Abstract
Primary cilia are antenna-like structures developed on the cell surface of mammalian cells during the quiescent G0 phase. Primary cilia in mammalian cells receive extracellular signals for early development and cell tissue homeostasis. Ciliopathies characterized with congenital anomalies such as cerebellar hypoplasia, polycystic kidney and polydactyly are caused by germline mutations of ciliary structure- and function-related genes. Gene knock-out techniques in ciliated cultured cells with the uniformed genetic background are useful to evaluate the pathophysiological roles of ciliopathy-related gene products. Genome editing technology has been applied into the gene knock-out in many types of cultured cell lines. However, the frequency of genome editing varies according to cell species and cycle because of dependency on error-free homology-directed repair (HDR) activity. The human telomerase reverse transcriptase-immortalized retinal pigmented epithelial cell line (hTERT-RPE1) is well known for its suitability in cilia research. However, the efficacy of the HDR-mediated knock-out clone isolation was low. Here, we introduce the clustered regularly interspaced short palindromic repeats-obligate ligation-gated recombination (CRISPR-ObLiGaRe) system, which is a nonhomologous end-joining (NHEJ)-mediated gene targeting method, to generate the knock-out clones effectively even in the lower-HDR activity cell lines including hTERT-RPE1 cell. This CRISPR-ObLiGaRe system is a powerful tool for establishing ciliopathy model cell libraries and identifying each gene function in cilia-related phenotypes.