The recently described clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology has proven to be an exquisitely powerful and invaluable method of genetic manipulation and/or ...modification. As such, many researchers have realized the potential of using the CRISPR/Cas9 system as a novel screening method for the identification of important proteins in biological processes and have designed short guide RNA libraries for an in vitro screening. The seminal papers describing these libraries offer valuable information regarding methods for generating the short guide RNA libraries, creating cell lines containing these libraries, and specific details regarding the screening workflow. However, certain considerations are often overlooked that may be important when planning and performing a screen, including which CRISPR library to use and how to best analyze the resulting screen data. In this review, we offer suggestions to answer some of these questions that are not covered as deeply in the papers describing the available CRISPR libraries for an in vitro screening.
The recently discovered CRISPR/Cas9 technology allows for researchers to perform genome‐wide screens for many biological processes. However, for screens to generate high quality data, many decisions need to be considered during the design and progression of a CRISPR screen. In this review, we discuss important variables in the design, progression, and downstream steps of a CRISPR genome‐wide screen.
The recent discovery of the CRISPR/Cas system and repurposing of this technology to edit a variety of different genomes have revolutionized an array of scientific fields, from genetics and ...translational research, to agriculture and bioproduction. In particular, the prospect of rapid and precise genome editing in laboratory animals by CRISPR/Cas has generated an immense interest in the scientific community. Here we review current in vivo applications of CRISPR/Cas and how this technology can improve our knowledge of gene function and our understanding of biological processes in animal models.
CRISPR/Cas technology is a powerful tool for efficient sequence‐specific genome editing. Depending on the choice of target cell and mode of delivery, CRISPR can be used to generate loss‐of‐function germline and somatic mutations, as well as to insert new sequences in a site‐specific manner. Here we discuss current applications of CRISPR/Cas technology for modeling disease and perturbing gene function in vivo.
We present SplashRNA, a sequential classifier to predict potent microRNA-based short hairpin RNAs (shRNAs). Trained on published and novel data sets, SplashRNA outperforms previous algorithms and ...reliably predicts the most efficient shRNAs for a given gene. Combined with an optimized miR-E backbone, >90% of high-scoring SplashRNA predictions trigger >85% protein knockdown when expressed from a single genomic integration. SplashRNA can significantly improve the accuracy of loss-of-function genetics studies and facilitates the generation of compact shRNA libraries.
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.
It is early to fully reflect on the state of the art in high content screening (HCS), because it is still a relatively new approach in drug discovery. Although the development of the first ...microscopes are a century old and the first confocal microscope is only 20 yr old, the fluorescent probes used within HCS along with the combination of robotic automation and integrated software technologies are quite new. HCS will require a few more years to fully demonstrate its potential power in drug discovery. Within the last year, however, one has seen this ever-expanding field lure participants in from all areas of science, introducing newer versions of instruments and reagents such that the combined efforts result in platforms and tools that meet many organizational goals in multiple ways. The potential of HCS today lies in its versatility. HCS can be used for primary screening, basic research, target identification, biomarkers, cytotoxicity, and helping to predict clinical outcomes. HCS is being applied to stem cells, patient cells, primary hepatocytes, and immortalized cultured cells. We have noted for individual specialized assays, there are multiple solutions just as there are for those standardized universally accepted assays. Whether we have needed to query cellular processes under live conditions or wanted to follow kinetically the course of a compound's effects on particular cellular reactions, we have been hampered by only a few limitations. This chapter offers a glimpse inside the use of HCS in our drug discovery environment.
The recently described clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology has proven to be an exquisitely powerful and invaluable method of genetic manipulation and/or ...modification. As such, many researchers have realized the potential of using the CRISPR/Cas9 system as a novel screening method for the identification of important proteins in biological processes and have designed short guide RNA libraries for an in vitro screening. The seminal papers describing these libraries offer valuable information regarding methods for generating the short guide RNA libraries, creating cell lines containing these libraries, and specific details regarding the screening workflow. However, certain considerations are often overlooked that may be important when planning and performing a screen, including which CRISPR library to use and how to best analyze the resulting screen data. In this review, we offer suggestions to answer some of these questions that are not covered as deeply in the papers describing the available CRISPR libraries for an in vitro screening.
Ligand-activated G protein-coupled receptors (GPCRs) are known to regulate a myriad of homeostatic functions. Inappropriate signaling is associated with several pathophysiological states. GPCRs ...belong to a approximately 800 member superfamily of seven transmembrane-spanning receptor proteins that respond to a diversity of ligands. As such, they present themselves as potential points of therapeutic intervention. Furthermore, orphan GPCRs, which are GPCRs without a known cognate ligand, offer new opportunities as drug development targets. This chapter describes a systems-based biological approach, one that combines in silico bioinformatics, genomics, high-throughput screening, and high-content cell-based confocal microscopy strategies to (1) identify a relevant subset of protein family targets, (2) within the therapeutic area of energy metabolism/obesity, (3) and to identify small molecule leads as tractable combinatorial and medicinal chemistry starting points. Our choice of screening platform was the Transfluor beta-arrestin-green fluorescent protein translocation assay in which full-length human orphan GPCRs were stably expressed in a U-2 OS cell background. These cells lend themselves to high-speed confocal imaging techniques using the Evotec Technologies Opera automated microscope system. The basic assay system can be implemented in any laboratory using a fluorescent probe, a stably expressed GPCR of interest, automation-assisted plate and liquid-handling techniques, an optimized image analysis algorithm, and a high-speed confocal microscope with sophisticated data analysis tools.
We present SplashRNA, a sequential classifier to predict potent microRNA-based short hairpin RNAs (shRNAs). Trained on published and novel datasets, SplashRNA outperforms previous algorithms and ...reliably predicts the most efficient shRNAs for a given gene. Combined with an optimized miR-E backbone, >90% of high-scoring SplashRNA predictions trigger >85% protein knockdown when expressed from a single genomic integration. SplashRNA can significantly improve the accuracy of loss-of-function genetics studies and facilitates the generation of compact shRNA libraries.
Previous studies have demonstrated that the amino-terminal cytoplasmic domain of GLUT4 contains a phenylalanine-based targeting motif that determines its steady state distribution between the surface ...and the interior of cells. To directly measure the effect that the GLUT4 amino terminus has on internalization and subsequent recycling back to the cell surface, we constructed chimeras in which this sequence was substituted for the amino-terminal cytoplasmic domain of the human transferrin receptor. The chimeras were stably transfected into Chinese hamster ovary cells and their endocytic behavior characterized. The GLUT4-transferrin receptor chimera was recycled back to the cell surface with a rate similar to the transferrin receptor, indicating that the GLUT4 sequence was not promoting intracellular retention of the chimera. The GLUT4-transferrin receptor chimera was internalized at half the rate of the transferrin receptor. Substitution of an alanine for phenylalanine at position 5 slowed internalization of the chimera by twofold, to a level characteristic of bulk membrane internalization. However, substitution of a tyrosine increased the rate of internalization to the level of the transferrin receptor. Neither of these substitutions significantly altered the rate at which the chimeras were recycled back to the cell surface. These results demonstrate that the major function of the GLUT4 amino-terminal domain is to promote the effective internalization of the protein from the cell surface, via a functional phenylalanine-based internalization motif, rather than retention of the transporter within intracellular structures.
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