CRISPR/Cas9-mediated gene knockout in human adipose stem/progenitor cells

Area of Science:

• Cell Biology
• Molecular Biology
• Genetics

Background:

• CRISPR/Cas9 gene knockout (KO) is effective for loss-of-function studies but challenging in primary human cells.
• Human adipose stem/progenitor cells (ASCs) are a valuable cell source for regenerative medicine, requiring efficient genetic manipulation tools.

Purpose of the Study:

• To establish and validate a reliable CRISPR/Cas9 protocol for achieving functional gene KO in human ASCs.
• To demonstrate the efficacy of this protocol using Sprouty1 (SPRY1) as a model gene.

Main Methods:

• CRISPR/Cas9 target sequence selection and lentiviral vector delivery.
• PCR-based mutation detection assay and sequence analysis to confirm gene editing.
• RT-qPCR and Western blotting to assess gene and protein level changes.
• Evaluation of adipogenic differentiation following gene KO.

Main Results:

• A reliable protocol for CRISPR/Cas9 mediated gene KO in human ASCs was established.
• SPRY1 gene knockout was confirmed via mutation detection assays and sequence analysis.
• SPRY1 mRNA and protein levels were significantly reduced post-CRISPR/Cas9 treatment.
• CRISPR/Cas9 mediated SPRY1 KO effectively inhibited adipogenic differentiation, comparable to shRNA gene silencing.

Conclusions:

• The developed CRISPR/Cas9 protocol provides a reliable method for functional gene KO in human ASCs.
• This approach is potentially applicable to other primary cell types, advancing genetic studies and therapeutic applications.
• Effective SPRY1 depletion inhibits adipogenic differentiation in human ASCs.