CRISPR–Cas9 has been adopted as a powerful genome-editing technology in various species. By generating libraries of thousands of guide RNAs — which direct the Cas9 nuclease to chosen genomic loci — high-throughput genetic perturbations are now possible. This Review discusses the latest applications of CRISPR–Cas9 in mammalian functional genomics screens. It covers related genome-scale applications of Cas9 for either gene knockout or transcriptional modulation, and provides comparisons with complementary RNA interference (RNAi)-based approaches.

High-throughput functional genomics using CRISPR–Cas9

Opportunities and challenges for use of tumor spheroids as models to test drug delivery and efficacy.

We demonstrate for 24 metal oxide (MOx) nanoparticles that it is possible to use conduction band energy levels to delineate their toxicological potential at cellular and whole animal levels. Among the materials, the overlap of conduction band energy (Ec) levels with the cellular redox potential (−4.12 to −4.84 eV) was strongly correlated to the ability of Co3O4, Cr2O3, Ni2O3, Mn2O3, and CoO nanoparticles to induce oxygen radicals, oxidative stress, and inflammation. This outcome is premised on permissible electron transfers from the biological redox couples that maintain the cellular redox equilibrium to the conduction band of the semiconductor particles. Both single-parameter cytotoxic as well as multi-parameter oxidative stress assays in cells showed excellent correlation to the generation of acute neutrophilic inflammation and cytokine responses in the lungs of C57 BL/6 mice. Co3O4, Ni2O3, Mn2O3, and CoO nanoparticles could also oxidize cytochrome c as a representative redox couple involved in redox ho...

Use of Metal Oxide Nanoparticle Band Gap To Develop a Predictive Paradigm for Oxidative Stress and Acute Pulmonary Inflammation

Brass AL, Dykxhoorn DM, Benita Y, et al. Science . 2008;319(5865):921–926 PURPOSE OF THE STUDY. The HIV genome encodes only 15 proteins and, therefore, must use multiple host-cell collaborators for successful replication and transmission. Required host-derived proteins include CD4 as the primary virus receptor and chemokine receptors as coreceptors. This study identified multiple other host proteins required for HIV activity. METHODS. Human cells known to be susceptible to HIV were exposed in vitro to HIV. Using small interfering RNAs able to inhibit each known gene in the human genome 1 at a time, the investigators tested whether HIV could establish an infection and copy itself. HIV dependence on >21 000 human genes was examined. RESULTS. More than 250 human genes were identified to be required for efficient HIV replication. Termed “HIV-dependency factors,” the products of these genes are known to participate in a broad array of cellular functions and implicate unsuspected pathways in the virus life cycle. CONCLUSIONS. The extraordinary dependence of HIV on human host proteins for efficient transmission and replication provides many new potential targets for antiretroviral therapy. REVIEWER COMMENTS. An example of targeting host proteins is the use of chemokine receptor 5 (CCR5) inhibitors. Many people with CCR5 deficiency are very resistant to HIV infection yet have limited if any clinical consequences. Maraviroc CCR5 inhibitor is approved for treatment for HIV infection. This study identified many more such potential targets.

Identification of Host Proteins Required for HIV Infection Through a Functional Genomic Screen

MicroRNAs (miRNAs) repress cellular protein levels to provide a sophisticated parameter of gene regulation that coordinates a broad spectrum of biological processes. Bone organogenesis is a complex process involving the differentiation and crosstalk of multiple cell types for formation and remodeling of the skeleton. Inhibition of mRNA translation by miRNAs has emerged as an important regulator of developmental osteogenic signaling pathways, osteoblast growth and differentiation, osteoclast-mediated bone resorption activity and bone homeostasis in the adult skeleton. miRNAs control multiple layers of gene regulation for bone development and postnatal functions, from the initial response of stem/progenitor cells to the structural and metabolic activity of the mature tissue. This Review brings into focus an emerging concept of bone-regulating miRNAs, the evidence for which has been gathered largely from in vivo mouse models and in vitro studies in human and mouse skeletal cell populations. Characterization of miRNAs that operate through tissue-specific transcription factors in osteoblast and osteoclast lineage cells, as well as intricate feedforward and reverse loops, has provided novel insights into the supervision of signaling pathways and regulatory networks controlling normal bone formation and turnover. The current knowledge of miRNAs characteristic of human pathologic disorders of the skeleton is presented with a future goal towards translational studies.

/pdf/microrna-control-of-bone-formation-and-homeostasis-huwg3mj6bo.pdf

MicroRNA control of bone formation and homeostasis

RNA interference (RNAi) has become a powerful technique for reverse genetics and drug discovery, and in both of these areas large-scale high-throughput RNAi screens are commonly performed. The statistical techniques used to analyze these screens are frequently borrowed directly from small-molecule screening; however, small-molecule and RNAi data characteristics differ in meaningful ways. We examine the similarities and differences between RNAi and small-molecule screens, highlighting particular characteristics of RNAi screen data that must be addressed during analysis. Additionally, we provide guidance on selection of analysis techniques in the context of a sample workflow.

/pdf/statistical-methods-for-analysis-of-high-throughput-rna-271vo8s70u.pdf

Statistical methods for analysis of high-throughput RNA interference screens

Background 
Mesenchymal stem (MS) cells are excellent candidates for cell-based therapeutic strategies to regenerate injured tissue. Although human MS cells can be isolated from bone marrow and directed to differentiate by means of an osteogenic pathway, the regulation of cell-fate determination is not well understood. Recent reports identify critical roles for microRNAs (miRNAs), regulators of gene expression either by inhibiting the translation or by stimulating the degradation of target mRNAs.
Methodology/Principal Findings 
In this study, we employed a library of miRNA inhibitors to evaluate the role of miRNAs in early osteogenic differentiation of human MS cells. We discovered that miR-148b, -27a and -489 are essential for the regulation of osteogenesis: miR-27a and miR-489 down-regulate while miR-148b up-regulates differentiation. Modulation of these miRNAs induced osteogenesis in the absence of other external differentiation cues and restored osteogenic potential in high passage number human MS cells.
Conclusions/Significance 
Overall, we have demonstrated the utility of the functional profiling strategy for unraveling complex miRNA pathways. Our findings indicate that miRNAs regulate early osteogenic differentiation in human MS cells: miR-148b, -27a, and -489 were found to play a critical role in osteogenesis.

/pdf/functional-profiling-reveals-critical-role-for-mirna-in-s9ss3z193r.pdf

Functional profiling reveals critical role for miRNA in differentiation of human mesenchymal stem cells.

Influenza virus encodes only 11 viral proteins but replicates in a broad range of avian and mammalian species by exploiting host cell functions. Genome-wide RNA interference (RNAi) has proven to be a powerful tool for identifying the host molecules that participate in each step of virus replication. Meta-analysis of findings from genome-wide RNAi screens has shown influenza virus to be dependent on functional nodes in host cell pathways, requiring a wide variety of molecules and cellular proteins for replication. Because rapid evolution of the influenza A viruses persistently complicates the effectiveness of vaccines and therapeutics, a further understanding of the complex host cell pathways coopted by influenza virus for replication may provide new targets and strategies for antiviral therapy. RNAi genome screening technologies together with bioinformatics can provide the ability to rapidly identify specific host factors involved in resistance and susceptibility to influenza virus, allowing for novel disease intervention strategies.

Queta Smith

Papers

Statistical methods for analysis of high-throughput RNA interference screens

Functional profiling reveals critical role for miRNA in differentiation of human mesenchymal stem cells.

Host gene targets for novel influenza therapies elucidated by high-throughput RNA interference screens