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Related Experiment Video

Updated: Jul 19, 2025

Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation
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Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation

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PINE-TREE enables highly efficient genetic modification of human cell lines.

Carlye Frisch1, William W Kostes1, Brooke Galyon1

  • 1School of Biological and Health Systems Engineering, Arizona State University, Tempe, AZ 85287, USA.

Molecular Therapy. Nucleic Acids
|August 17, 2023
PubMed
Summary

Prime editing offers precise genome editing. A new fluorescence-based system, PINE-TREE, efficiently enriches prime-edited cells for various genetic modifications and stem cell line generation.

Keywords:
CRISPRMT: RNA/DNAgenome modificationhuman pluripotent stem cellsprime editing

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • Prime editing provides precise genome modification capabilities.
  • Current methods for isolating prime-edited cells are inefficient.
  • CRISPR nuclease-based methods have limitations.

Purpose of the Study:

  • To develop an efficient method for enriching prime-edited cell populations.
  • To establish a real-time, fluorescence-based system for prime editing enrichment.
  • To demonstrate the utility of the new system across various genomic applications.

Main Methods:

  • Development of a fluorescence-based reporter system: prime-induced nucleotide engineering using a transient reporter for editing enrichment (PINE-TREE).
  • Application of PINE-TREE for real-time enrichment of prime-edited cells.
  • Utilizing PINE-TREE for generating clonal isogenic human pluripotent stem cell lines.

Main Results:

  • PINE-TREE enables real-time enrichment of prime-edited cell populations.
  • The system demonstrates broad utility for introducing substitutions, insertions, and deletions.
  • Highly efficient generation of prime-edited cells, including in human pluripotent stem cells.

Conclusions:

  • PINE-TREE significantly improves the efficiency of isolating prime-edited cells.
  • This technology facilitates precise genome editing applications.
  • PINE-TREE is particularly valuable for editing challenging cell types like human pluripotent stem cells.