By Doug Macron
Researchers from Cold Spring Harbor Laboratory this week published details of a new method to identify potent shRNAs on a large scale, which they say also provides “new insights into sequence requirements of effective RNAi.”
While shRNAs are “powerful experimental tools” for gene silencing, the requirements for “efficient shRNA biogenesis and target suppression are largely unknown … [and as a result] many predicted shRNAs fail to efficiently suppress their target,” the team, lead by CSHL researchers Greg Hannon and Scott Lowe, wrote in Molecular Cell.
This, they noted, can lead to false-negative results in functional studies and screens. To address the issue, they developed a so-called sensor assay that enables the “biological detection” of effective shRNAs.
“Our approach measures the potency of shRNAs by monitoring their interaction with a surrogate target cloned into the 3' UTR of a fluorescent reporter, and thus integrates most aspects of shRNA biogenesis, target recognition, and repression,” the team stated in the paper.
“Combining on-chip synthesis of long oligonucleotides with a two-step cloning procedure, we generated a library of [about] 20,000 shRNA-sensor constructs representing almost every target site in nine mammalian transcripts,” they explained. “Using genetically distant avian reporter cells, we simultaneously evaluated the potency of every shRNA within this library via iterative cycles of FACS-based enrichment and deep sequencing-based quantification, and thereby established a straightforward protocol for identifying potent shRNAs in a multiplexed format.”
The team noted that the method accurately predicts the activity of shRNAs toward endogenous targets and “reliably” identifies hairpins that are effective when expressed from a single genomic integration. Their approach, they added, “vastly outperforms” existing siRNA-based algorithms, which miss more than 70 percent of the sensor-derived shRNAs “and generally necessitate the testing of many predictions to identify even a single potent shRNA.”
The CSHL investigators also wrote in Molecular Cell that the shRNA-sensor method, while designed with potency in mind, may also address RNAi specificity issues. “First, our assay helps to control for sequence-specific off-target effects by enabling the identification of multiple potent shRNAs against any gene,” they stated. “Second, it will reduce passenger-mediated off-target effects by selecting potent shRNAs with a bias for incorporating the intended guide strand into RISC.”
The identification parameters guiding Drosha/DGCR8 processing, a component of the microRNA pathway, will also help minimize off-target effects associated with aberrant guide strands, while the assay's identification of single-copy shRNAs “should further reduce off-target toxicities owing to saturation of the RNAi machinery,” they added.
“Our approach lays out a practical workflow for the rapid generation of functionally validated shRNA libraries, as well as the identification of potent RNAi triggers for biological studies and, eventually, RNAi therapeutics,” they concluded.
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