Gene Editing in Human Pluripotent Stem Cells: Choosing the Correct Path

Area of Science:

• Stem Cell Biology
• Molecular Biology
• Gene Editing Technologies

Background:

• Targeted nucleases enable precise genetic modifications in human pluripotent stem cells (hPSCs).
• The rapid development of new gene editing technologies presents choices for researchers.
• Selecting the appropriate nuclease (ZFNs, TALENs, CRISPR/Cas9) and methodology is crucial for experimental success.

Purpose of the Study:

• To provide an overview of current gene editing technologies for hPSCs.
• To discuss methodologies for generating precise point mutations.
• To compare the pros and cons of different nucleases and editing strategies.

Main Methods:

• Review of Zinc-Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and CRISPR/Cas9 systems.
• Discussion of strategies for homology-directed repair using single-stranded oligodeoxynucleotides (ssODNs) or donor vectors.
• Consideration of piggyBac™ transposase for seamless gene edits.

Main Results:

• Advancements like dimeric CRISPR RNA-guided Fok1 nucleases (RFNs) reduce off-target mutation rates.
• Different nucleases and repair strategies offer distinct advantages depending on the experimental goal.
• The choice of technology impacts the efficiency and accuracy of genetic modifications in hPSCs.

Conclusions:

• Researchers must carefully consider experimental objectives and target loci when selecting gene editing tools.
• Optimized nucleases and repair strategies are essential for successful and precise genetic engineering of hPSCs.
• This review serves as a guide to navigating the evolving landscape of gene editing technologies.