The goal of this proposal is to further a unique genetic interaction technology to facilitate the unveiling of the functions of the poorly characterized members of the human kinome. Our end goal is to generate game-changing reagents and protocols, and illuminate the """"""""Dark Matter"""""""" of the genome. Our strategy is based on ultra-high throughput gene interaction, a technology that we have developed for mammalian cells. Genetic interaction is a gold standard for shedding functional insight into gene function and is complimentary to protein:protein interaction and other standard protocols. Our proposed technologies represent a novel advance on cutting-edge approaches that use CRISPR and RNA interference. We stand ready to engage the project as we have already established a sound technical foundation having recently published the methodology. We describe pooled approaches as a readily scalable technology. This proposal describes our efforts to screen immense numbers of interactions surrounding the human kinome. This will give us insight into functions and pathways for the poorly characterized genes. A major goal is to create customized 'doubles libraries'where each of the 518 human kinase genes are co-ablated against every gene in the genome. This library constitutes millions of gene interaction pairs and is screened at high coverage in bioreactors. Our initial plan is to probe signaling pathways that lead to fundamental phenotypes that relate to loss of proliferation and cell death. However we reserve the option to perform additional screens for specific signaling pathways, using integrated fluorescent:antibiotic reporter genes. These resources are scalable and applicable to nearly any human gene and nearly every cell type. Future screens may scale to explore higher-order interactions, and extend beyond the kinome (i.e., GPCRs, nuclear receptors, and ion channels).

Public Health Relevance

The goal of this proposal is to further a unique cell-based technology to unveil gene functions for the poorly characterized druggable genome. Our end goal is to generate game-changing reagents and protocols, illuminating the Dark Matter of the genome.

National Institute of Health (NIH)
National Institute of Mental Health (NIMH)
Research Project--Cooperative Agreements (U01)
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Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Yao, Yong
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University of California San Francisco
Internal Medicine/Medicine
Schools of Medicine
San Francisco
United States
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Boettcher, Michael; Tian, Ruilin; Blau, James A et al. (2018) Dual gene activation and knockout screen reveals directional dependencies in genetic networks. Nat Biotechnol 36:170-178
Oprea, Tudor I; Bologa, Cristian G; Brunak, Søren et al. (2018) Unexplored therapeutic opportunities in the human genome. Nat Rev Drug Discov 17:317-332
Pappalardo, Zachary; Gambhir Chopra, Deeksha; Hennings, Thomas G et al. (2017) A Whole-Genome RNA Interference Screen Reveals a Role for Spry2 in Insulin Transcription and the Unfolded Protein Response. Diabetes 66:1703-1712
Boettcher, Michael; McManus, Michael T (2015) Choosing the Right Tool for the Job: RNAi, TALEN, or CRISPR. Mol Cell 58:575-85