientists Develop Cas9-based Method to Reprogram Gene Expression

NEW YORK (GenomeWeb) – Researchers from Harvard University have leveraged the Cas9 protein to activate genes, causing them to activate transcription to express or repress desired genetic traits.

By fusing the Cas9 nuclease to a hybrid triple-strength transcription factor, the scientists from the Wyss Institute at Harvard University and Harvard Medical School further were able to manipulate multiple genes to control expression, they reported in a paper published this week in Nature Methods.

"The ability to selectively upregulate gene expression provides a powerful means to reprogram cellular identity for regenerative medicine and basic research purposes," the authors wrote.

"In terms of genetic engineering, the more knobs you can twist to exert control over the expression of genetic traits, the better," George Church, a professor of genetics at HMS and a co-author on the study, said in a statement. "This new work represents a major, entirely new class of knobs that we could use to control multiple genes and, therefore, influence whether or not specific genetic traits are expressed, and to what extent we could essentially dial gene expression up or down with great precision."

Scientists have known that the RNA-guided, DNA cutting nuclease Cas9 can be reengineered into a toothless programmable transcription factor that has lost its ability to cleave. But prior designs have had underwhelming efficiency, so scientists in Church's lab, led by postdoc Alejandro Chavez, Jonathan Schieman, and Suhani Vora, set out to make a better one.

The researchers looked into a series of more than 20 candidate effectors with known transcriptional roles and fused them to the C terminus of the Streptococcus pyogenes dCas9 enzyme. VP64, an activation domain module known to work in CRISPR/Cas9 induced gene expression, proved to be the most efficient. Taking the dCas9-VP64 complex as a starting scaffold, the scientists added the transcription factors p65 and RTA to create a triple-pronged hybrid VP64-p65-Rta activator, which they dubbed VPR.

Using VPR, the team demonstrated the ability to manipulate gene expression in yeast, flies, mouse, and human cell cultures, with a fivefold to 300-fold improvement in activation levels over VP64-only Cas9 activator complexes.

The researchers also showed that VPR has the ability to activate genes in the "dark matter" regions of the genome that aren't translated into proteins themselves but which have a poorly understood effect on gene expression.

The scientists could even use multiple RNA guides to activate multiple genes at once. Running a pooled activation experiment, they induced four genes at the same time: MIAT, NEUROD1, ASCL1 and RHOXF2. "VPR allowed for robust multilocus activation, showing significantly (severalfold) higher expression levels than VP64 across the panel of genes," the authors said.

Finally, the researchers used VPR to induce "rapid and robust" differentiation in human induced pluripotent stem cells, growing neurons from the cells. They wrote that previous attempts to do so using dCas9-VP64-based activators had been unsuccessful.

The potential applications of improved CRISPR/Cas9 induced gene expression are both academic and clinical.

"In order to grow organs from stem cells, our understanding of developmental biology needs to increase rapidly," said Church, one of the senior authors. "This multivariate approach allows us to quickly churn through and analyze large numbers of gene combinations to identify developmental pathways much faster than has been previously capable."

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