title: CRISPR/Cas9-Mediated Generation of Niemann-Pick C1 Knockout Cell Line.
abstract: Generating a cholesterol storage phenotype of Niemann-Pick Type C (NPC) disease is important for investigating the mechanisms of intracellular cholesterol trafficking, as well as screening drugs for potential treatment of NPC disease. The use of the CRISPR/Cas9 technology to knockout specific genes within the genome of mammals has become routine in the past few years. Here, we describe a protocol for producing a cellular NPC cholesterol storage phenotype in HeLa cells using the CRISPR-Cas9 system to disrupt the NPC1 gene. The protocol details the steps for single guide RNA oligo cloning, cell colony selection, and cell line verification by filipin staining and immunoblotting.
authors.affiliation: School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW, 2052, Australia.
authors.forename: Ximing
authors.initials: X
authors.lastname: Du
authors.affiliation: School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW, 2052, Australia.
authors.forename: Ivan
authors.initials: I
authors.lastname: Lukmantara
authors.affiliation: School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW, 2052, Australia. h.rob.yang@unsw.edu.au.
authors.forename: Hongyuan
authors.initials: H
authors.lastname: Yang
mesh_terms: D064113:CRISPR-Cas Systems; D002352:Carrier Proteins / Q000235:genetics*; D055785:Gene Knockdown Techniques / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins / Q000235:genetics*; D008954:Models, Biological; D000077296:Niemann-Pick C1 Protein; D052556:Niemann-Pick Disease, Type C / Q000235:genetics / Q000378:metabolism / Q000473:pathology
chemical_list: D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; C106881:NPC1 protein, human; D000077296:Niemann-Pick C1 Protein
keywords: CRISPR; Cas9; Cholesterol; Filipin; NPC1; sgRNA