Significant progress has been made toward a better understanding of the axon guidance mechanisms during animal development. Several guidance pathways have been revealed where signaling initiated at the surface receptor level is relayed to the interior of neurons through numerous adaptor proteins. This leads to a reorganization of the cytoskeleton and other responses. We previously showed that the Drosophila protein Dunc-115, a homolog of the Unc-115 from the C. elegans, is involved in axon projection in the CNS and the visual system. Furthermore, our data also demonstrated that Dunc-115 binds to actin filaments, implying a possible mechanism for actin reorganization via Dunc-115. Dunc-115 is alternatively spliced resulting in the formation of three isoforms, and it has not previously been possible to dissect the functions of individual isoforms. Here, we report that by using the recently available CRISPR, we have generated mutant animals where the actin binding domain (VHD) of the longest isoform is deleted. The deletion of the VHD domain has severe phenotypic defects. The results suggest that the actin binding domain is essential and that distinct isoforms function differently. The outcome of the structural and functional analysis will be discussed.