TNRF has initiated a systematic evaluation and comparison of RNAi and CRISPR/Cas9-based gene-editing technologies. The objectives of this evaluation are to understand the utility and pitfalls of these newer technologies and to establish the experimental, automation and computational workflows required for focused, arrayed, small-scale CRISPR-based screens of approximately 400 to 600 genes. This work was initiated in June 2017.
TNRF’s evaluation plan has three components:
- Reagent selection,
- Robust screening workflow development, and
- Assessment of the relative strengths of each functional genomic approach to ensure selection of the most appropriate technologies needed to address the specific question under investigation.
See TNRF in action below.
TNRF Receives Deputy Director of Intramural Research (DDIR) Innovation Award (PDF - 604KB)
TNRF received a collaborative DDIR Innovation Award two years in a row, both in May 2017 and May 2018, to develop proof-of-concept pilot CRISPR screens (arrayed and population based) that bring in new investigator-driven expertise to develop complementary CRISPR-based approaches. These pilot screens are a mix of hypothesis-generating and hypothesis-driven studies to fully test different aspects of single-guide RNA-CRISPR-based screening platforms.
RNAi High-Throughput Screening of Single- and Multi-Cell-Type Tumor Spheroids: A Comprehensive Analysis in Two and Three Dimensions
The widespread use of two-dimensional (2-D) monolayer cultures for high-throughput screening (HTS) to identify targets in drug discovery has led to attrition in the number of drug targets being validated. Solid tumors are complex, aberrantly growing microenvironments that harness structural components from stroma, nutrients fed through vasculature, and immunosuppressive factors. Increasing evidence of stromally derived signaling broadens the complexity of our understanding of the tumor microenvironment while stressing the importance of developing better models that reflect these interactions. Three-dimensional (3-D) models may be more sensitive to certain gene-silencing events than 2-D models because of their components of hypoxia, nutrient gradients and increased dependence on cell-cell interactions and therefore are more representative of in vivo interactions.
Exploring Drug Dosing Regimens In Vitro Using Real-Time 3D Spheroid Tumor Growth Assays
Two-dimensional monolayer cell proliferation assays for cancer drug discovery have made the implementation of large-scale screens feasible but seem to reflect only a simplified view that oncogenes or tumor suppressor genes are the genetic drivers of cancer cell proliferation. However, there is now increased evidence that the cellular and physiological context in which these oncogenic events occur play a key role in how they drive tumor growth in vivo and, therefore, in how tumors respond to drug treatments. In vitro 3-D spheroid tumor models are being developed to better mimic the physiology of tumors in vivo, in an attempt to improve the predictability and efficiency of drug discovery for the treatment of cancer. Here we describe the establishment of a real-time 3-D spheroid growth, 384-well screening assay. The cells used in this study constitutively expressed green fluorescent protein (GFP), which enabled the real-time monitoring of spheroid formation and the effect of chemotherapeutic agents on spheroid size at different time points of sphere growth and drug treatment. This real-time 3-D spheroid assay platform represents a first step toward the replication in vitro of drug dosing regimens being investigated in vivo. We hope that further development of this assay platform will allow the investigation of drug dosing regimens, efficacy and resistance before preclinical and clinical studies.
Gene-Silencing Data Now Publicly Available to Help Scientists Better Understand Disease
On Dec. 11, 2013, NIH announced that for the first time, large-scale information on the biochemical makeup of small interfering RNA (siRNA) molecules is available publicly. NCATS researchers collaborated with Life Technologies Corporation of Carlsbad, California, which owns the siRNA information, to make it available to all researchers.
Gene-Silencing Study Finds New Targets for Parkinson’s Disease
On Nov. 25, 2013, NIH announced that NCATS and National Institute of Neurological Disorders and Stroke researchers had used RNAi technology to identify dozens of genes that may represent new therapeutic targets for treating Parkinson’s disease. The research was published online in Nature.
Functional Genomic Screening Identifies Dual Leucine Zipper Kinase as a Key Mediator of Retinal Ganglion Cell Death
On March 5, 2013, NCATS scientists published results of an RNAi screening study identifying a protein that could represent a therapeutic target for glaucoma.
Human Genome-Wide RNAi Screen Reveals a Role for Nuclear Pore Proteins in Poxvirus Morphogenesis
An article published on Feb. 26, 2013, revealed that a human genome-wide RNAi screen had identified candidate genes that modulate the activity of vaccinia virus, representing possible therapeutic targets.