The in vitro functions of thousands of cis-elements have been characterized using massively parallel reporter assays. However, the in vivo characterization of cis-element functions has been limited to a handful of mammalian enhancers using mouse models. Recently, genome-wide CRISPR/Cas9 knockout screening has proven to be an efficient approach for assessing the function of genomic DNAs and presenting an opportunity to study in vivo cis-element functions on a large scale. We have developed a statistical model to predict gRNA efficiency for the optimization of CRISPR/Cas9 screens, as well as a computational pipeline for the analysis of the screen data. In addition we have performed pilot genome- wide CRISPR/Cas9 knockout screens in several cell lines and have identified known genes essential cell growth. We have demonstrated expertise in studying gene transcriptional regulation through genome-wide analysis of protein-DNA interactions and chromatin accessibility. In this proposal, we hypothesize that the simultaneous lentiviral delivery of two guide RNAs (gRNA) flanking a cis-regulatory element can efficiently knockout (KO) the element, and that CRISPR/Cas9 KO screens are an efficient approach for the large-scale in vivo functional characterization of human cis-elements. We propose to develop the experimental and computational approaches for high-throughput cistrome CRISPR/Cas9 deletion screens to elucidate the regulatory mechanisms of mammalian cis-element in vivo functions and expand our knowledge on transcriptional regulation in normal physiology and diseases. Specifically we propose to 1) use CRISPR/Cas9 knockout screens to identify transcription factors and chromatin regulators in eight human cell lines that have strong effect on cell growth; 2) conduct CRISPR/Cas9 knockout screens on putative cis-regulatory elements to identify elements with strong effects on gene expression and cell growth or survival; 3) computationally model in vivo cistrome function, experimentally validate the model and create a Cistrome annotation web server. At the conclusion of these studies, we will have developed the experimental techniques and computational tools for continued investigation of in vivo cis-element functions, and expanded our knowledge on the mechanism of cell-specific gene transcriptional regulation. The resulting resource will improve interpretation of the function of disease associated somatic mutations or germline variants in the non-coding regions.

Public Health Relevance

In the past decade a very large number of potential gene regulatory regions have been identified in the human genome, however, the function of the vast majority of these regions remains unknown. This study will utilize a novel genome editing approach to efficiently test the function of a large number of these regions in a variety of human cell types. Having a more complete map of the functional gene regulatory regions will greatly increase our understanding of the impact of common genetic variations in the population that have been found to be associated with human health and disease.

National Institute of Health (NIH)
National Human Genome Research Institute (NHGRI)
Research Project (R01)
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Special Emphasis Panel (ZRG1)
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Fletcher, Colin F
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Dana-Farber Cancer Institute
United States
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