Claudins are cell adhesion molecules that form a crucial component of the epithelial cells and Blood-brain barrier in vertebrates, and are structurally conserved across phyla. In their canonical role, claudins form the integral components of tight junctions where they function to regulate barrier properties. However, their interaction with other non-tight junction proteins, such as actin cytoskeleton, EpCAM suggests the possibility of non-canonical roles for claudins. Recently claudins have also been discovered in glia and neurons. Further studies have indicated a link between the loss of claudins and several neurological disorders. However, their function in vertebrate neurons is still a mystery. We aim to understand Claudin function in rat primary cortical neuronal cultures. Initially, we conducted a screen using Reverse transcription PCR to analyze the expression of the 24 vertebrate claudins in primary neurons, and found that multiple Claudins appear to be expressed in the neuronal cultures. Further, we used a CRISPR-based gene knockout strategy followed by antibody staining of neuronal cytoskeletal markers to understand the effect of loss of claudins on neuronal structures. Compared to control cells, a decrease in neurite growth was observed in Claudin 11 and 20 individual knockout conditions. However, when a combined knockout of Claudin 11 and 20 was performed, it showed a decrease of 50% in neurite growth when compared to control primary neuronal cells, suggesting an additive effect of these claudins. Currently, we are rescuing the post-knockout phenotype and understanding the involvement of these proteins in neuronal structure and development via cytoskeletal regulation.