Search-and-remove genome editing allows selection of cells by DNA sequence

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Traditional cell selection relies on marker genes (drug resistance, fluorophores).
• Marker genes can cause unintended physiological effects.
• Existing methods are incompatible with single nucleotide edits.

Purpose of the Study:

• To present SNIPE, a novel marker-free cell selection method.
• To enable selection of cells with precise single nucleotide edits.
• To overcome limitations of current marker-based selection techniques.

Main Methods:

• SNIPE utilizes single nucleotide differences to distinguish edited from unedited cells.
• The method was validated using Cas9 and Cas12a gene editing tools.
• Experiments were conducted across diverse cell types and species.

Main Results:

• SNIPE achieves significant enrichment of precisely edited cells (median 7-fold).
• The approach was successfully validated for 42 distinct gene edits.
• SNIPE was used to revert modern human missense mutations to ancestral states.

Conclusions:

• SNIPE offers a powerful marker-free approach for precise gene editing selection.
• The method has broad applicability in various cell types and species.
• SNIPE demonstrates potential for therapeutic applications, including tumor cell ablation and mutation repair.