Efficient Installation of Heterozygous Mutations in Human Pluripotent Stem Cells Using Prime Editing

Area of Science:

• Stem Cell Biology
• Gene Editing Technologies
• Molecular Genetics

Background:

• Human pluripotent stem cells (hPSCs) are crucial for disease modeling and regenerative medicine.
• Precise genetic modification of hPSCs is essential but challenging, especially for heterozygous variants.
• Existing tools like Cas9 often lead to biallelic edits, limiting the study of dominant mutations.

Purpose of the Study:

• To evaluate prime editing (PE) for introducing heterozygous genetic edits in hPSCs.
• To assess PE's efficiency and applicability across diverse genetic loci and cell types.
• To demonstrate PE's utility in generating isogenic models for dominant mutations and gene-dosage effects.

Main Methods:

• Utilized prime editing (PE) to introduce site-specific heterozygous edits in over 10 genetic loci in hPSCs.
• Applied PE in combination with cellular reprogramming techniques.
• Generated heterozygous induced pluripotent stem cell clones directly from human fibroblasts and peripheral blood mononuclear cells.

Main Results:

• Achieved up to 40% knock-in efficiency for heterozygous edits using PE, without requiring selection steps.
• Successfully introduced heterozygous edits in paralogous genes, which are difficult with endonuclease-based methods.
• Demonstrated direct derivation of heterozygous iPSC clones from primary cells using PE and reprogramming.

Conclusions:

• Prime editing is a powerful tool for generating hPSCs with complex heterozygous edits.
• PE facilitates the creation of precise isogenic models for dominant mutations and gene-dosage effects.
• This technology advances the development of cellular models for studying genetic diseases.