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.

Public Health Relevance

Gene expression is precisely regulated and orchestrated in a spatio-temporal fashion and the aberrant regulation of gene function can lead to congenital disorders and cancer development. Light represents an ideal regulatory element to activate or deactivate - and thereby study - gene function as it can be easily controlled in a spatial and a temporal fashion. Within this project, new optical gene expression and editing methodologies for the zebrafish model system are being developed.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21HD085206-01A1
Application #
9112162
Study Section
Development - 2 Study Section (DEV2)
Program Officer
Coulombe, James N
Project Start
2016-09-23
Project End
2018-08-31
Budget Start
2016-09-23
Budget End
2017-08-31
Support Year
1
Fiscal Year
2016
Total Cost
$228,120
Indirect Cost
$78,120
Name
University of Pittsburgh
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Courtney, Taylor; Deiters, Alexander (2018) Recent advances in the optical control of protein function through genetic code expansion. Curr Opin Chem Biol 46:99-107
Ankenbruck, Nicholas; Courtney, Taylor; Naro, Yuta et al. (2018) Optochemical Control of Biological Processes in Cells and Animals. Angew Chem Int Ed Engl 57:2768-2798