ASSURED-optimized CRISPR protocol for knockout/SNP knockin in hiPSCs

Area of Science:

• Stem Cell Biology
• Gene Editing Technologies
• Genomics

Background:

• Human induced pluripotent stem cells (hiPSCs) are crucial for disease modeling.
• CRISPR-Cas9 technology offers precise gene modification capabilities.
• Existing CRISPR-Cas9 methods can be complex and time-consuming for generating modified hiPSC clones.

Purpose of the Study:

• To present an optimized CRISPR-Cas9 pipeline, named ASSURED, for efficient generation of gene-modified hiPSC clones.
• To provide a user-friendly, rapid, and efficient workflow for creating knockout or single-nucleotide-polymorphism-modified knockin hiPSCs.
• To facilitate precise disease modeling using precisely engineered hiPSC-derived cell types.

Main Methods:

• Detailed pipeline for analyzing targeted genomic sequences and designing guide RNAs and homology repair templates.
• CRISPR-Cas9 delivery workflow optimized for hiPSCs.
• Automated cell isolation and clone screening methods to evaluate editing efficiency.

Main Results:

• The ASSURED pipeline enables the production of single-cell-derived, gene-modified hiPSC clones.
• Demonstrated efficiency in generating both knockout and single-nucleotide-polymorphism-modified knockin hiPSCs.
• The workflow is designed to be affordable, successful, specific, user-friendly, rapid, and efficient.

Conclusions:

• The ASSURED pipeline provides a robust and streamlined approach for generating gene-edited hiPSCs.
• This optimized method supports precise disease modeling and the development of novel cell-based therapies.
• ASSURED enhances the accessibility and efficiency of CRISPR-Cas9 applications in stem cell research.