ABSTRACT: Purpose: Generate a large, high quality database of paired shRNA efficacy/sequence datapoints. Methods:Twelve shRNAs for each Refseq annotated human gene were selected based on the DSIR algorithm. Twelve batches of ~22K shRNAs (corresponding to 12 agilent chips) were then assessed for efficacy via the sensor method outlined in Fellmann et al, Mol Cell, 2011. Conclusions: Neighboring nucleotide combinations are best at predicting shRNA efficacy.
Project description:shRNAs selected with the shERWOOD algorithm were converted to have a U at the 5' end of their guide. When endogenous 1U shRNAs were compared to artificial shRNA via the sensor algorithm, the endogenous shRNAs were found to be more efficacious. Purpose: Structural studies have hinted that the 5' end of shRNA guides is engulfed in the RISC complex. It has also been reported that shRNAs with a 5' U are more efficacious than those with other 5' caps. We wished to determine whether replacement of shRNA guide 5' nucleotides with a U, regardless of the corresponding target base, would increase their efficacy. Method: For each gene in the "druggable genome" 10 shRNAs were selected with the shERWOOD algorithm. In each case the score was assessed as if the guide had a 5' U. Sensor constructs were designed pairing 1U-guide shRNAs with their endogenous target. shRNAs were assessed for efficacy via the shRNA sensor assay (Fellmann et al. Mol Cell 2011). Results: shRNAs with artificial 5' Us were found to be less efficacious than those with an endogenous 5' U,
Project description:Sub-genomewide shRNA libraries were constructed using the current RNAi consortium constructs as well as using the DSIR (siRNA algoirthm) and a novel shRNA specific algorithm (shERWOOD). All libraries were placed into mir30 expression vectors. The shERWOOD libraries were also placed in a vector harboring an optimized mir cassette (ultramir). Each library was screened using the pancreatic cell line A385. A concensus set of essential genes identified as the set for which two shRNAs depleted in each of the libries. For these genes, a great percentage of shERWOOD seletected shRNA depleted. In addition the placement of shERWOOD selected constructs into ultramir scaffoled increased the rate of shRNA depletion for essential genes further. Purpose: shRNA screens were carried out using various library construction strategies to identify the strategy that provides the best shRNA screening results. Method: Libraries were constructed using the TRC shRNA set as well as shRNAs identified using the DSIR and shERWOOD algorithms. shRNA libraries were cloned into mir30 expression vectors. shERWOOD shRNAs were also cloned into an expression vector harboring an optimized microRNA scaffold termed ultramir. Each resultant library was screened using the pancreatic cell line A385. Each library was analyzed separately to identify a set of genes where at least two shRNAs depleted. These gene sets were intersected to develop a set of essential genes. Results: The shERWOOD shRNA libraries provided the highest number depleting shRNAs for each essential gene. Further these shRNAs depleted to a greater extent than did the shRNAs from the other libraries. When shERWOOD libraries were placed into the ultramir cassette a greater number of shRNAs per essential gene depleted.
Project description:Two shRNAs were placed into expression vectors harboring mir30 microRNA scaffold and an optimized scaffold where the artificial restriction sights in mir30 have been removed. After infection and selection shRNA processing was assessed by small-RNA cloning. For both shRNAs, placement into the optimized scaffold resulted in a ~two-fold increase in processing (based on smallRNA levels). Purpose: Others have reported that the EcoRI site that was introduced to the mir30 scaffold results in decreased smallRNA processing and hence reduced target knockdown. We've developed an alternative scaffold (termed ultramir) where this site is removed. smallRNA cloning was used to determine if the movement of this sight resulted in an increase in shRNA processing. Method: Two shRNAs (one targeting Renilla Luciferase and one targeting Human RPA3) were cloned into the original mir30 cassette the ultramir cassette. Each of the 4 constructs were infected in duplicate at single copy into cells and the cells seltected unitil infection percentages reached >90% (the shRenilla hairpin was infected into HEK293T cells and the shRPA3 construts into the Gallus gallus cell line ERC. After selection smallRNA cloning was perfromed and the amount of smallRNAs corrresponding to the two shRNAs compared to the endogenous microRNA populatlon. Results: smallRNA levels of the two shRNAs doubled relative to the microRNA population when they were placed into the ultramir scaffold.
Project description:Short-hairpin RNA (shRNA)-induced RNAi is used for biological discovery and therapeutics. Dicer, whose normal role is to liberate endogenous miRNAs from their precursors, processes shRNAs into different biologically active siRNAs, affecting their efficacy and potential for off-targeting. We found that in cells, Dicer induced imprecise cleavage events around the expected sites based on the previously described 5'/3'-counting rules. These promiscuous non-canonical cleavages were abrogated when the cleavage site was positioned 2 nt from a bulge or loop. Interestingly, we observed that the ~1/3 of mammalian endogenous pre-miRNAs that contained such structures were more precisely processed by Dicer. Implementing a new "loop-counting rule", we designed potent anti-HCV shRNAs with substantially reduced off-target effects. Our results suggest that Dicer recognizes the loop/bulge structure in addition to the ends of shRNAs/pre-miRNAs for accurate processing. This has important implications for both miRNA processing and future design of shRNAs for RNAi-based genetic screens and therapies. Various shRNAs were expressed in Cell and processed by the RNase III enzyme Dicer. The profiles of the siRNA products were generated by deep sequencing with or without the Ago2-IP.
Project description:RNA interference (RNAi) is becoming routine in scientific discovery and treating human disease. However, its applications are hampered by unwanted effects. The RNA-Seq analyses from mouse liver samples expressing various anti-HCV shRNAs show that weak base-pairing in both seed and 3’ regions is required to achieve minimal off-targeting effect. We created nine shRNAs against the HCV genome, and transfected shRNA-expressing plasmids into mouse liver via a hydrodynamic tail vein infusion. A plasmid backbone lacking the shRNA sequence was used as a negative control. After 7 days, mouse liver RNAs were harvested and subject to RNA-Seq analysis (two biological repeats).
Project description:We analyzed miRNA-based shRNA off-target effects by transducing Trp53-/- MEFs at single- and high-copy with six well-characterized, potent and weak Trp53 shRNAs. To advance RNAi therapy for KRAS-mutant cancer, we developed a functionally validated library of siRNAs against RAS pathway genes that minimize off-target effects and enable combination gene silencing at low dose. We developed an in vivo model for real-time tracking of nanoparticle-based siRNA delivery and offer proof-of-principle that siRNA-mediated inhibition of a single gene (KRAS) or combinations of genes (A/B/C-RAF or KRAS+PIK3C-A/B) can impair the growth of KRAS-mutant colorectal cancer xenografts. Trp53-/- MEFs were transduced with LMP expressing Trp53 shRNAs at single copy (11-21% infection efficiency) and high copy (>98% infection efficiency), selected on puromycin and grown in absence of the selection agent before harvest. Uninfected Trp53-/- MEFs and Trp53-/- MEFs infected with an empty vector control served as “no shRNA” reference.
Project description:shRNAs were assessed for off-target effects by comparing the gene expression profiles of cells that they had been infected into. shRNAs designed with the shERWOOD algorithm and house in the ultramir microRNA scafold were found to have very little off targeting. Purpose: A major detriment to RNAi is off-targeting. We wished to assess the level of off targeting of microRNA (ultramiR) housed shERWOOD shRNAs as compared to similar shRNAs in the TRC collection. Methods: 5 shRNAs targeting each of two genes were infected into the 4T1 cell line. For each gene one shRNA was selected from the TRC collection and one based on the shERWOOD algorithm. For each gene, the exrpession profiles of the corresponding shRNA infected cells were compared using RNAseq. Conclusions: Highly similar profiles were observed between shERWOOD selected shRNAs. TRC shRNAs produced profiles indicative of off-targeting.
Project description:Transcriptional profiling of human BJ fibroblasts comparing control FF shRNA expressing cells vs. BRD7 shRNA expressing cells under two conditions, either untreated or treated with 8uM nutln-3a for 8 hours. This experiment was done using two independent shRNAs targeting BRD7. Nutlin-3a was used to stabilize p53 and induce its transcriptional activity. Two-condition experiment, FF shRNA cells vs. BRD7 shRNAs cells in two experimental conditions, either untreated or treated with nutlin-3a.
Project description:The transcription factor network in Hodgkin lymphoma (HL) represents a unique composition of proteins found in no other hematopoietic cell. Among these factors, an aberrant expression of the T cell transcription factor GATA-3 is observed in the B cell-derived Hodgkin and Reed/Sternberg (HRS) tumor cells. Herein, we elucidated the regulation and function of this factor in HL To obtain a more comprehensive overview of GATA-3 regulated genes in cHL cell lines, we performed a gene chip analysis comparing gene expression in GATA-3-specific shRNA-transduced L-1236 and U-HO1 cells compared to samples transduced with vectors encoding scrambled shRNAs. 12 Total samples were analyzed. Three independent samples were analyzed per condition and cell line.We generated the following pairwise comparisons using Bioconductor: shRNA Gata-3 UHO-1 < control shRNA UHO-1; shRNA Gata-3 L1236 < control shRNA L-1236. Genes with an FDR≤ 25% and a fold-change ≥ 1.5 and ≤ -1.5 were selected.
Project description:The aim of this study was to identify EZH2 targets in myeloid malignancies. RNA samples from control F-36P, MOLM-13 and OCI-M2 cells (secondary AML after MDS; EZH2 wild type) treated with random shRNA (n = 4, each) and test F-36P, MOLM-13 and OCI-M2 cells treated with either one of two EZH2-targeting shRNAs (n = 4, each) were screened for differential gene expression. RNA from EZH2 wild type (n = 12) and mutant (n = 12) MDS/MPN patients was also analyzed by microarray transcriptome analysis.