Generation of human Nup358 knock out cell line using CRISPR-CAS9 System
Nup358 is a member of the nucleoporin family, which constitutes the nuclear pore complex (NPC). It is present at the cytoplasmic side of the NPC. NPCs form the molecular gates through which macromolecules are transported between the nucleus and the cytoplasm. Studies involving the nucleoporin Nup358 suggest that it is involved in cell polarity, Wnt signalling, miRNA pathway, and SUMOylation; however, the mechanistic details are still to be understood. One of the methods to explore its functions in the cell and to gain mechanistic insight is by silencing or knocking-out Nup358. CRISPR-CAS9 system is a gene editing tool which can be used to knock-out Nup358 by co-expressing an endonuclease like CAS9 and Nup358 gene-specific single stranded guide RNA (sgRNA). A plasmid encoding Nup358-targeting sgRNA and CAS9 was commercially procured. Successful knock-out mammalian cell lines (HEK293T and HeLa S3) will be achieved for studying the morphological and physiological changes in the absence of Nup358 in cells. As the CRISPR/CAS9 plasmid contains a cassette for expressing red fluorescent protein (RFP), initially, transfected cells will be selected by fluorescence-assisted cell sorting (FACS) and later, single cell clones will be generated. The clones will be screened for knockout of Nup358 by immunostaining, western blotting and sequence confirmation of the PCR product obtained using primers designed to regions flanking the target site.