Utilizing CRISPR to visualize the dopamine receptors DOP-1 and DOP-4 in Caenorhabditis elegans

RM 216

Dopamine (DA) is a neurotransmitter with many important functions including movement, reward, and cognition. Studying dopamine signaling at multiple levels allows us to understand the underlying pathologies of dopamine-related disorders. We study dopamine in the nematode Caenorhabditis elegans because of its relatively simple and well-characterized nervous system. Prior studies have shown that DA is involved in regulating chemosensory behaviors in C. elegans. The purpose of this research project is to definitively answer the following question: are the dopamine receptors DOP-1 and DOP-4 expressed in chemosensory neurons? Previous research suggested that neither of these receptors are located in neurons in the chemosensory response circuit (Sugiura et al., 2005), although behavioral assays involving knockouts of the genes encoding these receptors showed behavioral deficits (Ezcurra et al., 2011). Classic transgenic techniques, such as those used originally to visualize the locations of dopamine receptors, involved injecting plasmid DNA containing promoter-reporter gene fusions into worm gonads to be expressed in offspring (Mello et al., 1991). However, these reporter genes may exhibit different expression patterns than endogenous genes. We will attempt to circumvent this problem with a gene knock-in (or gene replacement) approach. By using CRISPR/Cas9 technology to target the dop-1 and dop-4 genes encoding their respective dopamine receptors, I will replace the coding sequence of these genes with that of a reporter gene to visualize where exactly these genes are expressed in their native chromosomal context, with specific attention to neurons involved in chemosensory behavior.