Functional Identification of Optimized RNAi Triggers Using a Massively Parallel Sensor Assay

Abstract

Summary Short hairpin RNAs (shRNAs) provide powerful experimental tools by enabling stable and regulated gene silencing through programming of endogenous microRNA pathways. Since requirements for efficient shRNA biogenesis and target suppression are largely unknown, many predicted shRNAs fail to efficiently suppress their target. To overcome this barrier, we developed a “Sensor assay” that enables the biological identification of effective shRNAs at large scale. By constructing and evaluating 20,000 RNAi reporters covering every possible target site in 9 mammalian transcripts, we show that our assay reliably identifies potent shRNAs that are surprisingly rare and predominantly missed by existing algorithms. Our unbiased analyses reveal that potent shRNAs share various predicted and previously unknown features associated with specific microRNA processing steps, and suggest a new model for competitive strand selection. Together, our study establishes a powerful tool for large-scale identification of highly potent shRNAs and provides new insights into sequence requirements of effective RNAi.

Tiling genomes of pathogenic viruses identifies potent antiviral shRNAs and reveals a role for secondary structure in shRNA efficacy

“…The sensor assay enables pooled screening for the discovery of potent shRNAs from a large number of candidates (13). Our original study validated the assay by evaluating 20,000 shRNAs from tiling of nine mammalian genes (13).…”

“…The sensor assay enables pooled screening for the discovery of potent shRNAs from a large number of candidates (13). Our original study validated the assay by evaluating 20,000 shRNAs from tiling of nine mammalian genes (13). In the present study we apply this platform to identify potent antiviral shRNAs from four viral genomes, including two HIV strains: NL43 of clade B and 1084i of clade C (14), one HCV strain: JFH1 of Genotype 2a (15), and one H1N1 influenza A strain: influenza A virus A/Puerto Rico/8/34 (PR8).…”

“…We recently developed a multiplex high-throughput assay to identify potent shRNAs (13). This "sensor" assay uses a single vector to coexpress an shRNA and its target sequence fused to the 3′ UTR of a constitutively expressed fluorescence reporter.…”

A novel Atg5-shRNA mouse model enables temporal control of Autophagy in vivo

“…Using a sensor-based screening system [14], we first obtained a panel of shRNAs against Atg5 , encoding an essential component for autophagosome formation, and then selected an shRNA with the greatest knockdown in vitro, Atg5_1065 (#1 in Fig. S1, see Methods).…”

“…A panel of shRNAs in a microRNA backbone (miR30 design) [12] targeting Atg5 was obtained from Mirimus Inc. through a sensor-based screening system [14] in a pLMP [12] backbone and was used to generate retroviral supernatant, except for shAtg5 #4 (pMSCV), which was previously used (targeting both human ATG5 and mouse Atg5 ) [28]. We tested knockdown efficiency of those shRNAs in NIH3T3 cells, and an shRNA showing the strongest knockdown even with the highest dilution (1% v:v) viral supernatant was taken forward for LSL-ATG5i mouse generation by Mirimus Inc.…”