Biallelic genome engineering to create isogenic induced pluripotent stem cells modeling Huntington's disease

Area of Science:

• Stem Cell Biology
• Genetics
• Neurodegenerative Diseases

Background:

• Huntington's disease (HD) is a fatal autosomal dominant neurodegenerative disorder.
• Accurate modeling of HD pathogenesis requires precise genetic manipulation of relevant cell types.
• Induced pluripotent stem cells (iPSCs) offer a valuable platform for disease modeling.

Purpose of the Study:

• To develop a robust method for generating Huntington's disease (HD) modeling induced pluripotent stem cells (iPSCs).
• To establish a genome engineering technique enabling precise biallelic modification of iPSCs.
• To create isogenic iPSC lines with varying polyQ repeat lengths for studying HD.

Main Methods:

• Development of the allele-specific universal knock-in system (asUKiS), a homologous recombination-based technique.
• Application of asUKiS for scarless, allele-by-allele substitution of the polyQ-repeat region and surrounding genetic modifiers in healthy donor iPSCs.
• Generation of five distinct iPSC lines with controlled polyQ repeat numbers and validated by allele-specific genotyping.

Main Results:

• Successful generation of five Huntington's disease (HD) modeling induced pluripotent stem cell (iPSC) lines.
• Demonstration of the asUKiS system's capability for precise biallelic gene editing.
• Validation of engineered cell lines confirming accurate modification of polyQ repeat lengths.

Conclusions:

• The asUKiS system provides a powerful tool for generating precisely engineered isogenic iPSC lines for disease modeling.
• Biallelic modification in iPSCs minimizes genetic background noise, offering an advantage for studying autosomal dominant diseases like HD.
• This approach facilitates the investigation of genotype-phenotype correlations in Huntington's disease and potentially other genetic disorders.