CRISPR/Cas9-Mediated Generation of Niemann-Pick C1 Knockout Cell Line

Ximing Du; Ivan Lukmantara; Hongyuan Yang

doi:10.1007/978-1-4939-6875-6_7

CRISPR/Cas9-Mediated Generation of Niemann-Pick C1 Knockout Cell Line

Methods Mol Biol. 2017:1583:73-83. doi: 10.1007/978-1-4939-6875-6_7.

Authors

Ximing Du¹, Ivan Lukmantara¹, Hongyuan Yang²

Affiliations

¹ School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW, 2052, Australia.
² School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW, 2052, Australia. [email protected].

PMID: 28205168
DOI: 10.1007/978-1-4939-6875-6_7

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.

Keywords: CRISPR; Cas9; Cholesterol; Filipin; NPC1; sgRNA.

MeSH terms

CRISPR-Cas Systems*
Carrier Proteins / genetics*
Gene Knockdown Techniques / methods*
HeLa Cells
Humans
Intracellular Signaling Peptides and Proteins
Membrane Glycoproteins / genetics*
Models, Biological*
Niemann-Pick C1 Protein
Niemann-Pick Disease, Type C* / genetics
Niemann-Pick Disease, Type C* / metabolism
Niemann-Pick Disease, Type C* / pathology

Substances

Carrier Proteins
Intracellular Signaling Peptides and Proteins
Membrane Glycoproteins
NPC1 protein, human
Niemann-Pick C1 Protein