Explicit Modeling of siRNA-Dependent On- and Off-Target Repression Improves the Interpretation of Screening Results

Riba, Andrea; Emmenlauer, Mario; Chen, Amy; Sigoillot, Frederic; Cong, Feng; Dehio, Christoph; Jenkins, Jeremy L.; Zavolan, Mihaela

doi:10.1016/j.cels.2017.01.011

Cited by 20 publications

(13 citation statements)

References 92 publications

(154 reference statements)

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“…The resulting observations of hairpin abundance at each time point were used to model the importance of each targeted gene during the process of erythropoiesis. A linear mixed model was implemented to account for the longitudinal nature of the time course data (Li et al, 2015) and to handle the confounding off-target and efficiency effects inherent to the shRNA modality (Riba et al, 2017; Tsherniak et al, 2017). Since we wanted our model to be able to detect significant changes in hairpin abundance at any time point throughout the differentiation process, we converted the absolute hairpin abundances at each of the six time points to a log 2 fold change relative to the initial hairpin abundances at the start of the differentiation.…”

Section: Resultsmentioning

confidence: 99%

Gene-centric functional dissection of human genetic variation uncovers regulators of hematopoiesis

Nandakumar

McFarland

Mateyka

et al. 2019

eLife

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Section: Resultsmentioning

confidence: 99%

Gene-centric functional dissection of human genetic variation uncovers regulators of hematopoiesis

Nandakumar

McFarland

Mateyka

et al. 2019

eLife

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“…Although it is versatile, RNAi has relatively high off-target effects [ 69 ], caused by the RNAi machinery, not requiring full complementarity to the target sequence [ 70 ]. Recent developments aim to computationally predict on- and off-target efficiencies to achieve optimal editing efficiency [ 71 , 72 ].…”

Section: Genetic Screensmentioning

confidence: 99%

System-Based Approaches to Delineate the Antiviral Innate Immune Landscape

Krey

Babnis

Pichlmair

2020

Viruses

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Viruses pose substantial challenges for society, economy, healthcare systems, and research. Their distinctive pathologies are based on specific interactions with cellular factors. In order to develop new antiviral treatments, it is of central importance to understand how viruses interact with their host and how infected cells react to the virus on a molecular level. Invading viruses are commonly sensed by components of the innate immune system, which is composed of a highly effective yet complex network of proteins that, in most cases, mediate efficient virus inhibition. Central to this process is the activity of interferons and other cytokines that coordinate the antiviral response. So far, numerous methods have been used to identify how viruses interact with cellular processes and revealed that the innate immune response is highly complex and involves interferon-stimulated genes and their binding partners as functional factors. Novel approaches and careful experimental design, combined with large-scale, high-throughput methods and cutting-edge analysis pipelines, have to be utilized to delineate the antiviral innate immune landscape at a global level. In this review, we describe different currently used screening approaches, how they contributed to our knowledge on virus–host interactions, and essential considerations that have to be taken into account when planning such experiments.

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“…The resulting longitudinal observations of hairpin abundance at each time point were used to model the importance of each targeted gene during the process of erythropoiesis. A linear mixed model was implemented to account for the longitudinal nature of the time course data (Li et al, 2015) and to handle the confounding off-target and efficiency effects inherent to the shRNA modality (Riba et al, 2017;Tsherniak et al, 2017). Since we wanted our model to be able to detect significant changes in hairpin abundance at any time point throughout the differentiation process, we converted the absolute hairpin abundances at each of the six time points to a log2 fold change relative to the initial hairpin abundances at the start of the differentiation.…”

Section: Screenmentioning

confidence: 99%

Gene-centric functional dissection of human genetic variation uncovers regulators of hematopoiesis

Nandakumar

McFarland

Mateyka

et al. 2018

Preprint

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Genome-wide association studies (GWAS) have identified thousands of variants associated with human diseases and traits. However, the majority of GWAS-implicated variants are in noncoding regions of the genome and require in depth follow-up to identify target genes and decipher biological mechanisms. Here, rather than focusing on causal variants, we have undertaken a pooled loss-of-function screen in primary hematopoietic cells to interrogate 389 candidate genes contained in 75 loci associated with red blood cell traits. Using this approach, we identify 77 genes at 38 GWAS loci, with most loci harboring 1-2 candidate genes.Importantly, the hit set was strongly enriched for genes validated through orthogonal genetic approaches. Genes identified by this approach are enriched in specific and relevant biological pathways, allowing regulators of human erythropoiesis and modifiers of blood diseases to be defined. More generally, this functional screen provides a paradigm for gene-centric follow up of GWAS for a variety of human diseases and traits.We applied a gene-centric loss-of-function screening approach to GWAS of RBC traits. We focused on 75 loci associated with RBC traits that were identified by a GWAS performed in up to 135,000 individuals (van der Harst et al., 2012) spanning 6 RBC traits ( Figure S1A). Importantly, these 75 loci have been robustly replicated and show large effect sizes in more recently reported association studies performed on larger cohorts and thus represent ideal targets for perturbation studies (Astle et al., 2016;Lareau et al., 2018). We endeavored to select all genes that could potentially underlie these 75 GWAS signals. To do this, each of the 75 sentinel SNPs was first expanded to a linkage disequilibrium (LD) block including all SNPs in high LD (r 2 > 0.8, Figure 1A, Figure S1B), then further to the nearest genomic recombination hotspot. Based upon insights from previous expression quantitative trait locus (eQTL) studies (Montgomery and Dermitzakis, 2011; Rossin et al., 2011;Veyrieras et al., 2008), each gene annotated in the genome was expanded to include a wingspan encompassing 110 kb upstream and 40 kb downstream of the transcriptional start and end sites, respectively, to also capture potential functional regulatory elements. This resulted in selection of 389 genes overlapping or in the vicinity of the LD blocks to be tested in the pooled loss-of-function screen. These were distributed at a median of 4 genes per loci ( Figure S1C).Since the majority of common genetic variation underlying RBC traits appears to act in a cellintrinsic manner within the erythroid lineage, we decided to perturb the candidate genes during the process of human erythropoiesis Sankaran et al., 2012;Sankaran et al., 2008;Ulirsch et al., 2016). We chose a pooled short hairpin RNA (shRNA) based loss-of-

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Explicit Modeling of siRNA-Dependent On- and Off-Target Repression Improves the Interpretation of Screening Results

Cited by 20 publications

References 92 publications

Gene-centric functional dissection of human genetic variation uncovers regulators of hematopoiesis

Gene-centric functional dissection of human genetic variation uncovers regulators of hematopoiesis

System-Based Approaches to Delineate the Antiviral Innate Immune Landscape

Gene-centric functional dissection of human genetic variation uncovers regulators of hematopoiesis

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