Deciphering the genetics of retinal ganglion cell development using CRISPT/Cas9 genome editing in zebrafish.

Retinal ganglion cells are specialized nerve cells in the eye. These specialized nerve cells develop from progenitor cells following an intricate pattern of gene expression. In this project, I intended to elucidate the roles of several genes in the development of retinal ganglion cells in zebrafish. Specifically we have chosen genes from our single cell expression data that were found in developing ganglion cells and encode proteins which modify histones. These modifications are known to affect gene transcription, but it is unclear how these proteins and their histone modifications influence gene expression in retinal ganglion cells. To address these questions, we first set out to determine the retinal expression of different SET domain containing proteins using in-situ hybridization. I generated a large number of different probes, but to date we have only observed widespread expression of the genes. Second, I utilized the CRISPR/Cas9 system to generate mutations in different Set domain proteins with the goal of assessing the retinal phenotype of the mutants. I designed and generated numerous gRNAs, which were injected into fish. However, our mutation rates were mostly low in F0 fish, not allowing for any phenotypic analysis. These fish will need to be raised and crossed for generations to produce homozygote fish before the role of these proteins in retinal ganglion cell development can be ascertained.