Marker-free co-selection for successive rounds of prime editing in human cells

Prime editing enables the introduction of precise point mutations, small insertions, or short deletions without requiring donor DNA templates. However, efficiency remains a key challenge in a broad range of human cell types. In this work, we design a robust co-selection strategy through coediting of the ubiquitous and essential sodium/potassium pump (Na(+)/K(+) ATPase). We readily engineer highly modified pools of cells and clones with homozygous modifications for functional studies with minimal pegRNA optimization. This process reveals that nicking the non-edited strand stimulates multiallelic editing but often generates tandem duplications and large deletions at the target site, an outcome dictated by the relative orientation of the protospacer adjacent motifs. Our approach streamlines the production of cell lines with multiple genetic modifications to create cellular models for biological research and lays the foundation for the development of cell-type specific co-selection strategies.