Modeling and correction of structural variations in patient-derived iPSCs using CRISPR/Cas9

Area of Science:

• Genomics
• Molecular Biology
• Stem Cell Biology

Background:

• Genome engineering advances enable simple mutation correction.
• Precise correction of large structural variations (SVs) like inversions remains challenging.
• Short nucleotide repeat expansions cause genetic disorders.

Purpose of the Study:

• To detail a procedure for modeling and correcting large chromosomal rearrangements and short nucleotide repeat expansions.
• To utilize engineered nucleases in human induced pluripotent stem cells (hiPSCs) for SV correction.
• To establish a method for generating gene-manipulated hiPSC clones with corrected genetic defects.

Main Methods:

• Delivery of engineered nucleases without a donor template into hiPSCs.
• Genotyping and characterization of gene-manipulated hiPSC clones.
• Application of the protocol to correct large inverted segments and short nucleotide repeat expansions.

Main Results:

• Engineered nucleases enabled identification of genomic inversions, reversions, and deletions of short nucleotide expansions within 2 weeks.
• Desired hiPSC clones were generated in 3-4 weeks.
• The protocol successfully corrected large inverted segments and short nucleotide repeat expansions.

Conclusions:

• This protocol provides an efficient method for correcting large chromosomal rearrangements and repeat expansions in hiPSCs.
• The technology holds promise for treating genetic disorders like hemophilia A, fragile X syndrome, Hunter syndrome, and Friedreich's ataxia.
• The method facilitates the generation of corrected hiPSC lines for disease modeling and therapeutic development.