CRISPR/Cas9-mediated knockout and endogenous complex analysis of Cluap1/ IFT38

CRISPR/Cas9-mediated gene-editing allows manipulation of a gene of interest in its own chromosomal context. When applied to the analysis of protein interaction, and in contrast to an exogenous expression of a protein, these can be studied maintaining physiological stochiometries, topology and context. We have used CRISPR/Cas9-mediated recombination to Cluap1/ IFT38, a component of the intraflagellar transport complex B (IFT-B). Cluap1 has been implicated in human development as well as in cancer progression. Cluap1 loss of function results in early developmental defects with neural tube closure, sonic hedgehog signaling and left-right defects. Herein, we generated an endogenously tagged Cluap1 for protein complex analysis which was then correlated to the corresponding interactome determined by ectopic expression.