In a pilot experiment to find the most potent RNAi triggers for nine target genes—including a few hard-to-suppress cancer genes—the team generated genetic codes for about 20,000 shRNAs, each with the potential to shut down any one of the nine target genes. Each genetic code was then inserted into a retrovirus that was also engineered to carry the target gene—or the “sensor”—and a gene for a fluorescent protein, called a “marker.” This design ensured that when cells growing in dishes were infected with the genetically engineered viruses, both the sensor and the marker would always be co-produced within each cell along with one shRNA.
ShRNAs that were inefficient at triggering RNAi failed to spur the destruction of their target (or sensor) genes’ RNA and that of the fluorescent marker as well. So these cells produced a fluorescent protein whose brightness could easily be detected. But shRNAs that were potent RNAi triggers caused the efficient destruction of the target gene’s RNA and that of the fluorescent marker as well. Those host cells therefore lacked all traces of fluorescence.
“All we had to do then was to sort these cells out, pull out each cell’s genetic material and sequence the short hairpin RNA,” explains graduate student Christof Fellmann, who together with post-doctoral fellow Johannes Zuber led these efforts. “This gave us the identity of the RNAi trigger that was most potent in shutting down the target gene.”
The team found that for each of the nine genes, only about 2.5% of all possible shRNAs against that gene could shut down its activity efficiently. The success of their approach also means that scientists might no longer have to rely on current algorithms that are often not accurate in predicting which shRNA would work best at shutting down any given gene.
“There is still very little that is known about small RNA biogenesis,” says Hannon. “But this assay has now allowed us to explore this process at a scale that wasn’t feasible before.” The scientists’ analysis of the 20,000 small hairpin RNAs, and especially those recovered from cells in which the target gene had been efficiently shut off has revealed new insights into this process and drastically improved the recipe for creating a potent RNAi trigger. “Our assay has given us a strong framework for rational design of small hairpin RNAs,” says Lowe.
About Cold Spring Harbor Laboratory Founded in 1890, Cold Spring Harbor Laboratory (CSHL) has shaped contemporary biomedical research and education with programs in cancer, neuroscience, plant biology and quantitative biology. CSHL is ranked number one in the world by Thomson Reuters for impact of its research in molecular biology and genetics. The Laboratory has been home to eight Nobel Prize winners. Today, CSHL’s multidisciplinary scientific community is more than 400 scientists strong, and its Meetings & Courses program hosts more than 12,000 scientists from around the world each year. The Laboratory’s education arm also includes a graduate school and programs for undergraduates as well as middle and high school students and teachers. CSHL is a private, not-for-profit institution on the north shore of Long Island. For more information, visit www.cshl.edu.
“Functional identification of optimized RNAi triggers using a massively parallel Sensor assay,” appears online ahead of print in Molecular Cell on February 24. The full citation is: Christof Fellmann, Johannes Zuber, Katherine McJunkin, Kenneth Chang, Colin D. Malone, Ross A. Dickins, Qikai Xu, Michael O. Hengartner, Stephen J. Elledge, Gregory J. Hannon, and Scott W. Lowe. The paper is available for download at http://www.cell.com/molecular-cell/fulltext/S1097-2765(11)00091-8.
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