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

Ximing Du1, Ivan Lukmantara1, Hongyuan Yang2

  • 1School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW, 2052, Australia.

Methods in Molecular Biology (Clifton, N.J.)
|February 17, 2017
PubMed

Insights

Researchers developed a CRISPR/Cas9 method to create a Niemann-Pick Type C (NPC) cellular model. This technique disrupts the NPC1 gene in HeLa cells, enabling cholesterol metabolism studies and drug screening for NPC disease.

Area of Science:

  • Cell Biology
  • Genetics
  • Molecular Biology

Background:

  • Niemann-Pick Type C (NPC) disease is characterized by intracellular cholesterol accumulation.
  • Understanding cholesterol trafficking is crucial for NPC disease research and therapeutic development.
  • CRISPR/Cas9 gene editing technology offers precise genetic manipulation capabilities.

Purpose of the Study:

  • To establish a reliable protocol for generating a cellular model of NPC disease.
  • To utilize CRISPR/Cas9 technology to disrupt the NPC1 gene in HeLa cells.
  • To create a cellular phenotype mimicking NPC disease for research and drug screening.

Main Methods:

  • CRISPR/Cas9 system employed for targeted gene disruption.
  • NPC1 gene knockout in HeLa cells using single guide RNA (sgRNA) oligo cloning.
  • Cell colony selection and isolation of genetically modified cells.
  • Verification of the NPC phenotype through filipin staining and immunoblotting.

Main Results:

  • Successful disruption of the NPC1 gene in HeLa cells using CRISPR/Cas9.
  • Generation of a cellular phenotype exhibiting cholesterol storage, characteristic of NPC disease.
  • Validated the protocol's efficacy in creating a reliable cellular model.

Conclusions:

  • The described protocol effectively generates a cellular NPC cholesterol storage phenotype using CRISPR/Cas9.
  • This method provides a valuable tool for investigating NPC disease mechanisms.
  • The established cell line is suitable for high-throughput drug screening and therapeutic development for NPC disease.
Keywords:
CRISPRCas9CholesterolFilipinNPC1sgRNA

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