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

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.