Generation of CRISPR/Cas-mediated mutants of IROC genes and protein tagging for endogenous expression analysis in drosophila

Abstract

In the olfactory system, correct olfactory receptor neuron (ORN) development depends on proper olfactory receptor (OR) gene expression and proper projection of axons to higher brain centers. Thus, during development, ORNs go through two important decisions: selecting one OR gene from a large repertoire of OR genes and project their axons to a particular location in the brain. The availability of genetic tools and its relative simplicity make Drosophila an important model organism to uncover the molecular basis of the olfactory system development and function. Our studies focus on the function of IroC (Iroquois complex), a transcription factor family, in ORN specification. The Iro family includes three genes called araucan (ara), caupolican (caup) and mirror (mirr), which are conserved as clusters in all multi-cellular organisms. Previous analysis of enhancer trap lines showed that IroC is expressed in two olfactory organs, antennae and maxillary palp and using a triple mutant of IroC, RNA Sequencing analysis was performed on olfactory organs and iroC target genes were characterized. In the framework of this study, to investigate IroC genes individually we wanted to generate novel tools, single knock-outs and individually-tagged proteins of iroC using CRISPR/Cas9 technology. To identify and compare target gene repertoires, I have generated single IroC mutants which will be used for RNASeq analysis in future studies. Also, I have generated individual fluorescently-tagged iro proteins to study their endogenous expression patterns. Overall, the generated tools will help to study iroC function in general and help to clarify the role of these proteins in olfactory system development.