Viral vector-mediated gene activation to facilitate large-scale genetic analysis in Caenorhabditis elegans.

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Summary Page: Administrative Supplement Request to Promote Diversity in Health-Related Research (R21GM148859) Request: We are seeking an administrative supplement to promote diversity in health-related research for grant number R21GM148859. This funding would support Ms. Sydney Purcell, a 1st year Biotechnology and Molecular Biosciences major at the Rochester Institute of Technology (RIT}. Qualifications: Sydney qualifies for this supplement as a student from a group that is underrepresented in health-related research. Sydney identifies as a member of Caribbean American community. Overview of Sydney’s goals: This is a collaborative R21 grant and Sydney is working in Dr. Ferran’s laboratory at RIT. This research group is responsible for the research proposed in the first aim of the grant; therefore Sydney’s efforts will focus on the experiments underlined in the grant abstract below. As Sydney gains expertise she will help the next generation of students working on this project in the lab, therefore her trainees will also contribute to this work. Grant Abstract: Fulfilling the promise of modern systems biology and grasping the underlying complexity of biological systems requires a foundation built upon the development of high-throughput functional genomic approaches capable of generating large datasets. Large scale sequencing efforts reveal correlations, but lacks causal interactions best provided via genetic approaches. Caenorhabditis (C.) elegans has been a workhorse for gene discovery and pathway analysis, and is the only established system where high-throughput genetic analysis can be conducted in the context of a living multi-cellular organism (i.e. feeding based RNAi). Despite the power of this model system, no high-throughput methods to achieve targeted gene overexpression in C. elegans have been developed. This project will explore how recombinant strains of two different viruses can be adapted as vectors to enable large-scale genetic analysis of gene overexpression in C. elegans. The objective of Specific Aim 1 is to achieve promoter- specific gene activation using CRISPRa. This variant form of CRISPR relies on a cleavage defective isoform of Cas9 (dCas9) fused with a transcriptional activator to drive overexpression of a gene targeted by the single gene RNA (sgRNA). Specifically, we propose to generate proof-of-principle evidence that recombinant vesicular stomatitis virus (rVSV) can deliver a sgRNA into transgenic C. elegans that express the CRISPRa machinery in intestinal cells to induce sgRNA-directed overexpression of a reporter gene. Ultimately our goal is to develop a comprehensive sgRNA VSV library directed to promoter regions to allow high-throughput functional genomic screening in C. elegans. The objective of Specific Aim 2 is to develop Orsay virus (OV) as a vector to deliver functional mRNA exogenously into C. elegans. The use of OV as a gene delivery system is straightforward as this virus readily enters the animal via the intestinal lumen, and C. elegans expressing integrated segments of the OV genome have been validated. Briefly, we will use these existing strains as “packaging lines” to express C. elegans genes of interest capable of being incorporated in newly generated virion to infect recipient nematodes. These studies represent an initial step towards the use of OV as an overexpression vector and would accelerate the development large- scale genetic analysis in this multicellular organism. These viral-based expression tools would integrate easily with existing approaches widely used by the C. elegans community, which could potentially transform multiple areas of scientific investigation, and has implications for understanding of many diseases.