Optical Control of Translation and Gene Editing in Zebrafish Embryos

University Of Pittsburgh At Pittsburgh, Pittsburgh PA

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Abstract

? DESCRIPTION (provided by applicant): Gene expression is precisely regulated and orchestrated in a spatio-temporal fashion and the activity of multiple genes is often interlinked, creating highly complex genetic networks. The aberrant regulation of gene function can induce disease phenotypes and has been connected with congenital disorders and cancer development. In order to investigate genes, gene networks, and downstream biological functions, the ability to regulate gene expression with high spatio-temporal resolution is desirable. Light represents an ideal regulatory element as it can be easily controlled in a spatial and a temporal fashion, conveying spatio-temporal control of biological activity to the system under study. In the context of dissecting vertebrate gene function, the zebrafish has been established as a powerful model organism. Several methods have been developed for examining gene function in zebrafish embryos, the most common ones being gene overexpression through mRNA injection and gene silencing through morpholino injection. Optically controlled antisense function in zebrafish embryos has been reported; however, no generally applicable, tightly controlled methodology exists for optical gene activation or gene editing. This project addresses this methodology-gap by developing a light-activated RNA approach that directly interfaces with established zebrafish techniques by site-specifically introducing caging groups into RNA molecules. Specifically, two aims will be completed: (1) Develop an mRNA light-activation methodology through the site- specific introduction of caging groups, in order to achieve rapid and precisely regulated gene activation in zebrafish embryos. (2) Develop an optically controlled CRISPR/Cas9 genome editing methodology through the site-specific introduction of caging groups into guide RNA. Cas9-mediated gene editing in zebrafish embryos is rapidly emerging as a versatile research tool and optical control will provide unprecedented spatial and temporal resolution over genome editing. The expected outcomes of the described research are general methods for the spatio-temporal control of gene function through optical activation of gene expression and gene editing in zebrafish embryos. The proposed methodologies directly interface with well-established RNA transcription and injection protocols that are standard in the zebrafish community. The development of tight, non-leaky, and fully predictable optical control of mRNA and sgRNA function in zebrafish embryos will enable a plethora of biological investigations that will have a long-term impact on the field and will be the enabling methodologies for a wide range of future studies.