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Updated: May 24, 2025
Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System
Published on: November 8, 2017
Novel CRISPR-Cas9 iPSC knockouts for PCCA and PCCB genes: advancing propionic acidemia research
Emilio M García-Tenorio1,2, Mar Álvarez1, Mónica Gallego-Bonhomme3
1Centro de Biología Molecular Severo Ochoa UAM-CSIC, Universidad Autónoma de Madrid, 28049, Madrid, Spain.
Insights
Researchers created gene-edited stem cells to model propionic acidemia (PA), a rare metabolic disorder. These induced pluripotent stem cell (iPSC) models offer a platform for studying PA's mechanisms and developing new therapies.
Area of Science:
- Biochemistry
- Genetics
- Cell Biology
Background:
- Propionic acidemia (PA) is a rare autosomal recessive metabolic disorder.
- It results from mutations in PCCA and PCCB genes, leading to propionyl-CoA carboxylase (PCC) enzyme deficiency.
- This deficiency causes toxic metabolite accumulation and severe metabolic dysfunction.
Purpose of the Study:
- To generate in vitro disease models of PA with isogenic controls.
- To establish a robust platform for therapeutic research in PA.
- To create induced pluripotent stem cell (iPSC) lines with knockout (KO) mutations in PCCA and PCCB genes.
Main Methods:
- CRISPR-Cas9 gene editing was used to introduce KO mutations in PCCA and PCCB genes.
- A healthy control iPSC line was utilized as the starting material.
- Generated KO iPSC lines were characterized for genetic mutations, transcript/protein expression, and enzyme activity.
Main Results:
- Successfully established PCCA and PCCB KO iPSC lines.
- Confirmed frameshift mutations, loss of transcript and protein expression, and reduced enzyme activity.
- Verified pluripotency of the generated iPSC lines, including differentiation potential into three germ layers.
Conclusions:
- The PCCA and PCCB KO iPSC models are valuable tools for studying PA's molecular mechanisms.
- These models hold potential for advancing novel therapeutic strategies for PA.
- The study provides a foundation for future research into rare metabolic disorders.
Abstract:
Propionic acidemia (PA) is a rare autosomal recessive metabolic disorder caused by mutations in the PCCA and PCCB genes, which encode subunits of the mitochondrial enzyme propionyl-CoA carboxylase (PCC). This enzyme deficiency leads to the accumulation of toxic metabolites, resulting in severe metabolic dysfunction. To create ideal in vitro disease models of PA with isogenic controls and provide a robust platform for therapeutic research, we generated two induced pluripotent stem cell (iPSC) lines with knockout (KO) mutations in the PCCA and PCCB genes using CRISPR-Cas9 gene editing in a healthy control iPSC line. The KO iPS cells were successfully established and characterized, confirming the presence of frameshift insertions and deletions in each target gene, as well as the loss of the corresponding transcript, protein expression, and activity. Additionally, the generated iPSC lines exhibit hallmark characteristics of pluripotency, including the potential to differentiate into all three germ layers. Our PCCA and PCCB KO iPSC models provide a valuable tool for studying the molecular mechanisms underlying PA and hold potential for advancing new therapeutic approaches.
