Simultaneous reprogramming and gene editing of human fibroblasts

Area of Science:

• Stem Cell Biology
• Gene Editing Technologies
• Molecular Biology

Background:

• Human induced pluripotent stem cells (iPSCs) offer significant potential for regenerative medicine and disease modeling.
• Current methods for generating gene-edited iPSCs are often multi-step, time-consuming, and require selection or enrichment.
• Precise genomic modification of iPSCs is crucial for advancing their therapeutic and research utility.

Purpose of the Study:

• To develop a simplified, one-step protocol for generating clonally derived gene-edited human iPSC lines.
• To enhance the efficiency and reduce the time required for iPSC gene editing.
• To provide a cost-effective and reliable method for introducing specific genetic modifications into iPSCs.

Main Methods:

• Utilized enhanced episomal-based reprogramming combined with CRISPR/Cas9 gene editing.
• Employed a Cas9 variant with reduced nonhomologous end-joining (NHEJ) activity to minimize off-target mutations.
• Performed a single electroporation of human fibroblasts, bypassing drug selection or FACS enrichment.

Main Results:

• Successfully generated clonally derived, gene-edited iPSC lines in a single step.
• Achieved both monoallelic and biallelic gene editing, including base changes and transgene insertions.
• Obtained gene-edited and passage-matched unmodified iPSC lines efficiently and rapidly (approximately 6-8 weeks).

Conclusions:

• The developed one-step protocol significantly streamlines the generation of gene-edited iPSCs.
• This method offers an efficient, rapid, and cost-effective approach for precise genomic modification of iPSCs.
• The protocol facilitates the creation of valuable research tools for various biological and medical applications.