From sequence to function: using RNAi to elucidate mechanisms of human disease

Wolters, Natalie; MacKeigan, Jeffrey P.

doi:10.1038/sj.cdd.4402311

Cited by 27 publications

(23 citation statements)

References 82 publications

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“…This natural process has been exploited as a research tool to target mRNA transcripts by introducing into cells homologous synthetic siRNAs, siRNA precursors such as short-hairpin RNAs (shRNAs), or double stranded RNA (dsRNA) [70–72]. Delivery methods include lipid-based transfection, electroporation and viral transduction with retroviruses and lentiviruses [73]. As some mammalian cell types are resistant to transfection with synthetic siRNAs, they may be alternatively transduced with viruses (e.g.…”

Section: Functional Genomics By Rna Interference (Rnai)mentioning

confidence: 99%

“…The phenotype may be viability or a cell marker selected by fluorescent activated cell sorting. In the arrayed format, cells are transfected with individual siRNAs or shRNAs in separate wells, exposed, and assayed in parallel by a variety of methods, including high-content microscopy or viability screening, or reporter assays [73]. In HCS, cells are labeled with multiple fluorescent markers, which can be measured in multiple channels in a highly automated manner [77].…”

Section: Functional Genomics By Rna Interference (Rnai)mentioning

confidence: 99%

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Functional genomic screening approaches in mechanistic toxicology and potential future applications of CRISPR-Cas9

Shen¹,

McHale²,

Smith³

et al. 2015

Mutation Research/Reviews in Mutation Research

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Characterizing variability in the extent and nature of responses to environmental exposures is a critical aspect of human health risk assessment. Chemical toxicants act by many different mechanisms, however, and the genes involved in adverse outcome pathways (AOPs) and AOP networks are not yet characterized. Functional genomic approaches can reveal both toxicity pathways and susceptibility genes, through knockdown or knockout of all non-essential genes in a cell of interest, and identification of genes associated with a toxicity phenotype following toxicant exposure. Screening approaches in yeast and human near-haploid leukemic KBM7 cells, have identified roles for genes and pathways involved in response to many toxicants but are limited by partial homology among yeast and human genes and limited relevance to normal diploid cells. RNA interference (RNAi) suppresses mRNA expression level but is limited by off-target effects (OTEs) and incomplete knockdown. The recently developed gene editing approach called clustered regularly interspaced short palindrome repeats-associated nuclease (CRISPR)-Cas9, can precisely knock-out most regions of the genome at the DNA level with fewer OTEs than RNAi, in multiple human cell types, thus overcoming the limitations of the other approaches. It has been used to identify genes involved in the response to chemical and microbial toxicants in several human cell types and could readily be extended to the systematic screening of large numbers of environmental chemicals. CRISPR-Cas9 can also repress and activate gene expression, including that of non-coding RNA, with near-saturation, thus offering the potential to more fully characterize AOPs and AOP networks. Finally, CRISPR-Cas9 can generate complex animal models in which to conduct preclinical toxicity testing at the level of individual genotypes or haplotypes. Therefore, CRISPR-Cas9 is a powerful and flexible functional genomic screening approach that can be harnessed to provide unprecedented mechanistic insight in the field of modern toxicology.

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Section: Functional Genomics By Rna Interference (Rnai)mentioning

confidence: 99%

Section: Functional Genomics By Rna Interference (Rnai)mentioning

confidence: 99%

Functional genomic screening approaches in mechanistic toxicology and potential future applications of CRISPR-Cas9

Shen¹,

McHale²,

Smith³

et al. 2015

Mutation Research/Reviews in Mutation Research

View full text Add to dashboard Cite

show abstract

“…RNA interference (RNAi) is a well-established powerful tool for identifying novel genes involved in a wide range of biological phenomena (Fire et al, 1998;Wolters and MacKeigan, 2008). In C. elegans, large-scale RNAi screens have been used to define key sets of genes involved in processes such as fat regulation (Ashrafi et al, 2003), longevity (Hamilton et al, 2005) and axon guidance (Schmitz et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

C. elegans bicd-1, homolog of theDrosophiladynein accessory factorBicaudal D, regulates the branching of PVD sensory neuron dendrites

2011

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SUMMARYThe establishment of cell type-specific dendritic arborization patterns is a key phase in the assembly of neuronal circuitry that facilitates the integration and processing of synaptic and sensory input. Although studies in Drosophila and vertebrate systems have identified a variety of factors that regulate dendrite branch formation, the molecular mechanisms that control this process remain poorly defined. Here, we introduce the use of the Caenorhabditis elegans PVD neurons, a pair of putative nociceptors that elaborate complex dendritic arbors, as a tractable model for conducting high-throughput RNAi screens aimed at identifying key regulators of dendritic branch formation. By carrying out two separate RNAi screens, a small-scale candidate-based screen and a large-scale screen of the ~3000 genes on chromosome IV, we retrieved 11 genes that either promote or suppress the formation of PVD-associated dendrites. We present a detailed functional characterization of one of the genes, bicd-1, which encodes a microtubule-associated protein previously shown to modulate the transport of mRNAs and organelles in a variety of organisms. Specifically, we describe a novel role for bicd-1 in regulating dendrite branch formation and show that bicd-1 is likely to be expressed, and primarily required, in PVD neurons to control dendritic branching. We also present evidence that bicd-1 operates in a conserved pathway with dhc-1 and unc-116, components of the dynein minus-end-directed and kinesin-1 plus-enddirected microtubule-based motor complexes, respectively, and interacts genetically with the repulsive guidance receptor unc-5.

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“…Development of libraries of chemically synthesized siRNAs or plasmid-encoded short hairpin RNAs (shRNAs) has enabled the execution of many genome-wide screens in mammalian cells (for reviews, see (refs. 21-25). Despite the transformative potential of this new technology, it is important to recognize that suppression of gene expression by RNAi is not equivalent to inactivation of a gene by mutation.…”

Section: Introductionmentioning

confidence: 99%

Vigilance and Validation: Keys to Success in RNAi Screening

2010

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In the twelve years since the process of RNA interference (RNAi) was first discovered, great progress has been made in understanding its mechanism and exploiting its ability to silence gene expression to study gene function at a genome-wide level. Its extensive use as a screening method has yielded many published lists of genes that play novel roles in higher eukaryotes. However, the usefulness of this information is potentially limited by the occurrence of unintended off-target effects. Here we review the potential causes of off-target effects, and the impact of this phenomenon in interpreting the results of high-throughput RNAi screens. In addition to targeting the intended gene product, artificial short interfering RNAs (siRNAs) can produce off-target effects by down-regulating the expression of multiple messenger RNAs through microRNA-like targeting of the 3′ untranslated region. We examine why this phenomenon can produce high hit rates in siRNA screens, and why independent validation of screening results is critical for the approach to yield new biological insights.

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From sequence to function: using RNAi to elucidate mechanisms of human disease

Cited by 27 publications

References 82 publications

Functional genomic screening approaches in mechanistic toxicology and potential future applications of CRISPR-Cas9

Functional genomic screening approaches in mechanistic toxicology and potential future applications of CRISPR-Cas9

C. elegans bicd-1, homolog of theDrosophiladynein accessory factorBicaudal D, regulates the branching of PVD sensory neuron dendrites

Vigilance and Validation: Keys to Success in RNAi Screening

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