CRISPR/Cas9-mediated Precise SNP Editing in Human iPSC Lines.

Area of Science:

• Stem cell biology
• Genetics
• Molecular biology

Background:

• Human induced pluripotent stem cells (hiPSCs) are vital for developmental biology and disease modeling.
• CRISPR/Cas9 gene editing in hiPSCs often shows low efficiency, limiting precise allele editing for single nucleotide polymorphisms (SNPs), particularly in noncoding genomic regions.

Purpose of the Study:

• To develop an efficient workflow for engineering isogenic hiPSC lines with precise SNP editing.
• To facilitate the generation of homozygous or heterozygous disease risk/non-risk alleles for advanced disease modeling.

Main Methods:

• A unique, transient, and transfection-based protocol was employed for SNP editing in hiPSCs.
• The workflow enables editing of heterozygous SNPs to homozygous risk or non-risk alleles.

Main Results:

• The protocol successfully generated pure and clonal isogenic hiPSC lines.
• All three possible genotypes (homozygous risk, homozygous non-risk, heterozygous) for a target SNP site were obtained simultaneously.
• The entire process was completed within approximately 4 to 5 weeks.

Conclusions:

• This straightforward workflow significantly enhances the ability to create precisely engineered isogenic hiPSC lines.
• It overcomes the limitations of low editing frequency in CRISPR/Cas9 applications for SNP allele editing.
• The method provides a valuable tool for disease modeling and genetic studies using hiPSCs.