Impact of RNA-Guided Technologies for Target Identification and Deconvolution

Abstract

For well over a decade, RNA interference (RNAi) has provided a powerful tool for investigators to query specific gene targets in an easily modulated loss-of-function setting, both in vitro and in vivo. Hundreds of publications have demonstrated the utility of RNAi in arrayed and pooled-based formats, in a wide variety of cell-based systems, including clonal, stem, transformed, and primary cells. Over the years, there have been significant improvements in the design of target-specific small-interfering RNA (siRNA) and short-hairpin RNA (shRNA), expression vectors, methods for mitigating off-target effects, and accurately interpreting screening results. Recent developments in RNAi technology include the Sensor assay, high-efficiency miR-E shRNAs, improved shRNA virus production with Pasha (DRGC8) knockdown, and assessment of RNAi off-target effects by using the C9-11 method. An exciting addition to the arsenal of RNA-mediated gene modulation is the clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas) system for genomic editing, allowing for gene functional knockout rather than knockdown.

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

“…lentivirus) carrying expression vectors (normally plasmid) that encode shRNAs to express gene-specific siRNAs within the targeted cells, which can achieve stable and highly effective gene suppression in a variety of mammalian cell types [74–76]. Various aspects of RNAi approaches, technical limitations and improvements have been reviewed recently [77]. …”

“…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]. …”

Modern Lead Generation Strategies

“…As biologists have developed several new powerful target validation alternatives to chemical probes, for example, based on small interfering RNA (siRNA) [19], genetic expression analysis (genome-wide expression analysis, GWAS) [20], or as observed in phenotype changes within inducible pluripotent stem cells (iPS) [21], many of the new and strongly validated biological targets lead generation medicinal chemists work on today are considered "difficult to drug" if not "undrugable." A few decades ago, most lead generation medicinal chemists would create leads for G-protein-coupled receptors and kinases (among the most popular target classes), whereas lead generation chemists of today may have to tackle protein-protein interactions, phosphatases, allosteric modulation, or phenotypic screening outcomes where several or unknown tar gets could contribute to the observed desired effect.…”

Versatile Viral Vector Strategies for Postscreening Target Validation and RNAi ON-Target Control

“…Recent refinement of the clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas) gene-editing technology for eukaryotic cells 22 will likely add a further dimension to the optimization of gene target identification in screening applications. 23 Modifying or adding to the complexity of future screening methods will help increase their effectiveness; however, it is a dangerous notion to suggest that it would justify omission of stringent postscreening validation of their top-hit candidates. On the contrary: With the awareness of past shortcomings, better methods to challenge top-hit gene candidates post screening have gained in importance, to consolidate findings as early as possible within the drug development process and base subsequent developments on solid ground.…”